Vol 12 No 1 June 2013
EGYPTIAN PHARMACEUTICAL JOURNAL
Editorial board
Editor-in-Chief
Aida El-Azzouny
Egypt
[email protected]
+202 33371362/33371433
+2 0100 52 54 161
+202 33370931/3601877
Complete professional affiliations:
Pharmaceutical and Drug Industries Research Division,
National Research Centre (NRC), Dokki-Cairo,
12622-Egypt
Specialization: Medicinal and Pharmaceutical Chemistry
Deputy-Editors
Abdel-Hamid Zaki Abdel-Hamid Amer
Egypt
[email protected].
+201002020747
Complete professional affiliation:
Pharmaceutical and Drug Industries Research Division,
National Research Centre (NRC), Dokki-Cairo,
12622-Egypt
Specialization: Applied Biochemistry
Mohamed Ahmed Abdel-Naby
Egypt
+202 24708049
+201149921388
+202 3370931/3601877
[email protected]
Complete professional affiliations:
Pharmaceutical and Drug Industries Research Division,
National Research Centre (NRC), Dokki-Cairo,
12622-Egypt
Specialization: Professor of Biochemistry
Editorial Assistants
Hassan Abdel Zaher Mohamed Mohamed AMER
Egypt
[email protected]
00201227341899
Complete professional affiliations:
National Research Centre
Center of Excellence for Advanced Sciences,
Dept of Natural and Microbial Products Chemistry
Division of Pharmaceutical and Drug Industries
Dokki, El-behoos Street
Cairo, Egypt
Tel: +201227341899
Specialization:
Associate professor of bioorganic Chemistry
Mohammad H. A. Ibrahim
Egypt
[email protected]
+201150935326
Complete professional affiliations:
Chemistry of Natural and Microbial Products Dept.,
Pharmaceutical & Drugs Industries Research Division,
National Research Centre, Al-Bohoos st., Dokki,
12622 Cairo, Egypt
Specialization: Microbial Biotechnology, Fermentation
Technology, Bioplastics
Mona E. Aboutabl
Egypt
[email protected]
+2011155 330 72
Complete professional affiliations:
Researcher of Pharmacology, Room # 374, Medicinal and
Pharmaceutical Chemistry Department, Pharmaceutical and
Drug Industries Research Division, National Research Center,
El-bohous St., Dokki, Cairo, 12311 Egypt
Specialization: Pharmacology and Toxicology
Vol 12 No 1 June 2013
EGYPTIAN PHARMACEUTICAL JOURNAL
Table of contents
Review article
1
D2-dopaminergic receptor and 5-HT3 serotoninergic receptor antagonists having antiemetic profile
Mohamed N. Aboul-Enein, Aida A. EL-Azzouny, Yousreya A. Maklad, Mohamed I. Attia, Mohamed Abd EL-Hamid Ismail and
Walaa H.A. Abd EL-Hamid
Original articles
11
Synthesis and DPPH radical-scavenging activity of some new 5-(N-substituted-1H-indol-3-yl)-5H-thiazolo[4,3-b]-1,3,
4-thiadiazole derivatives
Heba M. Abo-Salem, Manal Sh. Ebaid, Eslam R. El-Sawy, Abd El-Nasser El-Gendy and Adel H. Mandour
20
Synthesis and antihypertensive activity of certain substituted dihydropyridines and pyrimidinones
Wageeh S. El-Hamouly, Kamelia M. Amine, Hanaa A. Tawfik and Dina H. Dawood
28
Immobilization of Mucor racemosus NRRL 3631 lipase and characterization of silica-coated magnetite (Fe3O4)
nanoparticles
Abeer A. El-Hadi, Hesham I. Saleh, Samia A. Moustafa and Hanan M. Ahmed
36
Extracellular polysaccharides produced by the newly discovered source Scopularis spp.
Siham A. Ismail
40
Biotransformation of soybean saponin to soyasapogenol B by Aspergillus parasiticus
Hala A. Amin, Yousseria M. Hassan and Soad M. Yehia
46
Characterization of ternary solid dispersions of nimesulide with Inutec SP1 and b-cyclodextrin and evaluation of
anti-inflammatory efficiency in rats
Rawia M. Khalil, Mamdouh M. Ghorab, Noha Abd El Rahman and Silvia Kocova El-Arini
57
DNA fingerprinting and profile of phenolics in root and root calli of Arctium lappa L. grown in Egypt
Elsayed A. Aboutabl, Mona El-Tantawy, Nadia Sokkar and Manal M. Shams
63
Influence of formulation parameters on the physicochemical properties of meloxicam-loaded solid lipid nanoparticles
Rawia M. Khalil, Ahmed Abd El-Bary, Mahfoz A. Kassem, Mamdouh M. Ghorab and Mona Basha
73
Effect of pollution on the chemical content and secondary metabolites of Zygophyllum coccineum and Tamarix nilotica
Hanan E. Osman and Reham K. Badawy
83
Optimization of growth conditions and continuous production of inulinase using immobilized Aspergillus niger cells
Nagwa A. Atwa and Enas N. Danial
Short communication
90
Chemical constituents from the aerial parts of Salsola inermis
Fatma S. Elsharabasy and Ahlam M. Hosney
Vol 12 No 1 June 2013
EGYPTIAN PHARMACEUTICAL JOURNAL
Instructions to Authors
Papers must be submitted with the understanding that they have not been published elsewhere (except in the form of an abstract or
as part of a published lecture, review, or thesis) and are not currently under consideration by another journal or any other publisher.
The submitting author is responsible for ensuring that the article’s publication has been approved by all the other coauthors. It is also
the authors’ responsibility to ensure that the articles emanating from a particular institution are submitted with the approval of the
necessary institution. Only an acknowledgment from the editorial office officially establishes the date of receipt. Further
correspondence and proofs will be sent to the author(s) before publication, unless otherwise indicated. All manuscripts must be
written in clear and correct English. Corrections of spelling, grammar and typing are the responsibility of the author.
All enquiries concerning the publication of accepted papers should be addressed to: [email protected] and/or
[email protected]
Electronic submission:
Manuscripts may be submitted by e-mail to: [email protected] and/or [email protected]
E-mail submission should come as files attached to the e-mail, in a format that can be read by MS word 98. Figures or illustrations
should be sent in a high resolution format.
The Egyptian Pharmaceutical Journal (EPJ) accepts manuscripts via e-mail as attachment. It is the condition for the acceptance
of manuscripts that they have not been previously published elsewhere nor are under consideration by any other periodical. Decision
about acceptance of a manuscript for publication in EPJ is based on a double-blinded peer review process.
Manuscript preparation:
Manuscripts should be written in Word file format, double spaced, Times New Roman, size 12, regular style. All of the pages of the
manuscript should be numbered consecutively, beginning with the title page.
It is strongly recommended that authors ensure that the words used in manuscripts convey the intended meaning, both accurately
and clearly. Failure to do so may delay reviewing and publication of the manuscript. For a manuscript submitted for the sole purpose
of publication in EPJ, each of the following components of a manuscript should begin on a new page.
(a) Abstract
(b) Manuscript Text
(c) Figures & Table (if applicable)
(d) Acknowledgements (if any)
(e) References
Title page: should carry the following information:
a: The title of the article should be concise and informative.
b: Authors’ names in order. These should be written in the form of first name, middle initial(s) and surname. Institutions of the authors
should be included. The authors are numbered sequentially according to institutions using upper case Arabic numerical.
c: Affiliation: Name of department/ institution of author(s).
d: Corresponding Author: Full Name, postal address, telephone/fax number with country code, and valid e-mail address.
e: Running Title: A running title of maximum 50 characters.
Abstract: A maximum of 300 words drawn from the study briefly describing the aim, methods used, the results obtained and the
principal conclusions. Do not include any reference in the abstract and use few abbreviations if necessary.
Keywords: A list of 3-10 keywords in English for indexing purposes.
Manuscript Text
For a complete manuscript the following applies:
Introduction: To acquaint the reader with the area under investigation. It should cite related important and recent work by others.
Subjects and Methods: These should be sufficiently described. References for applied methods should be cited.
Results: Should be briefly presented in text, tables and figures.
Discussion: The discussion should focus on the interpretation and significance of the findings and their relation to other work in the
field. Numbers of References, Tables and Figures mentioned in the text should be written in Arabic numerals.
Conclusions: Should focus on final outcomes and future applications.
Acknowledgements: Financial grant from institutes or technical support should be mentioned, and similarly each author’s conflict
of interest contributed to the work.
References: It is the authors’ responsibility to check all references very carefully for accuracy and completeness. They should be in
Vancouver style and numbered in the order in which they appear in the manuscript text, do not alphabetize.These numbers should be
inserted in brackets [ ] each time the author cited, for example, [6]. Please keep in mind that ‘‘unpublished observations’’ and
‘‘personal communications’’ cannot be used as references. Examples of references used in a manuscript exclusively for EPJ are
given below.
Reference to an article
Duflos A, Redoules F, Fahy J, Jacquesy JC, Jouannetaud MP. Hydroxylation of yohimbine in superacidic media: One-step access to
human metabolites 10 and 11-hydroxyyohimbine. J Nat Prod 2001; 64: 193-5.
Mention up to 6 authors in the manner described above; if more than 6, list the first 6 followed by et al. The title of the journals should
be abbreviated according to the style used in Index Medicus, while complete name of non–indexed journals should be written.
Personal communications and unpublished data should not feature in the reference list, but should appear in parenthesis in the text.
Unpublished work accepted for publication should be listed in the reference list with the words (In Press) in parenthesis with the
name of the journal concerned.
References must be checked individually and verified by the author against original documents.
Reference to a book
William JG, Cindy LS. Principles of Human Physiology. 2nd ed. Benjamin Cummings, San Francisco: 2005.
Reference to a book chapter
Aaron T. Megakaryocytopoisis Kinetics and Platelet Radiolabeling. In Rossi’s Principles of Transfusion Medicine. 3rd ed.
pp179-184, Lippincott Williams NY: 2002.
Tables
Acceptable tables as part of manuscript text submitted for EPJ include Word; and Excel. Please, make sure that tables are
appended to the end of the manuscript text file. Tables should neither be submitted as separate file, nor as photographs. They
should be numbered consecutively in the order they appear in the text with brief titles. Place explanations of table in the footnotes.
Do not use internal horizontal and vertical rules. If data from another published source are used, the author should obtain permission
for publication prior to submission of the manuscripts and acknowledge fully.
Figures
All figures (max 4) must be submitted in a finished form, ready for reproduction. They are required to show the essential features
described in the text. A separate file should be submitted for each Figure or Figure part. Line figures should be professionally drawn
and submitted electronically, or scanned using a 600 DPI (or better) printer. A separate image is required for each figure part.
Letters, numbers and symbols should be clear and even throughout and of sufficient size so that when they are reduced in size for
publication, each item will be legible.
Figures should be numbered consecutively in Arabic numerals according to the order in which they have been first cited in the text.
All figure parts relating to one subject should have the same figure number. Symbols, arrows, or letters used in photomicrographs
should contrast with the background.
Units of measurements
Measurements of length, height, weight and volume should be reported in metric units (meters, kilograms or liters) or their decimal
multiples. Temperatures should be given in degrees Celsius. Blood pressure should be given in millimeters mercury. All
hematological and clinical chemistry measurements should be reported in the metric system in terms of International system of units.
Legends for Illustrations
Present the legends for illustrations using double-spacing, with Arabic numerals corresponding to the Illustrations. When symbols,
arrows, numbers or letters are used to identify parts of the illustrations, explain each one clearly in the legend. Explain the internal
scale and identify the method of staining and original magnification for photomicrographs.
Abbreviations and Symbols
Use only standard abbreviations. Avoid abbreviations and symbols in the title and abstract. The full term for which an abbreviation
stands should proceed, unless it is a standard unit of measurement.
Ethics committee approval
Submission of a manuscript to EPJ implies that all authors have read and agreed to its content and that any experimental research
that is reported in the manuscript has been performed with the approval of an appropriate ethics committee. Research carried out on
humans must be in compliance with the Helsinki Declaration. A statement to this effect should appear in the Methods section,
including the name of the body which gave approval. Informed consent should also be documented. Similarly, for experiments
involving animals you must state the care of animal and licensing guidelines under which the study was performed. If ethics
clearance was not necessary, or if there was any deviation from these standard ethical requests, please state why it was not
required. Please note that the editors may ask you to provide evidence of ethical approval. If you have approval from a National Drug
Agency (or similar), please state this and provide details, this can be particularly useful when discussing the use of unlicensed
drugs. Manuscripts may be rejected, if the editorial office considers that the research has not been carried out within an ethical
framework.
Copyright assignment
All submissions must be accompanied by a Copyright Assignment which should be signed by corresponding author. The copyright
can be submitted with the manuscript online, or mailed separately to:
Prof. Dr. Aida El-Azzouny
Editor in Chief
Egyptian Pharmaceutical Journal (EPJ)
Pharmaceutical and Drug Industries Research Division, National Research Centre (NRC), El Behouth St., Dokki, Cairo, Egypt.
Postal Code. 12311.
E-mail: [email protected]; [email protected]
Tel: (+202) 33371362/33371433
Fax: (+202) 33370931
Submitted manuscripts which do not meet the above guidelines will be returned to the corresponding author prior review process.
DO NOT submit the manuscript in PDF file. The Editorial board keeps unconditional right to determine the style and if necessary,
edit and shorten any material accepted for publication in EPJ.
All communications with the Editor/Editorial staff regarding manuscripts must be in WRITTEN form, preferably via e-mail:
[email protected] and/or [email protected]
The decision on the priority of publication is strictly determined by the Editorial Board.
Vol 12 No 1 June 2013
EGYPTIAN PHARMACEUTICAL JOURNAL
Journal Description
Egyptian Pharmaceutical Journal (EPJ) is a peer-reviewed journal, published biannually by the Pharmaceutical and Drug
Industries Research Division, National Research Centre (NRC), Cairo, Egypt. EPJ provides a forum for exploration of the rapidly
developing pharmaceutical sciences which are relevant to all pharmaceutical and health care professionals, as well as those
involved in the development and application of the advanced technology in the field of pharmaceutical research.
EPJ publishes high quality fundamental and applied research articles in all aspects of pharmaceutical sciences. The scopes of the
journal include the following topic areas: pharmaceutics, pharmaceutical technology, industrial pharmacy, phytochemistry, medicinal
and pharmaceutical chemistry, biochemistry, analytical pharmaceutical chemistry, microbiology, microbial biotechnology, molecular
pharmaceutics, pharmacology, toxicology, clinical pharmacy, nutraceuticals, aroma therapy, medicinal plants as well as all areas of
pharmaceutical sciences and related fields.
Authors are encouraged to submit review papers, full-length original research papers, short communications, and book reviewers,
which are not submitted to other journals. The journal also features annotations, short reports, and letters to the editor. The journal is
published in online version.
Review article 1
D2-dopaminergic receptor and 5-HT3 serotoninergic receptor
antagonists having antiemetic profile
Mohamed N. Aboul-Eneina, Aida A. EL-Azzounya, Yousreya A. Maklada,
Mohamed I. Attiaa,b, Mohamed Abd EL-Hamid Ismailc
and Walaa H.A. Abd EL-Hamidd
a
Department of Medicinal and Pharmaceutical
Chemistry, Pharmaceutical and Drug Industries
Research Division, National Research Centre, Giza,
b
Department of Pharmaceutical Chemistry, College of
Pharmacy, King Saud University, Riyadh, Saudi Arabia,
c
Department of Pharmaceutical Chemistry, Faculty
of Pharmacy, Ain Shams University, Cairo and
d
Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, Misr University for Science &Technology,
6th of October City, Egypt
Correspondence to Mohamed N. Aboul-Enein,
Department of Medicinal and Pharmaceutical
Chemistry, Pharmaceutical and Drug Industries
Research Division, National Research Centre,
12622 Dokki, Giza, Egypt
Tel: + 20 012 216 8624; fax: + 20 233370931;
e-mail: [email protected]
Received 15 January 2013
Accepted 11 March 2013
Metoclopramide is the prototype of the orthopramide family and is used clinically as a
stimulant of upper gastrointestinal motility and as an antiemetic. Its antiemetic potential
is attributed mainly to the antagonist activity at D2-dopaminergic receptors in the
chemoreceptor trigger zone of the central nervous system. Besides, ondansetron was
the first selective 5-HT3 serotoninergic receptor antagonist used in clinics as an
antiemetic. Herein, the antiemetic profile of different chemical classes of D2dopaminergic receptor and 5-HT3 serotoninergic receptor antagonists will be
discussed, which may be helpful in the development of potent antiemetic agents.
Keywords:
antagonists, antiemetic, D2-dopaminergic receptor, 5-HT3 serotoninergic receptor
Egypt Pharm J 12:1–10
& 2013 Division of Pharmaceutical and Drug Industries Research,
National Research Centre
1687-4315
Egyptian Pharmaceutical Journal
2013, 12:1–10
Introduction
Vomiting is the forceful repulsion of the contents of one’s
stomach through the mouth and sometimes the nose and
it may result from many causes, ranging from gastritis or
poisoning to brain tumor or elevated intracranial pressure.
It is generally considered to be a protective mechanism by
which undesirable substances are evacuated quickly from
the gastrointestinal tract. Vomiting is different from
regurgitation, although the two terms are often used
interchangeably. Regurgitation is the return of undigested
food back up the esophagus to the mouth, without the
force and displeasure associated with vomiting. Nausea,
which also has an impact on the gastrointestinal tract, is
the sensation of discomfort in the upper stomach with the
urge to vomit [1]. Persistent nausea may lead to loss of
appetite and reduction of food uptake until the point of
malnutrition and debilitation. Gastrointestinal infections
(37%) and food poisoning are the most common causes of
nausea and vomiting besides side effects from medications
(3%) and pregnancy [1,2]. In 10% of people the cause
remains unknown [2]. Prolonged and severe vomiting leads
to hypochloremia, hypokalemia, alkalosis, and dehydration;
it can even cause death, especially in children. Therefore,
treatment should be directed mainly toward eliminating the causes of illness. This review focuses on the
antiemetic agents that potentially act as antagonists to the
D2-dopaminergic and 5-HT3 serotoninergic receptors.
Mechanism of emesis
The act of emesis is very complicated and involves a
series of coordinated activities and changes in the
respiratory and gastrointestinal musculature. It is usually
preceded by salivation, nausea, malaise, lassitude, weakness, retching movements, and characteristic postures
of the head and body adopted to final expulsion of
vomitus [3,4]. This order of events indicates the
existence of at least two central areas concerned with
the vomiting act, namely, the chemoreceptor trigger zone
(CTZ), which can be stimulated by chemical agents such
as the dopaminergic apomorphine and transmits impulses
to the vomiting centre itself, which is located in the
reticular core of the medulla [2].
The latter center lies in proximity to the other centers
such as inspiratory and expiratory centers, the vasomotor
center, salivary nuclei, and vestibular nuclei. The action
of all these centers may manifest as the act of
vomiting [2]. Impulses from all these centers pass
through the CTZ to the vomiting center, resulting in
emesis [5–7].
Sites other than the CTZ may be effective in the
stimulation of emesis. Thus, visceral afferent impulses
mediated through the parasympathetic and sympathetic
routes transmit to the vomiting center impulses that
result in the genesis of vomiting [8,9].
Antiemetic agents
Antiemetic agents are drugs that are used for the
prophylaxis, control, and prevention of nausea and
vomiting. Emesis is the main symptom for motion
sickness, during the first trimester in pregnancy, in the
case of hyperemesis gravidarum, and of radiation sickness
1687-4315 & 2013 Division of Pharmaceutical and Drug Industries Research,
National Research Centre
DOI: 10.7123/01.EPJ.0000428875.20180.de
2 Egyptian Pharmaceutical Journal
Figure 1
S
O
N
NH
O
O
O
N
HN
S
N
N
Cl
H2N
O
Dompridone(Motillum ®)
Metopimazine
1
2
Examples of D2-receptor antagonist.
resulting during the treatment of tumors by irradiation
and using cytotoxic drugs. In addition, postoperative
vomiting can also occur, which may be due to the use of
general anesthetics and opiate analgesics after surgical
operations. Gastrointestinal irritation because of peptic
ulcer and ulcerative colitis also leads to nausea and
vomiting. Antiemetics include various classes and groups
of drugs with versatile pharmacological mechanisms.
In 1950, the first neuroleptic phenothiazine prototype,
chlorpromazine 4, was introduced. It possesses a large
number of pharmacological activities such as adrenolytic,
antidopaminergic, antihistaminic, antiserotonin, antimuscarinic, and antiemetic properties [18–22]. Subsequently,
numerous phenothiazines have been introduced such as
antiemetics with increased antiemetic potency, reduced
cardiovascular effects, milder tranquilizing action, and
decreased extrapyramidal side effects [23–25].
Dopaminergic antagonists
The difference in antiemetic activities between the
neuroleptic phenothiazines is because of differences in
the site of their sequestration in the central nervous
system; however, they act predominantly on the CTZ.
The neurotransmitter dopamine plays an important role
in neural functions involving reward processes, approach
behavior, economic decision making, adaptive behavior,
motion, and cognition [10]. Dopamine receptors belong
to two subclasses, with D1 and D5 receptors sharing
homology and coupling with Gs and D2, D3, and D4
receptors coupling with Gi.
Selective D1-receptor antagonists have been studied as
potential therapeutics for Parkinson’s disease, psychotic
behavior, substance abuse, and obesity [11] in animal
models and in human clinical trials [12].
D2-receptor antagonists were the first antiemetics used;
these drugs are currently primarily used as rescue
antiemetics. The primary reason for the ignorance of
these drugs is frequent induction of extrapyramidal side
effects such as akinesia, akathisia, and acute dystonic
reactions. Domperidone 1 and metopimazine 2 are
examples of D2-receptor antagonists that are effective
against nausea, which is a more troublesome chemotherapy-induced side effect than vomiting; these do not cause
extrapyramidal side effects (Fig. 1) [13].
Phenothiazines
Phenothiazine was first synthesized by Bernthsen in
1883. In 1934 [14], it was found to possess insecticidal
properties. Later, Hardwood et al. [15] discovered the
anthelmintic activity of this compound in swine ascaridosis. In 1946, Halpern and Ducrot [16] screened various
phenothiazine compounds for antihistaminic properties.
The first compound with definite therapeutic value was
promethazine 3, which has proven antihistaminic as well
as sedative and hypnotic properties [17].
Phenothiazines are classified into three groups according
to the substituents on nitrogen: (i) aliphatic analogues,
which bear acyclic groups; (ii) piperidines, which contain
piperidine-derived groups; (iii) piperazine, which incorporate piperazine-derived substituents. The most relevant antiemetic phenothiazines (3–15) are illustrated
in Table 1.
Butyrophenones
Neuroleptic drugs like droperidol 16 and haloperidol 17
are major tranquillizing drugs, which possess significant
antiemetic activity as a result of their D2-receptor
antagonist properties, especially when administered
through the intravenous and intramuscular routes [43].
However, side effects [44,45] such as drowsiness,
dysphoria, delayed discharge, extrapyramidal reactions,
restlessness, and anxiety after discharge have led to the
current reluctance to use these agents in the outpatient
setting. The structures of the commonly used antiemetic
butyrophenones are depicted in Table 2.
Benzamides
Metoclopramide hydrochloride monohydrate (Primperan
21; Table 3) is a benzamide derivative related to
orthopramides class that belongs to the neuropsychotropic, antipsychotic neuroleptics (Table 3). It is a
derivative of procainamide but it is virtually devoid of
antiarrhythmic or local anesthetic activity in clinical
doses [67]. It shows both central and peripheral
Receptors antagonists as antiemetics Aboul-Enein et al. 3
Table 1 Antiemetic phenothiazines (3–15)
S
N
R2
R1
Name
Aliphatic analogues Promethazine (3, Phenergan)
Chlorpromazine (4, Largactil)
Promazine (5, Sparine)
Triflupromazine (6, Vesprin)
Piperidines
Pipamazine (7, Mornidine)
R1
–CH2-CH(CH3)N(CH3)2
–(CH2)3N(CH3)2
–(CH2)3N(CH3)2
–(CH2)3N(CH3)2
O
(H2C)3
References
H
Cl
CF3
H
Cl
Couvoisier and colleagues [26,27]
Delay and colleagues [28,29]
Wirth [30]
Yale and colleagues [31,32]
Dobkin and Purkin [33]
C NH2
N
Mepazine (8, Pacatal)
R2
H
CH3
Bowes [34]
N
H2C
SO2CH3 Jacob and colleagues [35,36]
Metopimazine (9, Compazine)
Piperazines
(H2C)3
N
CONH2
(H2C)3
N
(H2C)3
N
(H2C)3
N
N CH2 CH2 OH
(H2C)3
N
N CH3
(H2C)3 N
N CH3
CF3
Trifluoperazine (10, Stelazine)
Tedeschi et al. [37]
N CH3
SCH2CH3 Bourquin et al. [38]
Thiethylperazine (11, Torecan)
N CH3
Perphenazine (12, Trilafon)
Prochlorperazine (13, Campizine )
Cl
Hotovy and Kapff-Walter [39]
Cl
Gralla et al. [40]
CF3
Fluphenazine (14, Prolixin)
Cl
Thiopropazate (15, Dartal)
(H2C)3 N
antiemetic activities. It is rapidly absorbed orally but has
a wide range of oral bioavailability. It has a plasma half life
of 4–6 h. In addition to its ability to block dopaminergic
receptors at the CTZ, metoclopramide increases lower
esophageal sphincter tone and enhances gastric and small
bowel motility, thereby preventing delayed gastric
emptying caused by opioid analgesics [68]. Although it
is not effective in controlling motion sickness, metoclopramide has some peripheral cholinergic actions.
High doses of metoclopramide (1–2 mg/kg) are effective
in managing chemotherapy-induced emesis [69]; however, this is associated with high incidence of dystonic
reactions and extrapyramidal side effects.
It has been successfully used to treat dyspepsia,
gastrointestinal disorders, including irritable colon syndrome, and spastic constipation [67].
N CH2 CH2
Kline and Simpson [41]
Toldy et al. [42]
COCH3
This gastroprokinetic activity is attributed to the release
of acetylcholine upon stimulation of 5-HT4 receptors,
whereas the antiemetic activity is attributed to the
antagonistic activity at both 5-HT3 serotoninergic and
D2-dopaminergic receptors in the CTZ of the central
nervous system. In addition, it stimulates orthograde
peristalsis, leading to suppression of the bile reflux, with
subsequent promotion of healing of gastric ulcers and
prevention of relapse. However, metoclopramide does not
accelerate healing of duodenal ulcers [70].
The antiemetic activity of metoclopramide as an
antiapomorphine drug is 35 times greater and more
selective than that of chlorpromazine [71]. Further, it
shows no sedative action at its antiemetic doses [71].
However, at large doses it produces extrapyramidal
side effects. It exerts its antiemetic activity through
4 Egyptian Pharmaceutical Journal
Table 2 The commonly used antiemetic butyrophenones (16–20)
Name
Structure
Benperidol 16
References
Bobon et al. [46]
O
N
F
N
O
N
H
Droperidol 17
H
N
Domino et al. [47]
O
N
O
N
F
Granger and Albu [48]
Haloperidol 18
N
Cl
F
O
OH
Lenperone 19
Nakra et al. [49]
O
O
N
F
F
Melperone 20
Grözinger et al. [50]
O
N
F
central [72,73] and peripheral [74] dopamine receptor
antagonism. Moreover, metoclopramide is ineffective
against motion sickness and emesis occurring in labyrinthine episodes [71].
Orthopramides possess three common structural elements
required for binding to the receptor site: an aromatic
moiety, a carbonyl group or carbonyl group bioisosteres,
and a basic nitrogen atom. The weak affinity and lack of
selectivity of metoclopramide for dopaminergic and
serotoninergic receptors can be explained by the large
number of permissible conformers because of the
flexibility of its amino chain. Accordingly, Aboul-Enein
and colleagues [75,76] studied certain molecular modifications of metoclopramide, which imply (i) a change in the
substituents of the aromatic ring, (ii) structural variations
in the amine moiety, and (iii) an increase in the
lipophilicity through a change in the vicinal carbon atom
of the basic nitrogen to a cyclohexane ring (22–24; Fig. 2).
These compounds were evaluated for their dopamine
D2-receptor antagonistic activity in vivo by measuring
their ability to inhibit apomorphine-induced chewing
‘‘Zwansgnagen’’ in rats. Among these compound, 24
possessed an ED50 of 5.94 mmol/kg, being nearly two-fold
more potent than the previously reported cyclohexanebased dopamine D2-receptor antagonist 23 (ED50 =
11.66 mmol/kg). Molecular simulation study of 24,
including fitting to the three-dimensional model of
dopamine D2-receptor antagonists using Discovery Studio 2.5 programs showed high-fit values [75]. The
experimental dopamine D2-receptor antagonistic activity
was consistent with the findings of the molecular
modeling study. Other substituted benzamides (Table 3)
that have been evaluated as antiemetics include
trimethobenzamide (25, Tigan), clebopride (27), cisapride (30), and alizapride (39) [77]. Trimethobenzamide
is an antiemetic having some structural similarities to
both reserpine and antihistamines [78] as well as to
orthopramides. It possesses one-tenth to one-twentieth
of the antiemetic activity of chlorpromazine. Its antiemetic
action is primarily on the CTZ. Trimethobenzamide does
not cause depression at very high doses. It has no sedative,
hypotensive, or extrapyramidal effects; moreover, it shows
no antihistaminic activity, and it proved effective against
vomiting from various causes [79,80].
Cisapride 30 has a greater ability than metoclopramide to
reverse morphine-induced gastric stasis and is not
associated with extrapyramidal side effects. However,
cisapride does not prevent the decrease in lower
esophageal tone following antagonism by neostigmine in
the form of neuromuscular blockade and has lesser
antiemetic activity than metoclopramide.
It is worth mentioning that metoclopramide and its
congeners, besides being potent antiemetics, show
neuroleptic, antidyskinetic, and antiulcer effects, also
Receptors antagonists as antiemetics Aboul-Enein et al. 5
Table 3 Antiemetic benzamides 25–39 (orthopramides)
Name
Structure
Trimethobenzamide 25
O
C N
H
H3CO
Bromopride 23
References
O
N
CH3
Report of the Workgroup on Vaccines [51]
CH3
OCH3
OCH3
C2H5
O
N
C N
C2H5
H OCH
3
Fontaine and Reuse [52]
Br
NH2
Clebopride 27
O
Cl
Cuena Boy and Macia Martinez [53]
N
N
H
OCH3
H2N
Tiapide 28
Fontaine and colleagues [52,54]
C2H5
O
N
C N
C2H5
H OCH3
O
H3C S
O
NH2
Dazopride 29
Lunsford and Cale [55]
C2H5
O
Cl
N
N CH
2 5
N
H
OCH3
H3CO
(CH2)3
N
H2N
Cisapride 30
Van Daele and colleagues [56,57]
O
CO N
H
OCH3
F
Cl
NH2
Burnton and colleagues [58,59]
Troxipide 31
NH
CONH
OCH3
OCH3
H3CO
Sulpiride 32
C2H5
CO NH CH2
OCH3
O
H2N S
Laville and Margarit [60]
N
O
Sultopride 33
C2H5
CO NH CH2
OCH3
Bruguerolle et al. [61]
N
O
C2H5 S
O
Amisulpride 34
C2H5
CO NH CH2
OCH3
O
C2H5 S
O
NH2
N
Florvall and Oegren [62]
6 Egyptian Pharmaceutical Journal
Name
Structure
Itopride 35
References
Florvall et al. [63]
O
OCH3
N
H
N
OCH3
O
Raclopride 36
Florvall and colleagues [63,64]
C2H5
HO
CO NH CH2
OCH3
Cl
Cl
N
Remoxipride 37
Florvall et al. [63]
C2H5
N
CO NH CH2
OCH3
H3CO
Br
Thominet et al. [65]
Veralipride 38
N
CO NH CH2
OCH3
O
H2N S
O
OCH3
Bleiberg et al. [66]
Alizapride 39
N
CO NH CH2
OCH3
N
N
N
H
Figure 2
O
Cl
C2H5
N
N
H
C2H5
OMe
H 2N
O
Cl
22
N R
O
H 2N
C2H5
OMe
H2N
Metoclopramide
21
Cl
C2H5
N
N
H
N
H
OMe
N
N
O
N
N
H
OCH3
OMe
OMe
Cl
R = C2H5
R = CH2C6H5
24
R = CH2C6H2(OCH3)3
R = CH(C6H5)2
23
Metoclopramide and structurally related compounds.
being useful as nonhormonal therapeutic agents in severe
cases of menopausal disorders [67,81].
5-HT3 serotoninergic receptor antagonists
5-HT3 antagonists are a class of medications that act as
receptor antagonists at the 5-HT3 receptor, a subtype of
the serotonin receptors found at the terminal ends of the
vagus nerve and in certain areas of the brain. They are
used as antiemetics in the prevention and treatment of
nausea and vomiting. They are particularly effective in
controlling nausea and vomiting caused by cancer
chemotherapy and are considered the gold standard for
this purpose [82].
5-HT3 receptors are present at several critical sites
involved in emesis, including vagal afferents, the solitary
Receptors antagonists as antiemetics Aboul-Enein et al. 7
Table 4 The 5-HT3 receptor antagonists commonly used as antiemetics
Name
Structure
Ondansetron (40, Zofran)
O
References
Gan [86]
N
N
N
Granisetron (41, Kytril)
N NCH3
O
NCH3
Dolasetron (42, Anzemet)
O
Gebbia et al. [87]
NH
N
Hainsworth et al. [88]
O
O
N
H
Ramosetron (43, Nasea)
O
H
N
Rabasseda [89]
N
N
Palonosetron (44, Aloxi)
N
O
Gebbia et al. [87]
N
H
tract nucleus, and the area postrema itself. Serotonin is
released by the enterochromaffin cells of the small
intestine in response to chemotherapeutic agents and
may stimulate the vagal afferents (through the 5-HT3
receptor) to initiate the vomiting reflux. 5-HT3 receptor
antagonists suppress vomiting and nausea by preventing
serotonin from binding to the 5-HT3 receptors. The
highest concentration of 5-HT3 receptors in the central
nervous system is found in the solitary tract nucleus and
CTZ, and 5-HT3 antagonists may also suppress vomiting
and nausea by acting at these sites [59].
and reduce them in the remaining 30% – they are only as
effective as other agents in controlling postoperative
nausea and vomiting.
Current evidence suggests that 5-HT3 antagonists are
ineffective in controlling motion sickness [85]. A randomized, placebo-controlled trial of ondansetron 40 to
treat motion sickness in air ambulance personnel showed
subjective improvement, but it was statistically insignificant.
Chemical structures of the first generation 5-HT3
receptor antagonists [86] can be categorized into three
main classes (Table 4).
5-HT3 serotoninergic receptor
5-HT3 antagonists are most effective in prevention and
treatment of chemotherapy-induced nausea and vomiting
(CINV), especially that caused by highly emetogenic
drugs such as cisplatin. When used for prevention and
treatment of CINV, they may be administered alone or,
more frequently, in combination with a glucocorticoid,
usually dexamethasone. They are usually administrated
intravenously, shortly before administration of the chemotherapeutic agent [60], although some authors have
argued that oral administration may be preferred [83].
The concomitant administration of an NK1 receptor
antagonist, such as aprepitant, significantly increases the
efficacy of 5-HT3 antagonists in preventing both acute
and delayed CINV [84].
5-HT3 antagonists are also indicated in the prevention
and treatment of radiation-induced nausea and vomiting,
when needed, and postoperative nausea and vomiting.
Although they are highly effective at controlling CINV –
they stop symptoms altogether in up to 70% of people
Carbazole derivatives
Ondansetron 40 was the first 5-HT3 antagonist; it was
developed by Glaxo around 1984. Its efficacy was first
established in 1987 in animal models [90]. Several
studies have demonstrated that ondansetron produces
an antiemetic effect equal to or superior to that of high
doses of metoclopramide; however, ondansetron has a
superior toxicity profile compared with dopaminergic
antagonist agents [88,91]. Ondansetron (0.15 mg/kg) is
administered intravenously 15–30 min before chemotherapy, and this dose is repeated every 4 h for two additional
doses.
Ondansetron is not approved for use in children younger
than 4 years. Its clearance is diminished in patients
with severe hepatic insufficiency; therefore, such
patients should receive a single injectable or oral dose
no higher than 8 mg. The major adverse effects of
ondansetron include headache, constipation or diarrhea,
fatigue, dry mouth, and transient asymptomatic elevation
8 Egyptian Pharmaceutical Journal
in liver function tests (alanine and aspartate transaminases), which may be related to concurrent cisplatin
administration.
Indole derivatives
Dolasetron 42 was first mentioned in the literature in
1989 [92]. Both oral and injectable formulations of dolasetron
are administered for the prevention of nausea and vomiting
associated with moderately emetogenic cancer chemotherapy, including initial and repeat courses. Dolasetron should
be administered intravenously or orally at 1.8 mg/kg as a
single dose B30 min before chemotherapy [87].
Indazole derivatives
Granisetron 41 was developed around 1988 [93]. It has
demonstrated the same efficacy and safety margin as
ondansetron in preventing and controlling nausea and
vomiting at broad-range doses (e.g. 10–80 mg/kg and
empirically 3 mg/dose) especially in patients receiving
emetogenic chemotherapy, including a high dose of
cisplatin [94].
Ramosetron 43 is only available in Japan and certain
Southeast Asian countries as of 2008. It has a higher affinity
for the 5-HT3 receptors than do the older 5-HT3
antagonists, and it maintains its effects over 2 days. It is
therefore significantly more effective against delayed
CINV [89]. In animal studies, ramosetron was also effective
against irritable bowel syndrome-like symptoms [95].
Palonosetron 44 is the newest 5-HT3 receptor antagonist. It shows antiemetic activity at both central and
gastrointestinal sites. In comparison with the older
5-HT3 antagonists, it has a higher binding affinity to
the 5-HT3 receptors, a higher potency, a significantly
longer half life (B40 h; four to five times longer than that
of dolasetron, granisetron, or ondansetron), and an
excellent safety profile. A dose finding study demonstrated that the effective dose was 0.25 mg or slightly
higher [87].
2 Britt H, Fahridin S. Presentations of nausea and vomiting. Aust Fam
Physician 2007; 36:682.
3 Wang SC. Physiological pharmacology. In: Root WS, Hofman FG, editors.
Emetic and antiemetic drugs. vol. II New York and London: Academic Press;
1965. pp. 225–328.
4 Golembiewski J, Chernin E, Chopra T. Prevention and treatment of
postoperative nausea and vomiting. Am J Health Syst Pharm 2005;
62:1247–1260.
5 Seigel LJ, Longo DL. The control of chemotherapy induced emesis. Ann
Intern Med 1981; 95:352–359.
6 Marty M. Future trends in cancer treatment and emesis control. Oncology
1993; 50:159–162.
7 Frytak S, Moertel CG. Management of nausea and vomiting in the cancer
patient. J Am Med Assoc 1981; 245:293–296.
8 Borison HL, Wang SC. Physiology and pharmacology of vomiting. Pharmacol
Rev 1953; 5:192–230.
9 Nasrallah HA, Brecher M, Paulsson B. Placebo-level incidence of extrapyramidal symptoms (EPS) with quetiapine in controlled studies of patients
with bipolar mania. Bipolar Disord 2006; 8 (5 Pt 1):467–474.
10 Schultz W. Multiple dopamine functions at different time courses. Annu Rev
Neurosci 2007; 30:259–288.
11 Olver JS, O’Keefe G, Jones GR, Burrows GD, Tochon-Danguy HJ, Ackermann U, et al. Dopamine D1 receptor binding in the striatum of patients
with obsessive–compulsive disorder. J Affect Disord 2009; 114:321–326.
12 Haney M, Ward AS, Foltin RW, Fischman MW. Effects of ecopipam, a
selective dopamine D1 antagonist, on smoked cocaine self-administration
by humans. Psychopharmacology (Berl) 2001; 155:330–337.
13 Herrstedt J, Sigsgaard T, Handberg J, Schousboe BM, Hansen M, Dombernowsky P. Randomized, double-blind comparison of ondansetron
versus ondansetron plus metopimazine as antiemetic prophylaxis during
platinum-based chemotherapy in patients with cancer. J Clin Oncol 1997;
15:1690–1696.
14 Campbell FL, Sullivan WN, Smith LE, Haller HL. Insecticidal tests of
synthetic organic compounds, chiefly tests of sulfur compounds against
culicine mosquito larvae. J Econ Entomol 1934; 27:1176–1185.
15 Harwood PD, Habermann RT, Roberts EH, Hunt WH. Preliminary observations on the effectiveness of crude, unconditioned phenothiazine for the
removal of worms from horses. Proc Helm Soc Washington 1940; 7:18–20.
16 Halpern BN, Ducrot R. Experimental research on a novel series of powerful
antihistamines: thiodiphenylamine derivatives. Comp Rend Soc Bio 1946;
140:361–364.
17 Tarkkila P, Torn K, Tuominen M, Lindgren L. Premedication with promethazine
and transdermal scopolamine reduces the incidence of nausea and vomiting
after intrathecal morphine. Acta Anaesthesiol Scand 1995; 39:983–986.
18 Schenker E, Herbst H. Progress in drug research. Progrès des recherches
pharmaceutiques 1963; 42:269–627.
19 Girault J, Greengard P. The neurobiology of dopamine signaling. Arch Neurol
2004; 61:641–644.
20 Courvoiries S, Fournel J, Ducrot R, Kolsky M, Koetschet P. Pharmacodynamic properties of 3-chloro-10-(3-dimethylaminopropyl)-phenothiazine
hydrochloride; experimental study of a new substance used in potentialized
anesthesia and in artificial hibernation. Arch Int Pharmacodyn Ther 1953;
92:305–361.
21 Holzbauer M, Vogt M. The action of chlorpromazine on diencephalic
sympathetic activity and on the release of adrenocorticotropic hormone. Br J
Pharmacol Chemother 1954; 9:402–407.
Conclusion
Antiemetics include various classes and groups having
versatile pharmacological mechanisms. This review deals
with D2-dopaminergic receptor and 5-HT3 serotoninergic
receptor antagonists possessing antiemetic potential,
which could be considered as biocandidates in the
development of new antiemetics or targets for extensive
molecular modifications in order to accentuate some
of their effects and attenuate or abolish side effects.
Acknowledgements
Conflicts of interest
There are no conflicts of interest.
22 Hudson RD. Effects of Chlorpromazine on motor reflexes of the chronic
spinal cat. Arch Int Pharmacodyn Ther 1968; 174:442–450.
23 Piala JJ, High JP, Hassert GLJ, Burke JC, Craver BN. Pharmacological and
acute toxicological comparisons of triflupromazine and chlorpromazine.
J Pharmacol Exp Therap 1959; 127:55–65.
24 Laffan RJ, Papandrianos DP, Burke JC, Craver BN. Antiemetic action of
fluphenazine (Prolixin): acomparison with other phenothiazines. J Pharmacol
Exp Ther 1961; 131:130–134.
25 Bhargava KP, Candra OM. Antiemetic activity of phenothiazine in relation to
their chemical structure. Br J Pharmacol Chemother 1963; 21:436–440.
26 Couvoisier S, Ducrot R, Julov L, Leau O. General pharmacological properties
of a new phenothiazine derivatives with powerful antihistaminic and antiallergic activity, 9,9-dioxo-10-(3-dimethylamino-2-methylpropyl)-phenothiazine. Arch Int Pharmacodyn Ther 1962; 135:364–375.
27 Jo S-H, Hong H-K, Chong SH, Lee HS, Choe H. H(1) antihistamine drug
promethazine directly blocks hERG K(+) channel. Pharmacol Res 2009;
60:429–437.
28 Delay J, Deniker P, Harl JM. Therapeutic psychiatric use of a selective centrally acting phenothiazine. Ann Med Psychol (Paris) 1952; 110:112–117.
29 Charpentier P. Phenothiazine derivatives. 1953. US 2645640.
30 Wirth W. The pharmacological action of promazine. Arzneimittelforschung
1958; 8:507–511.
References
1 Metz A, Hebbard G. Nausea and vomiting in adults-a diagnostic approach.
Aust Fam Physician 2007; 36:688–692.
31 Yale HL, Sowinski F, Bernstein J. 10-(3-dimethylaminopropyl)-2(trifluromethyl)-phenothiazine hydrochloride (Vesprin) and related compounds. J Am Chem Soc 1957; 79:4375–4379.
Receptors antagonists as antiemetics Aboul-Enein et al. 9
32 Feldmann PE. An analysis of the efficacy of diazepam. J Neuropsychiat
1962; 3 (Suppl 1):62–67.
33 Dobkin AB, Purkin N. The antisialogugue effect of phenpthiazine derivatives:
comparison of precazine, perphenazine, fluphenazine, thiopropazate, pipamazine and triflupromazine. Br J Anaesth 1960; 32:57–59.
34 Bowes HA. Ataractic composition comprising 10-(1-methyl-3-piperidylmethyl)phenothiazine and 10-(3-dimethyl-amino-propyl)-2-chlorophenothiazine. 1959.
US 2872376.
35 Jacob RM, Robert JG. Process for the preparation of phenthiazine derivatives. 1960; DE 1092476.
36 Goldenthal EIA. Compilation of LD50 values in new born and adult animals.
Toxicol Appl Pharmacol 1971; 18:185–207.
62 Florvall LG, Oegren SO. 2,6-Dialkoxybenzamides, process for their
preparation, composition and these compounds for use in treatment of
psychotic disorders. 1979;EP831.
63 Florvall GL, Lundstroem JOG, Raemsby SI, Oegren SO. BenzamidoDerivative. 1982; EP60235.
64 Kohler C, Hall H, Ogren SO, Gawell L. Specific in vitro and in vivo binding of
3H-raclopride. A potent substituted benzamide drug with high affinity for
dopamine D2 -receptors in the rat brain. Biochem Pharmacol 1985;
34:2251–2259.
65 Thominet M, Acher J, Monier JC. Heterocyclic substituted benzamides and
the process of their production. 1979; DE2901170.
37 Tedeschi DH, Tedeschi RE, Fellows EJ. The effects of tryptamine on the central
nervous system, including a pharmacological procedures for the evaluation of
iproniazid-like drugs. J Pharmacol Exp Ther 1959; 126:223–232.
66 Bleiberg H, Gerard B, Dalesio O, Crespeigne N, Rozencweig M. Activity
of a new antiemetic agent: alizapride. A randomized double-blind
crossover controlled trial. Cancer Chemother Pharmacol 1988; 22:
316–320.
38 Bourquin JP, Schward G, Gamboni G, Fischer R, Ruesch L, Theuss E, et al.
Synthses in the phenothiazine area - Part 1 Mercaptophenothiazine derivatives. J Helv Chim Acta 1958; 41:1061–1072.
67 Harrington RA, Hamilton CW, Brogden RN, Linkewich JA, Romankiewicz JA,
Heel RC, et al. Metoclopramide. An updated review of it’s pharmacological
properties and clinical use. Drugs 1983; 12:81–131.
39 Hotovy R, Kapff-Walter J. The pharmacological properties of perphenazine
sulphoxide. Arzneimittelforschung 1960; 10:638–650.
68 Rowbotham DJ. Current management of postoperative nausea and vomiting.
Br J Anaesth 1992; 69:46S–59S.
40 Gralla RJ, Osoba D, Kris MG, Kirkbride P, Hesketh PJ, Chinnery LW, et al.
Recommendations for the use of antiemetics: evidence-based, clinical practice guidelines. American Society of Clinical Oncology. J Clin Oncol 1999;
17:2971–2994.
69 Steward DJ. Cancer therapy, vomiting and antiemetics. Can J Physiol
Pharmacol 1990; 68:304–313.
70 Goodman LS, Gilman A. The pharmacological basis of therapeutics. New
York: Macmillan Publishing; 1980. pp. 997–1003.
41 Kline NS, Simpson GM. A long acting phenothiazine in office practice. Am J
Psychiatry 1964; 120:1012–1014.
71 Bowman WC, Rand MJ. Textbook of pharmacology. 2nd ed. London:
Blackwell Scientific Publications; 1980. p. 25.9.
42 Toldy L, Toth L, Fekete M, Borsy J. Phenothiazine derivatives. Acta Chim
Acad Sci Hung 1965; 44:301–325.
72 Pinnock RD, Ruff GHW, Turnbull MJ. Sulpiride blocks the action of
dopamine in the rat substantia nigra. Eur J Pharmacol 1979; 56:
413–414.
43 Loeser EA, Bennet EA, Stanley TH, Machin R. Comparison of droperidol,
haloperidol and prochlorperazine as postoperative antiemetic. Can Anaesth
Soc J 2007; 26:125–127.
44 Korttila K, Kauste A, Auvinen J. Comparison of domperidone, droperidol, and
metoclopramide in the prevention and treatment of nausea and vomiting after
balanced general anesthesia. Anaesth Analg 1979; 58:396–400.
45 Madej TH, Simpson KH. Comparison of the use of domperidone, droperidol
and metoclopramide in the prevention of nausea and vomiting following
gynaecological surgery in day cases. Br J Anaesth 1986; 58:879–883.
46 Bobon J, Collard J, Lecoq R. Benperidol and promazine: a ‘‘double blind’’
comparative study in mental geriatrics. Acta Neurol Belg 1963; 63:
839–843.
47 Domino KB, Anderson EA, Polissar NL, Posner KL. Comparative efficacy and
safety of ondansetron, droperidol, and metoclopramide for preventing
postoperative nausea and vomiting: a meta-analysis. Anesth Analg 1999;
88:1370–1379.
48 Granger B, Albu S. The haloperidol story. Ann Clin Psychiatry 2005;
17:137–140.
49 Nakra BR, Jones CJ, Majumdar AK, Gaind R. Preliminary evaluation of a new
psychotropic drug, lenperone, in the treatment of acute schizophrenia. Curr
Med Res Opin 1977; 4:529–534.
50 Grözinger M, Dragicevic A, Hiemke C, Shams M, Müller MJ, Härtter S.
Melperone is an inhibitor of the CYP2D6 catalyzed O-demethylation of
venlafaxine. Pharmacopsychiatry 2003; 36:3–6.
51 Report of the Workgroup on Vaccines. Report of the Second Public Health
Service AIDS Prevention and Control Conference. Public Health Rep 1988;
103 (Suppl 1):52–57.
52 Fontaine J, Reuse JJ. Comparative study on the action of some substituted
benzamides on the isolated ileum of the guinea pig. Arch Int Pharmacodyn
Ther 1975; 213:322–327.
53 Cuena Boy R, Macia Martinez MA. Extrapyramidal toxicity caused by metoclopramide and clebopride: study of voluntary notifications of adverse effects
to the Spanish Drug Surveillance System. Aten Primaria 1998; 21:289–295.
54 Bulteau G, Acher J. On the preparation of 2-alkoxy-4,5-substituted benzamides. 1973; DE 2327192.
55 Lunsford CD, Cale AD. N-(4-Pyrazolidinyl) benzamides and their salts in
pharmaceutical formulation. 1973; DE 2836062.
73 Alander T, Anden NE, Grabowska-Anden M. Metoclopramide
and sulpiride as selective blocking agents of pre- and postsynaptic
dopamine receptors. Naunyn Schmiedebergs Arch Pharmacol 1980;
312:145–150.
74 Hadley MS, King FD, McRitchie B, Turner DH, Watts EA. Substituted benzamides with conformationally restricted side chains. 1. Quinolizidine
derivatives as selective gastric prokinetic agents. J Med Chem 1985;
28:1843–1847.
75 Aboul-Enein MN, EL-Azzouny AA, Abdallah NA, Hegazy AY, Ebeid MY.
Synthesis and antiemetic profile of certain N-[1-[(diethylamino)methyl]
cyclohexyl] amides. Sci Pharm 1990; 58:273–280.
76 Aboul-Enein MN, EL-Azzouny AA, Attia MI, Maklad YA, Abd El-Hamid Ismail M,
Ismail NMS, Abd El-Hamid WHA. Dopamine D2 receptor antagonist activity
and molecular modeling of certain new cyclohexane derived arylcarboxamides
structurally related to metoclopramide. Dig J Nanomater Bios 2012; 7:
537–553.
77 Watcha MF, White PF. Postoperative nausea and vomiting. Its etiology,
treatment, and prevention. Anesthesiology 1992; 77:162–184.
78 Goldberg MW, Teitel S. Benzylamine derivatives. 1959; US 2879293.
79 Schallek W, Heise GA, Keith EF, Bagdon RE. Anti-emetic activity of 4-(2dimethylaminoethoxy)-N-(3,4,5-trimethoxybenzoyl)-benzylamine hydrochloride.
J Pharmacol Exp Ther 1959; 126:270–277.
80 Kolodny ALA. Controlled study of trimethobenzamide (Tigan), a specific
antiemetic. Am J Med Sci 1960; 239:682–689.
81 Van de Waterbeemd H, Carrupt PA, Testa B. Molecular electrostatic
potential of orthopramides: implications for their interaction with the D2
dopamine receptor. J Med Chem 1986; 29:600–606.
82 De Wit R, Aapro M, Blower PR. Is there a pharmacological basis for differences in 5-HT3-receptor antagonist efficacy in refractory patients? Cancer
Chemother Pharmacol 2005; 56:231–238.
83 Lindley C, Blower P. Oral serotonin type 3-receptor antagonists for
prevention of chemotherapy-induced emesis. Am J Health Syst Pharm 2000;
57:1685–1697.
84 Rolia F, Fatigoni S. New antiemetic drugs. Ann Oncol 2006; 17:
ii96–ii100.
1983.
85 Stott JR, Barnes GR, Wright RJ, Ruddock CJ. The effect on motion sickness
and oculomotor function of GR 38032 F, A 5-HT3 receptor antagonist with
antiemetic properties. Br J Clin Pharmacol 1989; 27:147–157.
57 Cooke HJ, Carey HV. The effects of cisapride on serotonin-evoked mucosal
responses in guinea-pig ileum. Eur J Pharmacol 1984; 98:147–148.
86 Gan TJ. Selective serotonine-5-HT3 receptor antagonists for postoperative
nausea and vomiting: are they all the same? CNS Drugs 2005; 19:
225–238.
56 Van Daele G. Novel N-(3-hydroxy-4-piperidinyl) benzamide.
EP76530.
58 Brunton LL, Lazo J, Paker K. Goddman & Gilman’s the pharmacological
basis of therapeutics. New York: Mc Graw-Hill; 2006. pp. 1000–1003.
59 Herrstedt J, Aapro MS, Rolia F, Kataja VV. ESMO minimum clinical
recommendations for prophylaxis of chemotherapy-induced nausea and
vomiting (NV). Ann Oncol 2005; 16:i77–i79.
60 Laville C, Margarit J. The influence of sulpiride on motor activity and vigilance
in the mouse. Pathol Biol 1968; 16:663–665.
61 Bruguerolle B, Jadot G, Valli M, Bouyard L, Fabregou-Bergier P, Perrot J,
et al. Four benzamides (metoclopramide, sulpiride, sultopride and tiapride)
effects on the oestrus cycle of the female rat: a comparative statistical study
(author’s transl). J Pharmacol 1981; 12:27–36.
87 Gebbia V, Cannata G, Testa A, Curto G, Valenza R, Cipolla C, et al. Ondansetron versus granisetron in the prevention of chemotherapy-induced
nausea and vomiting. Results of a prospective randomized trial. Cancer
1994; 74:1945–1952.
88 Hainsworth J, Harvey W, Pendergrass K, Kasimis B, Oblon D, Monaghan G,
et al. A single-blind comparison of intravenous ondansetron, a selective
serotonin antagonist, with intravenous metoclopramide in the prevention of
nausea and vomiting associated with high-dose cisplatin chemotherapy. J
Clin Oncol 1991; 9:721–728.
89 Rabasseda X. Ramosetron, a 5-HT3 receptor antagonist for the control of
nausea and vomiting. Drugs Today (Barc) 2002; 38:75–89.
10 Egyptian Pharmaceutical Journal
90 Hagan RM, Butler A, Hill JM, Jordan CC, Ireland SJ, Tyers MB, et al. Effect of
the 5-HT3 receptor antagonist, GR38032F, on responses to injection of a
neurokinin agonist into the ventral tegmental area of the rat brain. Eur J
Pharmacol 1987; 138:303–305.
91 Kaasa S, Kvaloy S, Dicato MA, Ries F, Huys JV, Royer E, et al. A comparison
of ondansetron with metoclopramide in the prophylaxis of chemotherapyinduced nausea and vomiting: a randomized, double-blind study. International Emesis Study Group. Eur J Cancer 1990; 26:311–314.
92 Cassidy J, Raina V, Lewis C, Adams L, Soukop M, Rapeport WG, et al.
Pharmacokinetics and anti-emetic efficacy of BRL43694, a new selective
5HT-3 antagonist. Br J Cancer 1988; 58:651–653.
93 Sorensen SM, Humphreys TM, Palfreyman MG. Effect of acute and chronic
MDL 73,147 EF, a 5-HT3 receptor antagonist, on A9 and A10 dopamine
neurons. Eur J Pharmacol 1989; 163:115–118.
94 Fauser AA, Duclos B, Chemaissani A, Del Favero A, Cognetti F, Diaz-Rubio
E, et al. Therapeutic equivalence of single oral doses of dolasetron mesilate
and multiple doses of ondansetron for the prevention of emesis after moderately emetogenic chemotherapy. European Dolasetron Comparative
Study Group. Eur J Cancer 1996; 32A:1523–1529.
95 Hirata T, Funatsu T, Keto Y, Nakata M, Sasamata M. Pharmacological profile
of ramosetron, a novel therapeutic agent for IBS. Inflammopharmacology
2007; 15:5–9.
Original article 11
Synthesis and DPPH radical-scavenging activity of some new
5-(N-substituted-1H-indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazole
derivatives
Heba M. Abo-Salema, Manal Sh. Ebaida, Eslam R. El-Sawya,
Abd El-Nasser El-Gendyb and Adel H. Mandoura
a
Chemistry Department of Natural Compounds
and bMedicinal and Aromatic Plants Department,
National Research Centre, Dokki, Giza, Egypt
Correspondence to Eslam R. El-Sawy, Chemistry
Department of Natural Compounds, National Research
Centre, Dokki 12311, Giza, Egypt
Tel: + 20 23 833 939 4; fax: + 20 33 370 931;
e-mail: [email protected]
Received 7 October 2012
Accepted 3 January 2013
Egyptian Pharmaceutical Journal
2013,12:11–19
Background and objectives
Heterocyclic systems with thiadiazole nucleus show a wide spectrum of biological
activities such as antioxidant, analgesic, antitumor, and anti-inflammatory activities.
The aim of this study is to describe the synthesis of some new 5-(N-substituted-1H-indol3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazole derivatives and to evaluate their antioxidant
activity using 2,20 -diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity.
Materials and methods
A one-pot reaction of N-substituted-1H-indol-3-carboxaldehyde 1a,b with thioglycolic
acid and thiosemicarbazide in concentrated sulfuric acid yielded novel 2-amino-5-(Nsubstituted-1H-indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazoles 2a,b. The reaction of
2a,b with some benzenesulfonyl chlorides and/or benzoyl chlorides yielded sulfonamides
3a,b and 4a,b and benzamide 5a,b and 6a,b derivatives, respectively, whereas, the
reaction of 2a,b with chloroacetyl chloride yielded chloroacetamide derivatives 7a,b,
which, on cyclization with potassium thiocyanate, yielded thiazolidinone derivatives 8a,b.
The reaction of 2a,b with sodium azide yielded tetrazole derivatives 9a,b. However, the
reaction of 2a,b with benzaldehyde yielded Schiff bases 10a,b, which cyclized with
chloroacetyl chloride and/or phenacyl bromide to yield azetidinone derivatives 11a,b and
12a,b, respectively. However, the reaction of 10a,b with sodium cyanide, followed by
acid hydrolysis yielded the a-amino acid derivatives 14a,b. Diazotization of 2a,b yielded
diazonium salt A, which, on coupling with sodium azide, yielded the azido derivatives
15a,b. Cyclization of 15a,b with ethylacetoacetate yielded tetrazole derivatives 16a,b,
whereas the coupling reaction of A with malononitrile yielded dicyano derivatives 17a,b,
which, on cyclization with hydrazine hydrate, yielded 3,5-diaminopyrazole derivatives
18a,b. The newly synthesized compounds were screened for their antioxidant activity
using 2,20 -diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity.
Results and conclusion
4-{5-[(1H-Indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl]diazo}-1H-pyrazole-3,5diamine (18a) was highly active with radical-scavenging activity (IC50 of 69.14 mg/ml)
compared with ascorbic acid (IC50 of 6.50 mg/ml).
Keywords:
DPPH radical-scavenging activity, indole-3-carboxaldehyde, synthesis, tetrazole,
thiazolo[4,3-b]-1,3,4-thiadiazole
Egypt Pharm J 12:11–19
& 2013 Division of Pharmaceutical and Drug Industries Research, National Research Centre
1687-4315
Introduction
Thiadiazole is a versatile moiety that shows a wide variety
of biological activities, viz, antioxidant, analgesic, anticonvulsant, anti-hepatitis B, antitubercular, antitumor, antidepressant, anti-inflammatory, antimicrobial, and antiHelicobacter pylori [1–6]. Besides these, fused 5H-thiazolo[4,3-b]-1,3,4-thiadiazoles have been prepared and become a substance among 1,3,4-thiadiazoles that has drawn
the attention of researchers [7–9]. Moreover, indole, which
is the potent basic pharmacodynamic nucleus, has been
reported to have a wide variety of biological properties, viz,
antioxidant [10], anti-inflammatory [11,12], anti-cancer
[13], and antimicrobial activities [12,14]. On the basis of
the above observations and as a part of our continuous work
on the preparation of new poly-heterocycles with pharmaceutical values [11–16], the present study focuses on the
synthesis of some new N-substituted-3-indolyl-5H-thiazolor-1,3,4-thiadiazoles for the evaluation of their antioxidant
activity using 2,20 -diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity starting from N-substituted indole3-carboxaldehyde.
Materials and methods
Chemistry
Melting points were determined in open capillary tubes on
an Electrothermal 9100 digital melting point apparatus
(Electrothermal Engineering Ltd, Serial No. 8694, Rochford,
1687-4315 & 2013 Division of Pharmaceutical and Drug Industries Research,
National Research Centre
DOI: 10.7123/01.EPJ.0000426585.93667.87
12 Egyptian Pharmaceutical Journal
United Kingdom) and were uncorrected. Elemental analyses
were carried out on a Perkin-Elmer 2400 analyzer (940
Winter Street, Waltham, Massachusetts, USA) and were
found to be within ± 0.4% of the theoretical values
(Table 1). IR spectra were recorded by Perkin-Elmer 1600
Fourier transform infrared spectroscopy against KBr discs.
The 1H NMR spectra were measured using a mass spectrometer (JEOL Ltd. 1-2, Musashino 3-chome Akishima,
Tokyo, Japan) 500 MHz in DMSO-d6, and chemical shifts
were recorded in d ppm relative to TMS as an internal
standard. Mass spectra (EI) were run at 70 eV using a
JEOL-JMS-AX500 mass spectrometer (Japan). All reagents
and solvents were of commercial grade. 1H-indole-3carboxaldehyde (1a) [17] and N-benzyl-1H-indole-3-carboxaldehyde (1b) have been prepared as reported [18].
2-Amino-5-(1H-indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazole
(2a) and 2-amino-5-(N-benzyl-1H-indol-3-yl)-5H-thiazolor1,3,4-thiadiazole (2b)
N-substituted-1H-indole-3-carboxaldehydes 1a or 1b
(0.02 mol) and thioglycolic acid (1.84 ml, 0.02 mol) were
mixed for 10–15 min. To the reaction mixture, thiosemicarbazide (1.82 g, 0.02 mol) was added with stirring and
then concentrated sulfuric acid (10 ml) was added in
portions upon cooling. The reaction mixture was homogenized and left for 24 h in a deep freezer (– 201C). The
reaction mixture was then treated with crushed ice (50 g)
and the suspension obtained was neutralized with an
aqueous sodium hydroxide solution (40%) to pHC7–8.
The precipitate that formed was filtered off, air dried, and
crystallized from aqueous dioxane (Scheme 1 and Table 1).
N-[5-(1H-Indol-3-yl1)-5H-thiazolor-1,3,4-thiadiazol-2-yl]
benzenesulfonamide (3a), N-[5-(N-benzyl-1H-indol-3-yl)-5Hthiazolo [4,3-b]-1,3,4-thiadiazol-2-yl]benzenesulfonamide
(3b), 4-chloro-N-[5-(1H-indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4thiadiazol-2-yl]benzene-sulfonamide (4a), and 4-chloro-N[5-(N-benzyl-1H-indol-3-yl)-5H-thiazolokr-1,3,4-thiadiazol-2yl]benzenesulfonamide (4b)
A mixture of compounds 2a or 2b (0.001 mol) and benzenesulfonyl chloride, or 4-chlorobenzenesulfonyl chloride (0.001 mol) in dry dioxane (10 ml) containing a few
drops of triethylamine was heated at reflux for 6 h. After
cooling, the reaction mixture was poured onto cold water
(10 ml). The solid that formed was filtered off, air dried,
and crystallized from dioxane (Scheme 1 and Table 1).
N-[5-(1H-Indol-3-yl)-5H-thiazolor-1,3,4-thiadiazol-2-yl]
benzamide (5a), N-[5-(N-benzyl-1H-indol-3-yl)-5Hthiazolo[4,3-b]-1,3,4-thiadiazol-2-yl]benzamide (5b), 2-chloroN-[5-(1H-indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazol-2yl]benzamide (6a), and 2-chloro-N-[5-(N-benzyl-1H-indol-3yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl]benzamide (6b)
A mixture of compounds 2a or 2b (0.001 mol) and
benzoyl chloride or 2-chlorobenzoyl chloride (0.001 mol)
in dry dioxane (10 ml) containing a few drops of
triethylamine was heated at reflux for 8 h. After cooling,
Table 1 Physical and analytical data of the newly synthesized compounds
Analysis (%; calculated/found)
Compound number
2a
2b
3a
3b
4a
4b
5a
5b
6a
6b
7a
7b
8a
8b
9a
9b
10a
10b
11a
11b
12a
12b
13a
13b
14a
14b
15a
15b
16a
16b
17a
17b
18a
18b
Formula (MW)
MP (1C)
Yield (%)
C
H
N
C12H10N4S2 (274.36)
C19H16N4S2 (364.49)
C18H14N4O2S3 (414.52)
C25H20N4O2S3 (504.65)
C18H13ClN4O2S3 (448.97)
C25H19ClN4O2S3 (539.09)
C19H14N4OS2 (378.47)
C26H20N4OS2 (468.59)
C19H13ClN4OS2 (412.92)
C26H19ClN4OS2 (503.04)
C14H11ClN4OS2 (350.85)
C21H17ClN4OS2 (440.97)
C15H11N5OS3 (373.48)
C22H17N5OS3 (463.60)
C13H9N7S2 (327.39)
C20H15N7S2 (417.51)
C19H14N4S2 (362.47)
C26H20N4S2 (452.59)
C21H15ClN4OS2(438.95)
C28H21ClN4OS2 (529.08)
C27H20N4OS2 (480.6)
C34H26N4OS2 (570.73)
C20H15N5S2 (389.50)
C27H21N5S2 (479.62)
C20H16N4O2S2 (408.5)
C27H22N4O2S2 (498.62)
C12H8N6S2 (300.36)
C19H14N6S2 (390.48)
C16H12N6O2S2 (384.44)
C23H18N6O2S2 (474.56)
C15H9N7S2 (351.41)
C22H15N7S2 (441.53)
C15H13N9S2 (383.45)
C22H19N9S2 (473.58)
146–148
86–88
111–113
76–78
212–214
187–189
165–168
136–138
300–302
300
135–137
127–129
175–177
162–164
252–254
175–177
170–172
158–160
100–102
70–72
159–161
172–174
234–236
106–108
196–208
130
86–88
61–3
126–128
92–94
144–146
123–125
201–202
130–132
94
88
70
65
83
76
84
77
84
81
91
95
90
91
81
82
86
83
78
65
75
71
79
80
76
70
40
30
46
36
67
55
83
85
52.53/52.33
62.61/62.44
52.15/52.01
59.50/59.36
48.15/48.01
55.70/55.54
60.30/60.16
66.64/66.48
55.27/55.04
62.08/62.16
47.93/47.98
57.20/57.28
48.24/48.11
57.00/57.23
47.69/47.73
57.53/57.40
62.96/62.76
69.00/69.11
57.46/57.66
63.56/63.44
67.48/67.50
71.55/71.60
61.67/61.62
67.61/67.69
58.80/58.77
65.04/65.00
–
–
49.99/49.75
58.21/58.00
51.27/51.33
59.85/59.92
46.98/47.01
55.80/55.71
3.67/3.58
4.42/4.26
3.40/3.27
3.99/3.81
2.92/2.76
3.55/3.41
3.73/3.61
4.30/4.21
3.17/3.06
3.81/3.66
3.16/3.20
3.89/3.76
2.97/3.00
3.70/3.55
2.77/2.64
3.62/3.58
3.89/3.99
4.45/4.33
3.44/3.68
4.00/4.28
4.19/4.32
4.56/4.44
3.88/3.90
4.41/4.46
3.95/3.90
4.45/4.35
–
–
3.15/3.00
3.82/3.78
2.56/2.35
3.42/3.33
3.42/3.37
4.04/4.15
20.42/20.31
15.37/15.20
13.52/13.41
11.10/10.99
12.48/12.32
10.39/10.22
14.80/14.66
11.96/11.77
13.57/13.41
11.14/11.02
15.97/15.99
12.71/12.68
18.75/18.80
15.11/15.22
29.95/29.80
23.48/23.40
15.46/15.54
12.38/12.55
12.76/12.56
10.59/10.72
11.66/11.45
9.82/9.78
17.98/17.93
14.60/14.58
13.72/13.69
11.24/11.44
–
–
21.86/21.66
17.71/17.69
27.90/27.93
22.21/22.30
32.87/32.66
26.61/26.45
Compounds 15a,b was decomposed slowly during the preparation of the samples analyzed.
3-Indolylthiazolothiadiazole Abo-Salem et al. 13
Scheme 1
CHO
O
H2 N
+ HS
OH
N
+
NH
S
NH2
R
H2 SO 4
1a,b
S
S
S
N
N
R
S
HC(OC2 H5 )3
N
N
N
N
NaN3
N
N
9a,b
Cl
CH
CO
X
N
N
R
Cl
NH2
SO 2 Cl
TEA
2a,b
2
COCl
Y
S
TEA
S
N
N
R
N
O
S
N
NH
S
N
Cl
S
7a,b
N
R
S
N
N
KSCN
NHSO 2
3a,b, X= H
4a,b, X= Cl
NHCO
N
R
X
Y
5a,b, Y= H
6a,b, Y= Cl
S
S
O
N
N
N
R
N
HN
S
8a,b
1- 9, R, a=H
, b=CH 2 Ph
Synthesis of compounds 1a,b to 9a,b.
the reaction mixture was poured onto cold water (20 ml).
The solid that formed was filtered off, air dried, and
crystallized from dioxane (Scheme 1 and Table 1).
(10 ml) was heated at reflux for 3 h. The solid that formed
was filtered off, air dried and crystallized from chloroform
(Scheme 1 and Table 1).
N-[5-(1H-Indol-3-yl)-5H-thiazolor-1,3,4-thiadiazol-2-yl]-2chloroacetamide (7a) and N-[5-(N-benzyl-1H-indol-3-yl)-5Hthiazolo [4,3-b]-1,3,4-thiadiazol-2-yl]-2-chloroacetamide (7b)
5-(1H-Indol-3-yl)-2-(1H-tetrazol-1-yl)-5H-thiazolor-1,3,4thiadiazole (9a) and 5-(N-benzyl-1H-indol-3-yl)-2-(1Htetrazol-1-yl)-5H-thiazolor-1,3,4-thiadiazole (9b)
To a solution of compounds 2a or 2b (0.02 mol) in dry
benzene (60 ml), a solution of chloroacetyl chloride (5 ml,
0.04 mol) in dry benzene (20 ml) was added dropwise
under vigorous stirring at 0–51C. After complete addition,
the reaction mixture was heated at reflux for 3 h. The
solvent was evaporated in vacuo and the solid that formed
was washed with sodium hydrogen carbonate (20 ml, 5%)
and then with water, air dried, and crystallized from
chloroform (Scheme 1 and Table 1).
A mixture of compounds 2a or 2b (0.001 mol), triethyl
orthoformate (0.15 ml, 0.001 mol), and sodium azide
(0.065 g, 0.001 mol) in glacial acetic acid (10 ml) was
stirred under reflux for 2 h. After cooling, the reaction
mixture was neutralized with concentrated hydrochloric
acid (10 ml). The solid that formed was filtered off,
washed with water, air dried, and crystallized from
absolute ethanol (Scheme 1 and Table 1).
3-[5-(1H-Indol-3-yl)-5H-thiazolor-1,3,4-thiadiazol-2-yl]-2iminothiazolidin-4-one (8a) and 3-[5-(N-benzyl-1H-indol-3-yl)5H-thiazolor-1,3,4-thiadiazol-2-yl]-2-iminothiazolidin-4-one (8b)
N-Benzylidene-(5-(1H-indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4thiadiazol-2-yl)-2-amine (10a) and N-benzylidene-[(5-(Nbenzyl-1H-indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl]2-amine (10b)
A mixture of compounds 7a or 7b (0.003 mol) and
potassium thiocyanate (0.58 g, 0.006 mol) in dry acetone
A mixture of compounds 2a or 2b (0.01 mol) and
benzaldehyde (1.06 g, 0.01 mol) in glacial acetic acid
14 Egyptian Pharmaceutical Journal
Scheme 2
S
S
S
N
N
N
R
S
N
ClCH2 COCl
TEA
dry dioxane
COOH
Cl
11a,b
50% H 2 SO 4
S
S
S
O
N
N
N
N
N
R
14a,b
S
O
N
NH
NH
N
R
CN
PhCH 2 COBr
TEA
dry dioxane
NaCN
gl.AcOH
S
13a,b
N
N
N
R
12a,b
S
N
N
N
R
N
10a,b
CHO
gl.AcOH
S
S
N
N
N
R
NH2
2a,b
NaNO2 /HCl
S
S
S
N
N
R
N
CH2 (CN)2
N
NH
N
17a,b
N
N
R
CN
NC
S
S
N N
Cl
N
N
R
A
S
N
NaN 3
N3
15a,b
CH3 COCH2 COOC2 H5
NH2 NH2
S
S
S
N
N
R
S
CH3
N
N
N
N
18a,b
H2 N
N
NH2
N
NH
N
R
N
COOH
N N
16a,b
2-18, R, a=H
, b=CH 2 Ph
Synthesis of compounds 10a,b to 18a,b.
(20 ml) was heated at reflux for 6 and 8 h. After cooling,
the reaction mixture was poured onto ice water (50 ml).
The solid that formed was filtered off, air dried, and
crystallized from benzene (Scheme 2 and Table 1).
1-[5-(1H-Indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl]3-chloro-4-phenylazetidin-2-one (11a), 1-[5-(N-benzyl-1Hindol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl]-3-chloro-4phenylazetidin-2-one (11b), 1-(5-(1H-indol-3-yl)-5H-
thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl)-3,4-diphenylazetidin-2one (12a), and 1-[5-(N-benzyl-1H-indol-3-yl)-5H-thiazolo[4,3b]-1,3,4-thiadiazol-2-yl]-3,4-diphenylazetidin-2-one (12b)
To a solution of Schiff bases 10a or 10b (0.01 mol) in dry
dioxane (5 ml), a solution of chloroacetyl chloride and/or
phenacyl bromide (0.01 mol) in dry dioxane (5 ml) and
triethylamine (0.59 ml, 0.01 mol) was added. The reaction mixture was heated at reflux for 12–14 h. The
reaction mixture was filtered off while hot and the
3-Indolylthiazolothiadiazole Abo-Salem et al. 15
solvent was removed in vacuo. The residue solid was
treated with water and filtered, air dried, and crystallized
from absolute ethanol (Scheme 2 and Table 1).
2-[5-(1H-Indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl
amino]phenylacetonitrile (13a) and 2-[5-(N-benzyl-1H-indol3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl
amino]phenylacetonitrile (13b)
To a solution of Schiff bases 10a or 10b (0.01 mol) in
glacial acetic acid (20 ml) sodium cyanide (0.49 g,
0.01 mol) was added and the reaction mixture was heated
at reflux for 6 h. After cooling, the reaction mixture
was poured onto cold water (10 ml) and the solid that
formed was filtered off, washed with water, air dried,
and crystallized from acetic acid–water (Scheme 2
and Table 1).
2-[5-(1H-Indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl
amino] phenyl acetic acid (14a) and 2-[5-(N-benzyl-1H-indol3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl amino]phenyl
acetic acid (14b)
A solution of compounds 13a or 13e (0.01 mol) in sulfuric
acid (30 ml, 50%) was heated at reflux for 10 h. After
cooling, the dark reaction mixture was poured onto cold
water (20 ml) and then neutralized with ammonia
solution (25%). The precipitate that formed was filtered
off, washed with water, air dried, and crystallized from
aqueous acetic acid (Scheme 2 and Table 1).
2-Azido-5-(1H-indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazole
(15a) and 2-azido-5-(N-benzyl-1H-indol-3-yl)-5Hthiazolo[4,3-b]-1,3,4-thiadiazole (15b)
To a cold solution of compounds 2a or 2b (0.02 mol) in a
mixture of concentrated hydrochloric acid (5 ml) and ice
water (5 ml), a cold aqueous solution of sodium nitrite
(1.73 g, 0.025 mol) in ice water (5 ml) was added dropwise
under stirring at 0–51C. After 10 min, the reaction
mixture was decanted. To the decanted solution of the
diazonium salt thus formed (A), sodium azide (1.3 g,
0.02 mol) in water (5 ml) was added dropwise. The
reaction mixture was left for 15 min at room temperature
and the azide was extracted by chloroform (3–10 ml) and
dried over anhydrous sodium sulfate. The solvent was
evaporated in vacuo and the residue was used without
subsequent purification, and used in the reaction
immediately after its formation because of its instability
(Scheme 2 and Table 1).
1-[5-(1H-Indol-3-yl)-5H-thiazolor-1,3,4-thiadiazol-2-yl]-5methyl-1H-1,2,3-triazole-4-carboxylic acid (16a) and 1-[5-(Nbenzyl-1H-indol-3-yl)-5H-thiazolor-1,3,4-thiadiazol-2-yl]-5methyl-1H-1,2,3-triazole-4-carboxylic acid (16b)
To a solution of sodium (0.23 g, 0.01 mol) in absolute
methanol (20 ml) ethylacetoacetate (1.34 g, 0.01 mol) and
compounds 15a or 15b (0.01 mol) were added dropwise
under cooling in an ice bath. The reaction mixture was
kept in an ice water bath for 30 min and then gradually
heated under reflux for 1 h. After cooling, the reaction
mixture was neutralized by diluted hydrochloric acid
(1 : 1). The solid that formed was filtered off, washed
with water, air dried, and crystallized from methanol
(Scheme 2 and Table 1).
2-{5-[(1H-Indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl]
hydrazono}malononitrile (17a) and 2-{5-[(N-benzyl-1H-indol3-yl)-5H-thiazolor-1,3,4-thiadiazol-2-yl]hydrazono}malononitrile (17b)
To a cold solution of compounds 2a or 2b (0.02 mol) in a
mixture of concentrated hydrochloric acid (5 ml) and ice
water (5 ml), a cold aqueous solution of sodium nitrite
(1.73 g, 0.025 mol) in ice water (5 ml) was added dropwise
under stirring at 0–51C. After 10 min, the reaction
mixture was decanted. To the decanted solution of the
diazonium salt thus formed (A), a cold solution of malononitrile (1.3 g, 0.02 mol) and sodium acetate trihydrate
(5.4 g, 0.04 mol) in ethanol (10 ml) was added under
stirring at 0–51C. The stirring was continued for an
additional 3 h at 0–51C, and then left overnight in the
refrigerator. The reaction mixture was poured onto water
(250 ml) and the solid that formed was filtered off, air
dried, and crystallized from absolute ethanol (Scheme 2
and Table 1).
4-{5-[(1H-Indol-3-yl)-5H-thiazolor-1,3,4-thiadiazol-2-yl]diazo}1H-pyrazole-3,5-diamine (18a) and 4-{5-[(N-benzyl-1H-indol3-yl)-5H-thiazolor-1,3,4-thiadiazol-2-yl]diazo}-1H-pyrazole3,5-diamine (18b)
A mixture of compounds 17a or 17b (0.01 mol) and
hydrazine hydrate (0.75 ml, 0.015 mol) in absolute
ethanol (20 ml) was heated at reflux for 6 h. The solvent
was evaporated in vacuo to half of its volume and the solid
that formed was filtered off, washed with water, air dried,
and crystallized from absolute ethanol (Scheme 2
and Table 1).
Biological assay
DPPH radical-scavenging activity
The antioxidant activity of the test compounds was
measured in terms of hydrogen-donating or radicalscavenging ability using the stable radical 2,20 -diphenyl-1picrylhydrazyl (DPPH) (Sigma Chemical Co., Steinheim,
Germany) [19]. A volume of 50 ml of a DMSO stock
solution of tested compounds at four different concentrations (50, 100, 200, and 300 mg/ml) was added to 2 ml
of 6 10–5 mol/l dimethylsulfoxide solution of DPPH
(2.3659 mg from DPPH/100 ml DMSO). The mixtures
were shacked in a vortex (2500 rpm) for 1 min and then
placed in a dark room. Ascorbic acid (Sigma-Aldrich
Chemie GmbH, Taufkirchen, Germany) was used as a
reference. The decrease in absorbance at 517 nm was
determined using a JENWAY 6315 spectrophotometer
(Keison Products, Chelmsford, England) after 1 h for all
samples. Dimethylsulfoxide was used to zero the spectrophotometer. The absorbance of the radical without a
sample was used as a negative control. The amount of
sample necessary to decrease the absorbance of DPPH
(IC50) by 50% was calculated graphically. The inhibition
percentage of the DPPH radical (scavenging activity) was
calculated according to the following formula:
% I¼½ðAB As Þ/AB 100;
16 Egyptian Pharmaceutical Journal
Table 2 Spectral characterization of the newly synthesized compounds
Compound
number
2a
2b
3a
3b
4a
4b
5a
5b
6a
6b
7a
7b
8a
8b
9a
9b
10a
10b
11a
11b
12a
12b
13a
13b
14a
IR (gmax/cm)
3410 (NH2), 3169 (NH), 1635
(C = N), 1575 (C = C)
1
H NMR (d, ppm)
Mass (m/z, %)
+
12.11 (s, 1H, NH), 11.11 (s, 1H, thiazolyl 5-H), 9.90 (s, 1H, 274 (M , 1), 256 (16), 192 (5),
thiazolyl 7-H), 8.25 (s, 1H, indolyl 2-H), 8.06 (d, 1H, indolyl 144 (34), 128 (14), 116 (16),
7-H), 7.48 (d, 1H, indolyl 4-H), 7.23-7.16 (m, 2H, indolyl
83 (47), 18 (100)
6-H and 5-H), 3.73 (s, 2H, NH2)
–
12.10 (s, 1H, thiazolyl 5-H), 9.93 (s, 1H, thiazolyl 7-H), 8.25
3336 (NH2), 1628 (C = N), 1543
(C = C)
(s, 1H, indolyl 2-H), 8.27-7.18 (m, 9H, Ar-H), 5.57 (s, 2H,
CH2-N), 3.85 (s, 2H, NH2)
3156 and 3111 (NH), 1636 (C = N), 12.14 (s, 1H, thiazolyl 5-H), 9.97 (s, 1H, thiazolyl 7-H), 8.95
–
1602 (C = C), 1354 and 1163
(s, 1H, NH), 8.31 (s, 1H, indolyl 2-H), 8.11-7.20 (m, 9H,
(SO2-N)
Ar-H), 5.08 (s, 1H, NH)
3125 (NH), 1631 (C = N), 1574
–
504 (M + , 21), 430 (12), 353
(C = C), 1363 and 1148 (SO2-N)
(10), 91 (100)
–
448/450 (M + /M + + 2, 33/11),
3232 and 3126 (NH), 1624 (C = N),
330 (2), 191 (20), 113 (37),
1575 (C = C), 1368 and 1136
111 (100)
(SO2-N), 745 (C-Cl)
–
12.01 (s, 1H, thiazolyl 5-H), 9.92 (s, 1H, thiazolyl 7-H), 8.69
3168 (NH), 1618 (C = N), 1610
(s, 1H, NH), 8.42 (s, 1H, indolyl 2-H), 8.21-7.18 (m, 13H,
(C = C), 1366 and 1134 (SO2-N),
747 (C-Cl)
Ar-H), 5.51 (s, 2H, CH2-N)
3327 and 3120 (NH), 1695 (C = O), 11.93 (s, 1H, thiazolyl 5-H), 9.90 (s, 1H, thiazolyl 7-H), 9.59 378 (M + , 23), 350 (10), 274
(20), 258 (1), 105 (100)
1640 (C = N), 1585 (C = C)
(s, 1H, NH), 8.56 (s, 1H, indolyl 2-H), 8.32-7.37 (m, 9H,
Ar-H), 4.18 (s, 1H, NH)
–
3154 (NH), 1710 (C = O),
12.24 (s, 1H, thiazolyl 5-H), 9.94 (s, 1H, thiazolyl 7-H), 8.26
1638 (C = N), 1563 (C = C)
(s, 1H, indolyl 2-H), 8.01-7.07 (m, 14H, Ar-H), 5.42 (s, 2H,
CH2-N), 3.75 (s, 1H, NH)
3260 and 3112 (NH), 1688 (C = O), 12.12 (s, 1H, thiazolyl 5-H), 9.95 (s, 1H, thiazolyl 7-H), 8.68
–
1644 (C = N), 1585 (C = C), 775
(s, 1H, NH), 8.37 (s, 1H, indolyl 2-H), 7.87-7.05 (m, 8H,
(C-Cl)
Ar-H), 3.96 (s, 1H, NH)
3212 (NH), 1759 (C = O),
–
503/505 (M + /M + + 2, 19/6),
391 (10), 113 (27), 111 (75),
1643 (C = N), 1578 (C = C),
91 (100)
773 (C-Cl)
–
3240 and 3163 (NH), 1722 (C = O), 11.85 (s, 1H, thiazolyl 5-H), 9.91 (s, 1H, thiazolyl 7-H), 8.26
1618 (C = N), 1521 (C = C),
(s, 1H, indolyl 2-H), 7.94-7.26 (m, 4H, Ar-H), 6.76 (s, 1H,
747 (C-Cl)
NH), 4.75 (s, 2H, CH2), 4.11 (s, 1H, NH)
3265 (NH), 1710 (C = O), 1588
–
440/442 (M + /M + + 2, 30/10),
349 (20), 318 (14), 91 (100)
(C = N), 1529 (C = C), 734 (C-Cl)
3186 and 3121 (NH), 1753 (C = O), 12.12 (s, 1H, thiazolyl 5-H), 9.93 (s, 1H, thiazolyl 7-H), 9.15 373 (M + , 34), 345 (10), 317
(20), 142 (100), 117 (15)
1616 (C = N), 1521 (C = C)
(s, 1H, NH), 8.29 (s, 1H, indolyl 2-H), 8.10-7.23 (m, 4H,
Ar-H), 6.08 (s, 1H, NH), 4.13 (s, 2H, CH2)
–
3265 (NH), 1725 (C = O),
12.15 (s, 1H, thiazolyl 5-H), 9.93 (s, 1H, thiazolyl 7-H), 8.72
1612 (C = N), 1522 (C = C)
(s, 1H, NH), 8.31 (s, 1H, indolyl 2-H), 8.26-7.36 (m, 9H,
Ar-H), 5.21 (s, 2H, CH2-N), 4.20 (s, 2H, CH2)
3160 (NH), 1643 (C = N),
12.10 (s, 1H, thiazolyl 5-H), 9.92 (s, 1H, thiazolyl 7-H), 8.86
–
1594 (C = C)
(s, 1H, tetrazolyl 5-H), 8.23 (s, 1H, indolyl 2-H), 7.76-7.24
(m, 4H, Ar-H), 6.90 (s, 1H, NH)
1635 (C = N), 1572 (C = C)
11.65 (s, 1H, thiazolyl 5-H), 9.92 (s, 1H, thiazolyl 7-H), 8.82 417 (M + , 17), 385 (2), 353 (21),
(s, 1H, tetrazolyl 5-H), 8.42 (s, 1H, indolyl 2-H), 8.06-7.15 117 (12), 91 (100)
(m, 9H, Ar-H), 5.92 (s, 2H, CH2-N)
3157 (NH), 1624 (C = N),
12.03 (s, 1H, thiazolyl 5-H), 10.11 (s, 1H, thiazolyl 7-H),
–
1565 (C = C)
9.90 (s, 1H, NH), 8.91 (s, 1H, CH = N), 8.50 (s, 1H,
indolyl 2-H), 8.34-7.40 (m, 9H, Ar-H)
–
1628 (C = N), 1571 (C = C)
12.11 (s, 1H, thiazolyl 5-H), 9.95 (s, 1H, thiazolyl 7-H), 9.01
(s, 1H, CH = N), 8.54 (s, 1H, indolyl 2-H), 8.23-7.11 (m,
14H, Ar-H), 5.66 (s, 2H, CH2-N)
3154 (NH), 1702 (C = O),
–
438/440 (M + /M + + 2, 12/4),
1633 (C = N), 1601 (C = C),
410 (1), 402 (3), 326 (10), 77
736 (C-Cl)
(100)
1724 (C = O), 1637 (C = N),
12.22 (s, 1H, thiazolyl 5-H), 9.93 (s, 1H, thiazolyl 7-H), 8.33
–
1563 (C = C), 745 (C-Cl)
(s, 1H, indolyl 2-H), 8.10-7.07 (m, 14H, Ar-H), 5.20 (d, 2H,
CH), 5.07 (d, 2H, CH)
3201 (NH), 1739 (C = O),
11.53 (s, 1H, thiazolyl 5-H), 9.81 (s, 1H, thiazolyl 7-H), 8.57 480 (M + , 2), 328 (10), 115 (14),
1640 (C = N), 1568 (C = C)
(s, 1H, NH), 8.25 (s, 1H, indolyl 2-H), 8.12-7.11 (m, 14H, 103 (100)
Ar-H), 5.20 and 4.81 (2d, 2H, 2CH)
–
1737 (C = O), 1635 (C = N),
12.23 (s, 1H, thiazolyl 5-H), 9.91 (s, 1H, thiazolyl 7-H), 8.65
1570 (C = C)
(s, 1H, indolyl 2-H), 8.32-7.01 (m, 19H, Ar-H), 5.51 (s, 2H,
CH2-N), 5.21 and 4.99 (2d, 2H, 2CH)
3240 and 3141 (NH), 2211 (CN), 12.15 (s, 1H, thiazolyl 5-H), 9.95 (s, 1H, thiazolyl 7-H), 9.34 389 (M + , 18), 349 (100), 333
(10), 103 (6)
1636 (C = N), 1583 (C = C)
(s, 1H, NH), 8.25 (s, 1H, indolyl 2-H), 8.11-7.25 (m, 9H,
Ar-H), 6.91 (s, 1H, NH)
3118 (NH), 2216 (CN),
–
479 (M + , 31), 388 (7), 312 (2),
91 (100)
1625 (C = N) 1587 (C = C)
3418 (OH), 3265 and 3152 (NH),
–
408 (M + , 46), 392 (1), 315 (20),
287 (2), 117 (50), 116 (100)
1700 (C = O), 1641 (C = N),
1573 (C = C)
3-Indolylthiazolothiadiazole Abo-Salem et al. 17
Table 2 (Continued)
Compound
number
14b
16a
16b
17a
17b
18a
18b
IR (gmax/cm)
1
H NMR (d, ppm)
Mass (m/z, %)
3400 (OH), 1715 (C = O),
1638 (C = N), 1524 (C = C)
13.45 (s, 1H, OH), 11.70 (s, 1H, thiazolyl 5-H), 9.65 (s, 1H,
–
thiazolyl 7-H), 8.81 (s, 1H, NH), 8.40 (s, 1H, indolyl 2-H),
8.19-7.01 (m, 14H, Ar-H), 5.51 (s, 2H, CH2-N), 2.3 (s, 1H,
CH)
–
3408 (OH), 3135 (NH),
13.23 (s, 1H, OH), 12.12 (s, 1H, thiazolyl 5-H), 9.93 (s, 1H,
1692 (C = O), 1631 (C = N),
thiazolyl 7-H), 8.40 (s, 1H, NH), 8.65 (s, 1H, indolyl 2-H),
1563 (C = C)
8.22-7.12 (m, 4H, Ar-H), 1.25 (s, 3H, CH3)
3368 (OH), 1707 (C = O),
–
474 (M + , 26), 460 (11), 431
(10), 389 (8), 91 (100)
1639 (C = N), 1585 (C = C)
3159 and 3112 (NH), 2195 (CN), 12.01 (s, 1H, thiazolyl 5-H), 9.92 (s, 1H, thiazolyl 7-H), 8.94
–
1628 (C = N), 1560 (C = C)
(s, 1H, NH), 8.26 (s, 1H, indolyl 2-H), 7.91-7.24 (m, 4H,
Ar-H), 6.91 (s, 1H, NH)
–
441 (M + , 45), 413 (3), 391 (2),
3160 (NH2), 2205 (CN),
1644 (C = N), 1615 (C = C)
381 (1), 244 (10), 91 (100)
–
3420 (NH2), 3192 and 3101 (NH), 11.65 (s, 1H, thiazolyl 5-H), 8.90 (s, 1H, thiazolyl 7-H), 8.53
1635 (C = N), 1620 (N = N), 1564
(s, 1H, NH), 8.32 (s, 1H, indolyl 2-H), 7.83-7.20 (m, 4H,
(C = C)
Ar-H), 6.50 (s, 1H, NH), 5.21 (s, 2H, NH2), 2.95 (s, 2H, NH2)
3363 and 3246 (NH2), 3133 (NH), 12.01 (s, 1H, thiazolyl 5-H), 9.91 (s, 1H, thiazolyl 7-H), 9.46 473 (M + , 66), 445 (2), 397 (21),
1638 (C = N), 1616 (N = N),
(s, 2H, NH2), 8.42 (s, 1H, indolyl 2-H), 8.05-7.17 (m, 9H, 115 (15), 91 (100)
Ar-H), 6.95 (s, 1H, NH), 5.37 (s, 2H, CH2-N), 3.91 (s, 2H,
1583 (C = C)
NH2)
where I is the DPPH inhibition %, AB the absorbance of
control (t = 0 h), and AS the absorbance of a tested
sample at the end of the reaction (t = 1 h). Each assay was
carried out in triplicate and the results were averaged.
Results and discussion
Chemistry
The reaction route for the synthesis of the newly
synthesized compounds has been described in Schemes
1 and 2. New 2-amino-5-(N-substituted-1H-indol-3-yl)5H-thiazolo[4,3-b]-1,3,4-thiadiazoles (2a,b) were prepared
by a one-pot reaction of N-substituted-1H-indole-3carboxaldehyde with thioglycolic acid and thiosemicarbazide in concentrated sulfuric acid according to the
procedure of Shukurov et al. [7] (Scheme 1). The IR
spectra of compounds 2a,b showed characteristic absorption bands at B3241–3410/cm for (NH2) and showed no
absorption band characteristic for C = O (Table 2). Their
1
H NMR (DMSO-d6) spectra showed two singlet signals
at d 12.12–9.90 ppm attributed to 5-H and 7-H of
thiazolo[4,3-b]-1,3,4-thiadiazole moiety, besides the other
aromatic protons located at their positions (Table 2).
The reaction of compounds 2a or 2b with benzenesulfonyl chloride and 4-chlorobenzenesulfonyl chloride in dry
dioxane and in the presence of triethylamine led to the
formation of N-[5-(N-substituted-1H-indol-3-yl)-5Hthiazolo[4,3-b]-1,3,4-thiadiazol-2-yl]benzenesulfonamide
derivatives 3a,b and 4a,b, respectively (Scheme 1).
However, the reaction of 2a,b with benzoyl chloride
and 2-chlorobenzoyl chloride yielded N-[5-(N-substituted-1H-indol-3-yl)-5H-thiazolor-1,3,4-thiadiazol-2-yl]benzamide derivatives 5a,b and 6a,b, respectively
(Scheme 1).
In contrast, the reaction of 2a or 2b with chloroacetyl
chloride in dry benzene yielded N-[5-(N-substituted-1H-indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl]-2-
chloroacetamides (7a,b). Cyclization of the latter compounds
through their reactions with potassium thiocyanate in
dry acetone yielded 3-[5-(N-substituted-1H-indol-3-yl)5H-thiazolor-1,3,4-thiadiazol-2-yl]-2-iminothiazolidin-4-ones
(8a,b) (Scheme 1).
The treatment of 2a or 2b with triethyl orthoformate and
sodium azide according to Abu-Hashem et al. [20] yielded
the new 5-(N-substituted-1H-indol-3-yl)-2-(1H-tetrazol-1yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazols (9a,b) (Scheme 1).
The acid-catalyzed reaction of 2a,b with benzaldehyde in
glacial acetic acid under reflux yielded the corresponding
Schiff bases, N-benzylidene-[5-(N-substituted-1H-indol3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl]-2-amines
(10a,b) (Scheme 2). Cyclocondensation of the latter
Schiff bases with chloroacetyl chloride and/or phenacyl
bromide under reflux in dry dioxane and in the presence
of triethylamine yielded 3-chloro-4-phenylazetidin-2-one
derivatives 11a,b and 3,4-diphenylazetidin-2-one derivatives 12a,b, respectively (Scheme 2).
However, the reaction of Schiff bases 10a or 10b with
sodium cyanide in glacial acetic acid yielded 2-[5-(Nsubstituted-1H-indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl amino]phenylacetonitriles (13a,b) (Scheme 2).
Acid hydrolysis of the latter compounds 13a or 13b
yielded the corresponding a-amino acid 14a,b (Scheme 2).
Diazotization of compounds 2a or 2b with concentrated
hydrochloric acid and sodium nitrite at 0–51C yielded the
corresponding diazonium salts (A), which, under coupling
with sodium azide, yielded the corresponding azides,
namely, 2-azido-5-(N-substituted-1H-indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazols (15a,b). The freshly prepared
azides 15a,b reacted with ethylacetoacetate in dry
methanol and in the presence of freshly prepared sodium
methoxide and yielded 1-[5-(N-substituted-1H-indol-3-yl)5H-thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl]-5-methyl-1H-1,2,3triazole-4-carboxylic acids (16a,b) (Scheme 2).
18 Egyptian Pharmaceutical Journal
Table 3 Scavenging activity % on DPPH radicals of the most
active synthesized compounds and IC50 values
Scavenging activity (%)a
Compound number
2a
6b
8a
9a
12a
12b
14a
16b
18a
Negative control
Ascorbic acid
50
100
200
300
IC50 (mg/ml)
11.39
8.49
6.15
6.33
1.63
4.15
25.67
14.64
45.56
0
83.79
18.62
13.74
8.67
6.87
2.35
5.06
34.9
19.71
56.05
0
88.99
30.19
18.67
9.40
13.02
5.06
8.13
45.26
36.34
79.56
0
85.41
44.42
22.24
17.00
16.64
64.19
11.21
84.61
47.55
80.83
0
91.25
368.59
1254.02
2243.39
1731.11
317.59
4221.33
164.15
327.21
69.14
0
6.50
with that of ascorbic acid of 91.25% at a concentration of
300 mg/ml, whereas at a concentration of 200 mg/ml, only
18a showed a radical-scavenging effect of 79.56%
compared with that of ascorbic acid of 85.41%. The
amount of sample necessary to decrease the absorbance
of DPPH by 50% (IC50) was calculated and it was found
that 4-{5-[(1H-indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl]diazo}-1H-pyrazole-3,5-diamine (18a) was
highly active with radical-scavenging activity (IC50 of
69.14 mg/ml) compared with ascorbic acid (IC50 of
6.50 mg/ml); this may be because of the presence of the
N–H moieties of the two primary aromatic amino groups
and secondary amine, which act as good hydrogen bond
donors (Table 3 and Fig. 1).
a
Results are the mean of three independent experiments.
Conclusion
Figure 1
(a)
100
Scavenging activity (%)
2a
6b
60
8a
%
80
9a
40
12a
20
VC
0
0
50
100
150
200
µg/mL
250
300
350
Scavenging activity (%)
(b)
100
Some new heterocycles derived from novel 2-amino-5-(Nsubstituted-1H-indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-thidiazoles (2a,b) were prepared and screened for their
antioxidant activity using 2,20 -diphenyl-1-picrylhydrazyl
(DPPH) radical-scavenging activity. 4-{5-[(1H-indol-3yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl]diazo}-1Hpyrazole-3,5-diamine (18a) was found to be highly active
with radical-scavenging activity (IC50 of 69.14 mg/ml)
compared with ascorbic acid (IC50 of 6.50 mg/ml); this
may be because of the presence of the N–H moieties of
the two primary aromatic amino groups and secondary
amine, which act as good hydrogen bond donors.
12b
80
14a
%
60
16b
40
Acknowledgements
Conflicts of interest
There are no conflicts of interest.
18a
20
0
VC
0
50
100
150
200
µg/mL
250
300
350
Scavenging activity % on DPPH radicals of the most active synthesized
compounds.
References
1 Soni BK, Singh T, Bhalgat CM, Kamlesh B, Kumar SM, Pavani M. In-vitro
antioxidant studies of some 1,3,4-thiadiazole derivatives. Int J Res Pharm
Biomed Sci 2011; 2:1590–1592.
2 Mishra G, Singh AK, Jyoti K. Review article on 1, 3, 4-thiadiazole
derivatives and its pharmacological activities. Int J ChemTech Res 2011;
3:1380–1393.
3 Bhuvaa H, Sahua D, Shaha BN, Modia DC, Patelb MB. Biological profile of
thiadiazole. Pharmacologyonline 2011; 1:528–543.
However, coupling of diazonium salts (A) with malononitrile in the presence of sodium acetate trihydrate
yielded 2-[(5-(N-substituted-1H-indol-3-yl)-5H-thiazolo[4,3-b]-1,3,4-thiadiazol-2-yl hydrazono] malononitriles
(17a,b). The reaction of the latter compounds with
hydrazine hydrate in absolute ethanol under reflux
yielded the corresponding pyrazoles (18a,b) (Scheme 2).
DPPH radical-scavenging activity
The preliminary DPPH radical-scavenging activity of the
newly synthesized compounds was determined using
ascorbic acid as a reference and IC50 of the most active
compounds were calculated (Table 3 and Fig. 1). From
the data obtained, compounds 14a and 18a showed free
radical-scavenging effects of 84.61 and 80.83% compared
4 Nelson JA, Rose LM, Bennett LL Jr.. Effects of 2 amino 1,3,4 thiadiazole on
ribonucleotide pools of leukemia L1210 cells. Cancer Res 1976; 36:
1375–1378.
5 Gupta JK, Dudhey R, Sharma PK. Synthesis and pharmacological activity of
substituted 1,3,4-thiadiazole derivatives. Medichemonline 2010; 1:1–10.
6 Kushwaha N, Kushwaha SKS, Rai AK. Biological activities of thiadiazole
derivatives: a review. Int J ChemTech Res 2012; 4:517–531.
7 Shukurov SSh, Kukaniev MA, Alibaeva AM. One-pot synthesis of
2-amino-5-aryl-5H-thiazolo[4,3-b]-1,3,4-thiadiazoles. Russ Chem Bull 1996;
45:724–725.
8 Karigar AA, Himaja M, Mali SV, Jagadeesh KP, Sikarwar MS. One-pot synthesis
and antitubercular activity of 2-amino-5-aryl-5H-thiazolo [4,3-b]-1,3,4-thiadiazoles. Int Res J Pharm 2011; 2:153–158.
9 Malipeddi H, Karigar AA, Malipeddi VR, Sikarwar MS. Synthesis and antitubercular activity of some novel thiazolidinone derivatives. Trop J Pharm Res
2012; 11:611–620.
10 Naik N, kumar HV, Shubhavathi T. Synthesis and antioxidant evaluation of
novel 5-methoxy indole analogues. Int J Curr Pharm Res 2011; 3:109–113.
11 Mandour AH, El-Sawy ER, Zahran MA, Ebaid MS, Mustafa MA. Anti-inflammatory analgesic, anticonvulsant and antimicrobial activities of some
new synthesized N-alkyl-3-indolyl pyrimidines and benzimidazolo(1,2-a)
pyrimidines. Biohealth SciBull (Malaysia) 2009; 1:57–67.
3-Indolylthiazolothiadiazole Abo-Salem et al. 19
12 Mandour AH, El-Sawy ER, Ebaid MS, Hassan SM. Synthesis and potential
biological activity of some novel 3-[(N-substituted indol-3-yl)methyleneamino]-6-amino-4-aryl-pyrano(2,3-c)pyrazole-5-carbonitriles and 3,6-diamino-4(N-substituted indol-3-yl)pyrano(2,3-c)pyrazole-5-carbonitriles. Acta Pharm
2012; 62:15–30.
13 El-Sawy E, Mandour A, Mahmoud K, Islam I, Abo-Salem H. Synthesis,
antimicrobial and anti-cancer activities of some new N-ethyl, N-benzyl and
N-benzoyl-3-indolyl heterocycles. Acta Pharm 2012; 62:157–179.
16 El-Sawy E, Bassyouni F, Abu-Bakr S, Rady H, Abdlla M. Synthesis and
biological activity of some new 1-benzyl and 1-benzoyl-3-heterocyclic indole
derivatives. Acta Pharm 2010; 60:55–71.
17 James PN, Snyder HR. Indole-3-aldehyde. Organic Syntheses 1959; 39:30–31.
18 Mndzhoyan AL, Papayan GL, Zhuruli LD, Karagezyan SG, Galstyan LS,
Sarafyan VG. Synthesis and biological study of hydrazinohydrazones of indole
aldehydes and ketones series. Arm Khim Zh (USSR) 1969; 22:707–713.
14 Abdel-Latif NA, El-Shihi TH, Islam IE, El-Sawy ER. Synthesis of some new
indole derivatives incorporated to heterocyclic systems and evaluation of
their antimicrobial activity. Egypt Pharm J (NRC) 2005; 4:313–329.
19 Viuda-Martos M, El Gendy AE-NGS, Sendra E, Fernández-López J,
El Razik KAA, Omer EA, Pérez-Alvarezj JA. Chemical composition and antioxidant and anti-Listeria activities of essential oils obtained from some
Egyptian plants. J Agric Food Chem 2010; 58:9063–9070.
15 Mandour A, El-Sawy E, Shaker K, Mustafa M. Synthesis, anti-inflammatory,
analgesic and anticonvulsant activities of 1,8-dihydro-1-ary1-8-alkyl
pyrazolo(3,4-b)indoles. Acta Pharm 2010; 60:73–88.
20 Abu-Hashem AA, Abu-Zied KM, El-Shehry MF. Synthetic utility of bifunctional
thiophene derivatives and antimicrobial evaluation of the newly synthesized
agents. Monatshefte für Chemie 2011; 142:539–545.
20 Original article
Synthesis and antihypertensive activity of certain substituted
dihydropyridines and pyrimidinones
Wageeh S. El-Hamoulya, Kamelia M. Amineb, Hanaa A. Tawfika
and Dina H. Dawooda
a
Department of Chemistry of Natural and Microbial
Products, National Research Centre and bDepartment
of Pharmaceutical Chemistry, Faculty of Pharmacy,
Cairo University, Giza, Egypt
Correspondence to Hanaa A. Tawfik, PhD, Department
of Chemistry of Natural and Microbial Products,
National Research Centre, Dokki, Giza 12311, Egypt
Tel: + 20 201 224 2709 16; fax: + 20 233 370 931;
e-mail: [email protected]
Received 17 July 2012
Accepted 10 October 2012
Egyptian Pharmaceutical Journal
2013,12:20–27
Background and objective
Some bulky substituted aromatic aldehydes reacted with urea and ethyl acetoacetate
in the presence of acetic acid as a catalyst to yield solely substituted dihydropyridines
(Hantzsch-type molecule). In the presence of p-toluene sulfonic acid as a catalyst, the
products were only dihydropyrimidines (Biginelli compounds). The same aldehydes
yielded dihydropyrimidinones on using acetyl acetone instead of ethyl acetoacetate
whatever the catalyst used. These two classes of molecules represent a heterocyclic
system of a remarkable antihypertensive effect. The aim of this study was to synthesize
certain dihydropyridine and pyrimidinone derivatives with aromatic moiety with bulky
substituents to be evaluated for their antihypertensive effect.
Methods
The aldehydes 3-(substituted-phenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde 3–5, 4oxo-4H-chromene-3-carbaldehyde (6), and substituted phenylazo-benzaldehyde 7–9
reacted with ethyl acetoacetate and urea in ethanol in the presence of acetic acid to
yield dihydropyridines 10–15. Aldehydes 3–9 reacted with ethyl acetoacetate and urea
in the presence of p-toluene sulfonic acid to yield dihydropyrimidinones 16–22.
Furthermore, the reaction of the aldehydes 3–9 with ethyl acetoacetate and urea in the
presence of either acetic acid or p-toluene sulfonic acid yielded the corresponding
dihydropyrimidinones 23–29.
Results and conclusion
The hypotensive activity of compounds 10–14 and 16–20 indicated that the 4-aryldihydropyridine derivatives 10–14 showed higher activity than the pyrimidinones
16–20. The most active compound was 4-(1,3-diphenyl-1H-pyrazol-4-yl)-2,6-dimethyl1,4-dihydropyridine-3,5-dicarboxylic acid diethyl ester (10) at dose levels of 0.6, 1.2,
and 2.4 mg/kg. It showed more or less similar hypotensive activity as the reference
drug nifedipene at doses of 1.2 and 2.4 mg/kg. Its LD50 = 298 mg/kg body weight.
Keywords:
antihypertensive activity, bulky substituted aldehydes, dihydropyridines,
dihydropyrimidinones
Egypt Pharm J 12:20–27
& 2013 Division of Pharmaceutical and Drug Industries Research, National Research Centre
1687-4315
Introduction
The one-pot acid-catalyzed Biginelli [1,2] condensation
is the most commonly used reaction to produce
dihydropyrimidines (DHPMs, 1). This very simple
reaction involves three component cyclocondensation of
urea, an aldehyde and a b-oxoester or 1,3-dicarbonyl
compound using ethanol as a solvent and catalytic
amounts of HCl, AcOH, or H2SO4 among other
acids [3–7]. In contrast, in the Hantzsch reaction
discussed, more than a century ago [8], the main way
to obtain dihydropyridines (DHPs, 2) and is commonly
carried out as a one-pot condensation of a b-dicarbonyl
compound with an aldehyde but with ammonia instead of
urea using ethanol as a solvent.
R
R
H
HN
O
COOEt
N
Me
H
1
Biginelli dihydropyrimidines
EtOOC
H
COOEt
Me
N
Me
H
2
Hantzsch dihydropyridines
These two classes of molecules (1 and 2) represent a
heterocyclic system with remarkable pharmacological
properties that include antiviral [9,10], antitumor [11,12],
antibacterial [13,14], and anti-inflammatory [15–18] activities. In addition, a number of these heterocyclic
1687-4315 & 2013 Division of Pharmaceutical and Drug Industries Research,
National Research Centre
DOI: 10.7123/01.EPJ.0000426587.41764.d4
Synthesis and antihypertensive activity El-Hamouly et al. 21
O2N
NH2
N
MeO
N
EtOOC
N
N
O
O
N
Me
O
O
MeO
O
Doxazosin
Me
N
H
Me
Nicardipine
systems have emerged as exerting orally active antihypertensive effects or to act as a-1A-adrenoceptor-selective
antagonists [19,20], for example nifedipene and amludepine. It is worth mentioning that several examples of
highly substituted DHPMs and DHPs are reported to
show high antihypertensive activity, for example doxazosin [20] and nicardipine [21,22].
pyrazole), 8.78 (s, 1H, NH, D2O exchangeable); Ms: m/z
(%): 469 [(M + -2, (62)], 441 (100%), 397 (83), 326 (71),
251 (93), 220 (90), 206 (22), 179 (32), 77 (99). Analysis:
for C28H29N3O4 (471.55), calcd: C, 71.32; H, 6.20; N,
8.91%. Found: C, 71.45; H, 6.30; N, 8.71%.
The aim of this work was to synthesize some DHPs and
pyrimidinones with the aromatic moiety bearing bulky
substituents to be evaluated for their antihypertensive
activity.
2,6-Dimethyl-4-[3-(4-nitrophenyl)-1-phenyl-1-Hpyrazole-4-yl]-1,4-dihydropyridine-3,5-dicarboxylic acid
diethyl ester (11)
Experimental
Chemistry
All melting points were determined in open capillary tubes
using silicon oil on a Gallen Kamp Apparatus (Finsbury,
London, England) and were uncorrected. 1H-NMR spectra
were determined using a JEOL EX-270 NMR spectrometer
(Musashino 3-chome, Akishima, Tokyo, Japan) with tetramethylsilane as an internal standard. Mass spectra were
performed using a GC-MS-QP 1000EX Schimadzu Gas
Chromatography MS Spectrometer (Columbia, Maryland,
USA). The infrared spectra were recorded on an FT/
IR330E infrared spectrophotometer using KBr discs.
Elemental analyses were carried out at the Micro analytical
Laboratory of the National Research Center, Dokki, Cairo,
Egypt. The reactions were followed up by thin layer
chromatography (TLC) using chloroform/methanol (9 : 1)
as an eluent and detected using a UV lamp.
General procedure for the preparation of substituted
dihydropyridine compounds (10–15)
A mixture of the appropriate aldehydes 3–9 (6 mmol), urea
(0.9 g, 15 mmol), ethyl acetoacetate (1.17 ml, 9 mmol), and
glacial acetic acid (2 ml) in absolute ethanol (50 ml) was
heated under reflux for several hours (12–18 h) (monitored
by TLC). After the completion of the reaction, the solvent
was removed under vacuum and the precipitated product
was treated with water, filtered off, washed with water,
dried, and crystallized from methanol.
4-(1,3-Diphenyl-1H-pyrazol-4-yl)-2,6-dimethyl-1,4dihydropyridine-3,5-dicarboxylic acid diethyl ester (10)
Yield 72%, m.p. 154–1561C, IR (KBr, cm – 1): 3343 (NH),
1682 (CO); 1H-NMR (d6-DMSO, d, ppm): 0.91 (t, 6H,
2CH3), 2.22 (s, 6H, 2CH3), 3.84 (q, 4H, 2CH2), 5.16 (s,
1H, C4-H), 7.26–7.88 (m, 10H, Ar-Hs), 8.00 (s, 1H,
Yield 75%, m.p. 110–1131C, IR (KBr, cm – 1): 3369 (NH),
1683 (CO); 1H-NMR (d6-DMSO, d, ppm) 0.87 (t, 6H,
2CH3), 2.24 (s, 6H, 2CH3), 3.87 (q, 4H, 2CH2), 5.18 (s,
1H, C4-H), 7.31–8.36 (m, 10H, 9Ar-Hs and 1H pyrazole),
8.81 (s, 1H, NH); Ms: m/z (%): 514 [M + -2, (22)], 486
(70), 442 (100), 251 (52). Analysis: for C28H28N4O6
(516.55), calcd: C, 65.11; H, 5.46; N, 10.85%. Found: C,
65.33; H: 5.19; N, 10.67%.
4-[3-(2-Hydroxy-phenyl)-1-phenyl-1H-pyrazol-4-yl]-2,
6-dimethyl-1,4-dihydro-pyridine-3,5-dicarboxylic acid
diethyl ester (12)
Yield 68%, m.p. 98–1001C, IR (KBr, cm – 1): 3357 (OH),
3249 (NH) and 1693 (CO); 1H-NMR (d6-DMSO, d,
ppm), 0.98 (t, 6H, 2CH3), 2.13 (s, 6H, 2CH3), 3.87 (q,
4H, 2CH2), 5.10 (s, 1H, C4-H), 6.91–7.77 (m, 9H, ArHs), 8.12 (s, 1H, pyrazole-H), 8.54 (s, 1H, NH) and 9.59
(s, 1H, OH); Ms: m/z (%): 485 [(M + -2, (94%)], 457 (24),
438 (100), 413 (43), 394 (20), 252 (16), 236 (27).
Analysis: for C28H29N3O5 (487.55), calcd: C, 68.98; H,
6.00; N, 8.62%. Found: C, 68.86; H, 5.79; N, 8.52%.
2,6-Dimethyl-4-(4-oxo-4H-chromen-3-yl)-1,4dihydropyridine-3,5-dicarboxylic acid diethyl ester (13)
Yield 65%, m.p. 213–2151C; 1H-NMR (d6-DMSO, d,
ppm) 1.10 (t, 6H, 2CH3), 1.12 (t, 6H, 2CH3), 2.22 (s,
6H, 2CH3), 2.25 (s, 6H, 2CH3), 3.96 (q, 4H, 2CH2), 4.02
(q, 4H, 2CH2), 4.82 (s, 1H, C4-H), 5.24 (s, 1H, C4-H),
7.43 (t, 1H, H-6), 7.50 (t, 1H, H-6), 7.55 (d, 1H, H-8),
7.57 (d, 1H, H-8), 7.64 (t, 1H, H-7), 7.73 (t, 1H, H-7),
7.93 (s, 1H, H-2), 8.14 (s, 1H, H-2), 8.00 (d, 1H, H-5),
8.02 (d, 1H, H-5), 8.82 (s, 1H, NH), 9.18 (s, 1H, NH);
Ms m/z (%) 397 (M + , 12%), 352 (7), 324 (100), 294 (10),
252 (32), 223 (17). Analysis: for C22H23NO6 (397.42),
calcd: C, 66.49; H, 5.83; N, 3.52%. Found: C, 66.80; H,
5.70; N, 3.41%.
22 Egyptian Pharmaceutical Journal
2,6-Dimethyl-4-(2-hydroxy-3-methoxy-5-phenylazophenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid diethyl
ester (14)
Yield 75%, m.p. 124–1261C; IR (KBr, cm – 1): 3448 (OH),
3344 (NH), 1693 (CO); 1H-NMR (d6-DMSO, d, ppm):
1.10 (t, 3H, CH3), 2.28 (s, 3H, CH3), 3.84 (s, 3H,
OCH3), 4.00 (q, 2H, CH2), 5.17 (s, 1H, C4-H), 7.03 (s,
1H, Ar-H), 7.22 (s, 1H, Ar-H), 7.56 (t, 3H, Ar-Hs), 7.80
(s, 1H, N3H, D2O exchangeable), 7.98 (d, 2H, Ar-Hs),
9.26 (s, 1H, N1H, D2O exchangeable), 10.99 (s, 1H,
OH); Ms: m/z (%): 477 [M + -2, (34)], 431 (12), 372 (38),
354 (32), 252 (81), 238 (41), 105 (55), 93 (86) and 77
(100). Analysis: for C26H29N3O6 (479.52), calcd: C,
65.12; H, 6.10; N, 8.76%. Found: C, 65.29; H, 6.12; N,
8.95%.
2,4-Dimethyl-5-oxo-9-phenylazo-5H-chromeno[3,4c]pyridine-1-carboxylic acid ethyl ester (15)
Yield 66%, m.p. 203–2061C; IR (KBr, cm – 1): 1730 (CO),
1684 (CO); 1H-NMR (d6-DMSO, d, ppm): 1.33 (t, 3H,
CH3), 2.69 (s, 3H, CH3), 2.93 (s, 3H, CH3), 4.58 (q, 2H,
CH2), 7.61 (t, 3H, Ar-Hs), 7.64 (d, 1H, Ar-H), 7.88 (d,
1H, Ar-H), 8.22 (d, 2H, Ar-Hs), 8.31 (s, 1H, Ar-H); Ms:
m/z (%), 400 [M + -1, (27)], 356 (10), 329 (17), 268 (37),
250 (60), 224 (24), 169 (91), 105 (55), 77 (100). Analysis:
for C23H19N3O4 (401.41), calcd: C, 68.82; H, 4.78; N,
10.47%. Found: C, 68.63; H, 4.91; N, 10.60%.
4-(Aryl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5carboxylicacid ethyl ester (16–22)
General procedure
A mixture of the appropriate aldehydes 3–9 (10 mmol),
urea (1.5 g, 25 mmol), ethyl acetoacetate (1.95 ml, 15 mmol),
and p-toluene sulfonic acid (1.72 g, 10 mmol) in absolute
ethanol (35 ml) was heated under reflux for 6–8 h
(monitored by TLC). After completion of the reaction,
the solvent was removed under vacuum and the precipitated product was treated with water, filtered, washed
with water, and dried. Crystallization from the appropriate
solvent yielded the desired compounds 16–22.
4-[1,3-Diphenyl-1H-pyrazole-4-yl]-6-methyl-2-oxo1,2,3,4-tetrahydropyrimidine-5-carboxylic acid ethyl
ester (16)
Yield 74%, m.p. 178–1801C (methanol); IR (KBr, cm – 1):
3349 (NH), 3222 (NH), 1693 (CO), 1642 (CO); 1HNMR (d6-DMSO, d, ppm): 0.82 (t, 3H, CH3), 2.23 (s,
3H, CH3), 3.80 (q, 2H, CH2), 5.38 (s, 1H, C4-H),
7.27–7.90 (m, 11H, 10Ar-Hs and 1H pyrazole), 8.35 (s,
1H, N3H) and 9.16 (s, 1H, N1H). Analysis: for
C23H22N4O3 (402.45), calcd: C, 68.64; H, 5.51; N,
13.92%. Found: C, 68.80; H, 5.34; N, 13.71%.
6-Methyl-4-[3-(4-nitro-phenyl)-1-phenyl-1H-pyrazol-4yl]-2-oxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylic acid
ethyl ester (17)
Yield 83%, m.p. 190–1931C; IR (KBr, cm – 1): 3439 (OH),
3210 (NH), 3122 (NH), 1713 (CO), 1657 (CO); 1HNMR (d6-DMSO, d, ppm): 0.87 (t, 3H, CH3), 2.25 (s,
3H, CH3), 3.82 (q, 2H, CH2), 5.44 (s, 1H, C4-H),
6.87–7.89 (m, 10H, 9Ar-Hs and 1H pyrazole), 8.34 (s, 1H,
N3H), 9.20 (s, 1H, N1H). Analysis: for C23H21N5O5
(447.44), calcd: C, 61.74; H, 4.73; N, 15.65%. Found: C,
61.96; H, 4.53; N, 15.85%.
4-[3-(2-Hydroxy-phenyl)-1-phenyl-1H-pyrazol-4-yl]-6methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic
acid ethyl ester (18)
Yield 79%, m.p. 201–2041C; IR (KBr, cm – 1): 3223 (NH),
3109 (NH), 1698 (CO), 1649 (CO); 1H-NMR (d6DMSO, d, ppm): 0.83 (t, 3H, CH3), 2.26 (s, 3H, CH3),
3.82 (q, 2H, CH2), 5.44 (s, 1H, C4-H), 7.13–8.50 (m,
10H, 9Ar-Hs and 1H pyrazole), 7.85 (s, 1H, N3H, D2O
exchangeable), 9.23 (s, 1H, N1H, D2O exchangeable).
Analysis: for C23H22N4O4 (418.45), calcd: C, 66.02; H,
5.30; N, 13.39%. Found: C, 66.37; H, 5.49; N, 13.21%.
6-Methyl-2-oxo-4-(4-oxo-4H-chromen-3-yl)-1,2,3,4tetrahydropyrimidine-5-carboxylic acid ethyl ester (19)
Yield 78%, m.p. 287–2901C, IR (KBr, cm – 1): 3386 (NH),
3281 (NH), 1710 (CO), 1669 (CO), 1638 (CO); 1HNMR (d6-DMSO, d, ppm): 1.00 (t, 3H, CH3), 2.23 (s,
3H, CH3), 3.98 (q, 2H, CH2), 5.23 (s, 1H, C4-H), 7.24
(s, 1H, H-2), 7.45 (t, 1H, H-6), 7.63 (d, 1H, H-8), 7.78
(t, 1H, H-7), 8.12 (d, 1H, H-5), 8.23 (s, 1H, N3H), 9.31
(s, 1H, N1H); Ms: m/z (%): 328 (M + , 12), 269 (17%),
255 (100%), 169 (18%); Analysis: for C17H16N2O5
(328.32), calcd: C, 62.19; H, 4.91; N, 8.53%. Found: C,
62.37; H, 4.79; N, 8.37%.
4-(2-Hydroxy-3-methoxy-5-phenylazo-phenyl)-6-methyl2-oxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylic acid
ethyl ester (20)
Yield 74%, m.p. 210–2121C, IR (KBr, cm – 1): 3357 (OH),
3214 (NH), 3198 (NH), 1689 (CO), 1640 (CO); 1HNMR (d6-DMSO d, ppm): 1.10 (t, 3H, CH3,), 2.28 (s,
3H, CH3), 3.84 (s, 3H, OCH3), 4.00 (q, 2H, CH2), 5.17
(s, 1H, C4-H), 7.03 (s, 1H, Ar-H), 7.22 (s, 1H, Ar-H),
7.56 (t, 3H, Ar-Hs), 7.80 (s, 1H, N3H, D2O exchangeable), 7.98 (d, 2H, Ar-Hs), 9.26 (s, 1H, N1H, D2O
exchangeable), 10.99 (s, 1H, OH, D2O exchangeable);
Ms: m/z (%), 410 [M + (12)], 302 (44), 210 (32), 105
(42), 93 (52), 77 (100). Analysis: for C21H22N4O5
(410.43), calcd: C, 61.46; H, 5.40; N, 13.65%. Found: C,
61.35; H, 5.35; N, 13.68%.
4-(2-Hydroxy-5-phenylazo-phenyl)-6-methyl-2-oxo1,2,3,4-tetrahydropyrimidine-5-carboxylic acid ethyl
ester (21)
m.p. 167–1701C, IR (KBr, cm – 1): 3455 (OH), 3220
(NH), 3210 (NH), 1690 (CO), 1662 (CO); 1H-NMR (d6DMSO, d, ppm) 1.05 (t, 3H, CH3), 2.24 (s, 3H, CH3),
3.98 (q, 2H, CH2), 5.52 (s, 1H, C4-H), 6.93 (d, 1H, ArH), 7.37 (s, 1H, N3H, D2O exchangeable), 7.51 (t, 3H,
Ar-Hs), 7.63 (s, 1H, Ar-H), 7.75 (d, 1H, Ar-H), 7.84 (d,
2H, Ar-Hs), 9.23 (s, 1H, N1H, D2O exchangeable), 10.61
(s, 1H, OH, D2O exchangeable); Ms: m/z (%), 380 (M + ,
20), 183 (22), 105 (21), 93 (28), 77 (100). Analysis: for
C20H20N4O4 (380.40), calcd: C, 63.15; H, 5.30; N,
14.73%. Found: C, 63.38; H, 5.40; N, 14.87%.
Synthesis and antihypertensive activity El-Hamouly et al. 23
Hs and 1H pyrazole), 7.83 (s, 1H, N3H, D2O exchangeable), 9.20 (s, 1H, N1H, D2O exchangeable); MS, m/z
(%): 416 (M + -1, 41), 373 (40), 326 (17), 266 (72), 235
(15), 153 (100) and 124 (50). Analysis: for C22H19N5O4
(417.42), calcd: C, 68.03; H, 5.19; N, 14.42%. Found: C,
68.23; H, 5.32; N, 14.61%.
4-[2-Hydroxy-5-(4-nitrophenylazo)phenyl]-6-methyl-2oxo-1,2,3,4-tetrahydro-pyrimidine-5-carboxylic acid
ethyl ester (22)
Yield 74%, m.p. 158–1611C, IR (KBr, cm – 1): 3356 (OH),
3234 (NH), 3114 (NH), 1687 (CO), 1651 (CO); 1H-NMR
(d6-DMSO, d, ppm): 1.07 (t, 3H, CH3), 2.30 (s, 3H, CH3),
3.97 (q, 2H, CH2), 5.50 (s, 1H,C4-H), 7.00 (d, 1H, Ar-H),
7.38 (s, 1H, N3H), 7.69 (s, 1H, Ar-H), 7.72–8.07 (m, 5H,
Ar-Hs), 9.21 (s, 1H, N1H), 10.90 (s, 1H, OH). Analysis: for
C20H19N5O6 (425.39), calcd: C, 56.47; H, 4.50; N, 16.46%.
Found: C, 56.66; H, 4.23; N, 16.64%.
5-Acetyl-6-methyl-4-(4-oxo-4H-chromen-3-yl)-3,4dihydro-1H-pyrimidin-2-one (26)
Yield 75%, m.p. 218–2201C; IR (KBr, cm – 1): 3340 (NH),
3273 (NH), 1703 (CO), 1671 (CO), 1645 (CO); 1HNMR (d6-DMSO, d, ppm): 2.15 (s, 3H, CH3), 2.31 (s,
3H, COCH3), 5.34 (s, 1H, C4-H), 7.25 (s, 1H, H-2), 7.45
(t, 1H, H-6), 7.63 (d, 1H, H-8), 7.78 (t, 1H, H-7), 8.12
(d, 1H, H-5), 8.25 (s, 1H, N3H), 9.32 (s, 1H, N1H); Ms:
m/z (%), 255 (100), 239 (8), 153 (18), 146 (26), 121 (31),
105 (35). Analysis: for C16H14N2O4 (298.29), calcd: C,
64.42; H, 4.73; N, 9.39%. Found: C, 64.56; H, 4.42; N,
9.61%.
Preparation of 5-acetyl-4-(3-aryl-1-phenyl-1H-pyrazole4-yl)-6-methyl-3,4-dihydro-1H-pyrimidin-2-one (23–29)
General procedure
A mixture of the selected aldehyde, 3–9 (10 mmol), urea
(1.5 g, 25 mmol) and acetylacetone (1.5 ml, 15 mmol) in
ethanol (50 ml) acidified with glacial acetic acid (2 ml) or
p-toluene sulfonic acid (1.72 g, 10 mmol) was heated
under reflux for 5–6 h. The solvent was then evaporated
under reduced pressure and the residue formed was
treated with water, filtered off, washed with water, dried,
and crystallized from methanol.
5-Acetyl-4-(2-hydroxy-3-methoxy-5-phenylazo-phenyl)6-methyl-3,4-dihydro-1H-pyrimidin-2-one (27)
Yield 74%, m.p. 228–2301C, IR (KBr, cm – 1): 3383 (OH),
3255 (NH), 3112 (NH), 1707 (CO), 1663 (CO). 1HNMR (d6-DMSO, d, ppm); MS, m/z (%): 379 (M + _1, 7),
350 (52), 335 (21), 322 (27), 258 (39), 244 (9), 153 (17),
93 (100), 124 (43). Analysis: for C20H20N4O4 (380.40),
calcd: C, 63.15; H, 5.30; N, 14.73%. Found: C, 63.40; H,
5.31; N, 14.55%.
5-Acetyl-4-(1,3-diphenyl-1H-pyrazol-4-yl)-6-methyl-3,4dihydro-1H-pyrimidin-2-one (23)
Yield 70%, m.p. 218–2201C, IR (KBr, cm – 1): 3327 (NH),
3222 (NH), 1696 (CO), 1671 (CO); 1H-NMR (d6DMSO, d, ppm): 2.16 (s, 3H, CH3), 2.25 (s, 3H,
COCH3), 5.43 (s, 1H, C4-H), 7.30–7.87 (m, 11H, 10ArHs and 1H pyrazole), 8.28 (s, 1H, N3H), 9.12 (s, 1H,
N1H); MS: m/z (%): 372 (M + , 93), 357 (38), 329 (36),
254 (8), 221 (100), and 153 (43). Analysis: for
C22H20N4O2 (372.42), calcd: C, 70.95; H, 5.41; N,
15.04%. Found: C, 70.79; H, 5.51; N, 15.19%.
5-Acetyl-4-(2-hydroxy-5-phenylazo-phenyl)-6-methyl3,4-dihydro-1H-pyrimidin-2-one (28)
Yield 78%, m.p. 202–2051C, IR (KBr, cm – 1): 3400 (OH),
3235 (NH), 3150 (NH), 1681 (CO), 1621 (CO); 1HNMR (d6-DMSO, d, ppm): 2.11 (s, 3H, CH3), 2.33 (s,
3H, COCH3), 5.63 (s, 1H, C4-H), 7.00 (d, 1H, Ar-H),
7.04 (s, 1H, N3H, D2O exchangeable), 7.53 (t, 3H, ArHs), 7.62 (s, 1H, Ar-H), 7.72 (d, 1H, Ar-H), 7.82 (d, 2H,
Ar-Hs), 9.27 (s, 1H, N1H, D2O exchangeable), 10.59 (s,
1H, OH, D2O exchangeable); MS m/z (%): 350 (M + , 13),
307 (10), 198 (23), 153 (16), 93 (100). Analysis: for
C19H18N4O3 (350.37), calcd: C, 65.13; H, 5.18; N,
15.99%. Found: C, 65.33; H, 5.28; N, 16.25%.
5-Acetyl-6-methyl-4-[3-(4-nitrophenyl)-1-phenyl-1Hpyrazol-4-yl]-3,4-dihydro-1H-pyrimidin-2-one (24)
Yield 67%, m.p. 178–1801C, IR (KBr, cm – 1): 3402 (OH),
3235 (NH), 3165 (NH), 1655 (CO), 1620 (CO); 1HNMR (d6-DMSO, d, ppm); MS: m/z (%): 386 (M + -2,
10), 345 (8), 235 (11), 221 (21), 154 (17) and 66 (100).
Analysis: for C22H19N5O4 (417.42), calcd: C, 63.30; H,
4.59; N, 16.78%. Found: C, 63.47; H, 4.68; N, 16.92%.
5-Acetyl-4-[2-hydroxy-5-(2-nitro-phenylazo)-phenyl]-6methyl-3,4-dihydro-1H-pyrimidin-2-one (29)
5-Acetyl-4-[3-(2-hydroxy-phenyl)-1-phenyl-1H-pyrazol-4yl]-6-methyl-3,4-dihydro-1H-pyrimidin-2-one (25)
Yield 70%, m.p. 213–2161C, IR (KBr, cm – 1): 3364 (OH),
3281 (NH), 3230 (NH), 1697 (CO), 1650 (CO); MS m/z
(%): 396 (M + + 1, 10), 350 (12), 337 (17), 257 (30), 243
(13), 337 (17), 257 (30), 243 (13), 226 (15), 153 (20%),
93 (100). Analysis: for C19H17N5O5 (395.37), calcd: C:
–1
Yield 82%, m.p. 193–1961C, IR (KBr, cm ): 3227 (NH),
3114 (NH), 1656 (CO), 1619 (CO); 1H-NMR (d6DMSO, d, ppm): 2.07 (s, 3H, CH3), 2.33 (s, 3H,
COCH3), 5.50 (s, 1H, C4-H), 7.12–8.36 (m, 10H, 9Ar-
CHO
CH3
NHNH2
+
O
CH3
R
H
N
N
R
POCl3
DMF
R
N
N
3) R = H; 4) R = 4-NO2;
5) R = 2-OH
24 Egyptian Pharmaceutical Journal
O
COCH3
CHO
POCl3/DMF
OH
O
6
R1
R1
OH
R
R
R1
OH
N2HCl.
OH
R
N
N
N
+
CHO
N
7, R = H, R1 = OMe
8, R = R1 = H,
9, R = NO2 R1 = H
MeO
R
N
N
N
O
N
HO
O
H
EtO2C
H3C
CO2Et
N
H
H
H
CH3
10) R = H
11) R = 4 -NO2
12) R = 2-OH
E tO2C
H3C
CO2 E t
N
H
13
CH3
E tO2 C
H3 C
CO2E t
N
H
CH3
14
57.72; H, 4.33; N, 17.71%. Found: C, 57.58; H, 4.41; N,
17.63%.
Chemistry
The aldehydes 3-(substituted-phenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde 3–5 [23] 4-oxo-4H-chromene-3carbaldehyde (6) [24] and substituted phenylazo-benzaldehyde 7–9 [25] reacted with ethyl acetaoaetate and
urea in ethanol in the presence of acetic acid to yield
DHPs 10–15.
Compound 8 reacted similarly but underwent intramolecular condensation and aromatization to yield 2,4dimethyl-5-oxo-9-phenylazo-5H-chromeno[3,4–c]pyridine1-carboxylic acid ethyl ester (15). Similar behavior has
been reported previously [26].
Also, compound 9 yielded a mixture of products that were
hardly separable; perhaps, decomposition occurred because of the long reaction time.
Moreover, aldehydes 3–9 reacted with urea and ethyl
acetoacetate in the presence of p-toluene sulfonic acid to
yield dihydropyrimidinones 16–22.
Furthermore, reaction of the aldehydes 3–9 with urea and
acetyl acetone in alcohol as a solvent in the presence of
either acetic acid or p-toluene sulfonic acid yielded the
corresponding dihydropyrimidinones 23–29.
Antihypertensive activity
Ten of the newly synthesized substituted DHPs 10–14
and tetrahydropyrimidines 16–20 were screened for their
hypotensive activity using normotensive cat models [27].
Materials and methods
Male cats of local strains weighing from 2.5 to 4.0 kg were
housed (one per cage) in the animal facility (Faculty of
Synthesis and antihypertensive activity El-Hamouly et al. 25
R
R1
N
R
N
O
N
N
HO
O
H
CO2E t
HN
N
H
O
CO2 E t
HN
O
Me
H
H
N
H
O
Me
N
H
19
16, R = H,
17, R = 4- NO2
18, R = 2-OH
CO2E t
HN
Me
20, R = H, R1 = OMe
21, R = R1 = H
22, R = NO2, R1 = H
R
R1
N
N
R
H O
O
O
Me
HN
N
H
Me
23, R = H,
24, R = 4-NO2
25, R = 2-OH
N
R2
O
O
O
Me
HN
26
The hypotensive effect of the tested DHP derivatives
10–14 and DHPMs 16–20 is shown in Table 1 in
comparison with nifedipine as a reference drug. In the
DHP series, the test compounds showed significant
hypotensive activity at all dose levels (0.6, 1.2, and
2.4 mg/kg). The 4-(1,3-diphenyl-1H-pyrazolyl) derivative
10 was the most active at all dose levels. Also, it had more
Me
N
H
Me
27, R = NO2 , R 1 = 2-OH, R 2 = H
28, R = H, R 1 = 2-OH, R 2 = H
29, R = H, R 1 = 4-OH, R 2 =3-OCH3
Table 1 Effect of tested compounds (10–14 and 16–20) on the
mean blood pressure of anesthetized normotensive cats
compared with the reference drug nifedipine
Dose (mg/kg)
0.6 mg/kg
1.2 mg/kg
2.4 mg/kg
Results and discussion
HN
O
N
H
Medicine, El-Azhar University) for 7 days before the
experiment. Animals were always kept at 22 ± 2 h and a
12 h light/12 h dark cycle. Stressful conditions or manipulation were avoided. Cats were divided into groups; each
group included four cats and one group was used as a
control. All cats were anesthetized with phenobarbital
sodium (35 mg/kg, intraperitoneally) and their blood
pressures (BP) were recorded from the carotid artery.
BP of each cat was measured before and 30 min after the
intravenous injection of the tested compounds. The
tested compounds were dissolved in DMSO and administered at different doses (0.6, 1.2, 2.4 mg/kg) in 0.5 ml
volume in the same way as the reference drug nifedipine.
The same volume of DMSO was administered to animals
in the control group. The reduction of BP between two
measurements was recorded as mmHg. These results
were expressed as mean ± SEM; analysis variance (twoway) was used for statistical analysis. LD50 was preformed
according to the procedure described in the study
conducted by Kerber [28].
N
Compounds
Mean reduction in BP
Control (DMSO)
Nifedipine
10
11
12
13
14
16
17
18
19
20
Nifedipine
10
11
12
13
14
16
17
18
19
20
Nifedipine
10
11
12
13
14
16
17
18
19
20
100.17 ± 1.82
44.17 ± 1.45
55.40 ± 1.40
79.00 ± 1.24
61.67 ± 2.23
65.00 ± 1.71
75.00 ± 1.59
76.23 ± 2.60
74.17 ± 1.60
76.50 ± 1.24
61.00 ± 1.10
89.50 ± 1.28
22.17 ± 1.42
27.17 ± 1.91
77.83 ± 0.87
51.50 ± 1.61
64.33 ± 1.71
67.00 ± 0.45
68.33 ± 1.89
70.00 ± 1.19
62.33 ± 0.87
58.00 ± 1.46
88.83 ± 1.71
15.17 ± 1.01
13.00 ± 0.82
62.00 ± 1.53
15.50 ± 0.92
60.33 ± 0.88
60.50 ± 1.18
64.50 ± 1.34
68.00 ± 1.39
57.00 ± 1.51
47.67 ± 1.09
85.67 ± 0.92
BP, blood pressure; DMSO, dimethyl sulfoxide.
26 Egyptian Pharmaceutical Journal
doses of 1.2 and 2.4 mg/kg. Its LD50 is 298 mg/kg body
weight, which would present a fruitful matrix for the
development of a potent antihypertensive agent.
Table 2 LD50 in male mice after an intraperitoneal
administration of compound 10
Group
number
1
2
3
5
6
7
Total
Oral doses
(mg/kg body
weight)
Number of
dead animals
240
260
280
300
320
340
–
1
3
5
7
10
Dose
difference Meana Productb
–
20
20
20
20
20
–
0.5
2
4
6
8.5
–
10
40
80
120
170
420
Number of animals/group = 10 mice.
LD50: 340 – (420/10) = 298 mg/kg body weight.
LD50, lethal dose, 50%.
a
Interval mean of the number of dead animals (mice).
b
Product of the interval mean and the dose difference.
or less similar potency as nifidipine (refrerence standerd)
at doses of 1.2 and 2.4 mg/kg. The other tested DHPs 11
and 12 bearing 3-aryl-1-phenyl-1H-pyrazolyl as well as
the chromonyl derivative 13 and 4-hydroxy-3-methoxy-5(phenylazo)-phenyl substituent at the 4-position 14
showed weak activities compared with the reference
drug. For tetrahydropyrimidine series 16–20, the evaluated data showed that the 4-chromonyl derivative 19
had significant hypertensive activity (61.00 ± 1.10),
which was higher than the 4-pyrazolyl analogous 16–18
at a dose of 0.6 mg/kg. A nonsignificant change was
observed in the presence of 4-[4-hydroxy-3-methoxy-5(phenylazo)-phenyl] derivative 20 when administered at
the same dose level. The hypotensive values of this series
were negligible compared with those of nifedipine at
doses of 0.6, 1.2, and 2.4 mg/kg.
Moreover, Table 2 shows that LD50 of the most active
compound 10 was equal to 298 mg/kg body weight.
Conclusively, the 4-aryl-DHP derivatives 10–14 showed
higher hypotensive activity than the tetrahydropyrimidines 16–20 carrying the same aryl substituents at the
same position. The most active compound was 4-(1,3diphenyl-1H-pyrazol-4-yl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylic acid diethyl ester 10 at dose levels
of 0.6, 1.2, and 2.4 mg/kg. It showed more or less similar
hypotensive activity as the reference drug nifedipine at
doses of 1.2 and 2.4 mg/kg.
Conclusion
The synthesis of substituted DHPs 10–15 and pyrimidinones 16–29 was achieved. The comparison of the
tested compounds 10–14 and 16–20 for their hypotensive activity using the nonselective cat models led to the
conclusion that the 4-aryl-DHP derivatives 10–14
showed higher hypotensive activity than the pyrimidinones derivatives carrying the same aryl substituent at
the same position. The most active compound was 4-(1,
3-diphenyl-1H-pyrazol-4-yl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylic acid diethyl ester 10 at dose levels
of 0.6, 1.2, and 2.4 mg/kg. It showed more or less similar
hypotensive activity as the reference drug nifedipine at
Acknowledgements
Conflicts of interest
There are no conflicts of interest.
References
1 Biginelli P. The first synthesis of dihydropyrimidinone by refluxing a mixture of
an aldehyde, a b-ketoester, and urea under strongly acidic condition. Gazz
Chim Ital 1893; 23:360–413.
2 Kappe CO. 100 years of the Biginelli dihydropyrimidine synthesis. Tetrahedron 1993; 49:6937–6963.
3 Folkers K, Harwood HJ, Johnson TB. Researches on pyrimidines. cxxx.
synthesis of 2-keto-1,2,3,4-tetrahydropyrimidines. J Am Chem Soc 1932;
54:3751–3758.
4 Folkers K, Johnson TB. Researches on pyrimidines. cxxxvi. The mechanism of
formation of tetrahydropyrimidines by the Biginelli reaction. J Am Chem Soc
1933; 55:3784–3791.
5 Dandia A, Saha M, Taneja H. Synthesis of fluorinated ethyl 4-aryl-6-methyl1,2,3,4-tetrahydropyrimidin-2-one/thione-5-carboxylates under microwave
irradiation. J Fluorine Chem 1998; 90:17–21.
6 Lu J, Bai Y, Wang Z, Yang B, Ma H. One-pot synthesis of 3,4-dihydropyrimidin-2(1H)-ones using lanthanum chloride as a catalyst. Tetrahedron Lett
2000; 41:9075–9078.
7 Ananda Kumar K, Kasthuraiah M, Suresh Reddy C, Devendranath Reddy C.
Mn(OAc)3 2H2O-mediated three-component, one-pot, condensation reaction: an efficient synthesis of 4-aryl-substituted 3,4-dihydropyrimidin-2-ones.
Tetrahedron Lett 2001; 42:7873–7875.
8 Hantzsch A. Hantzsch dihydropyridine synthesis. Just Leib Ann Chem 1882;
215:1–82.
9 Hilgeroth A, Billich A, Lilie H. Synthesis and biological evaluation of first Nalkyl syn dimeric 4-aryl-1,4-dihydropyridines as competitive HIV-1 protease
inhibitors. Eur J Med Chem 2001; 36:367–374.
10 Heys L, Moore CG, Murphy PJ. The guanidine metabolites of Ptilocaulis
spiculifer and related compounds; isolation and synthesis. Chem Soc Rev
2000; 29:57–67.
11 Yoshida J, Ishibashi T, Nishio M. Antitumor effects of amlodipine, a Ca2 +
channel blocker, on human epidermoid carcinoma A431 cells in vitro and
in vivo. Eur J Pharmacol 2004; 492 (2–3):103–112.
12 Haggarty SJ, Mayer TU, Miyamoto DT, Fathi R, King RW, Mitchison TJ,
Schreiber SL. Dissecting cellular processes using small molecules: identification of colchicine-like, taxol-like and other small molecules that perturb
mitosis. Chem Biol 2000; 7:275–286.
13 Fisher-Maliszewska L, Wieczorek J, Mordarski M. Biological activity of 1,4dihydropyridine derivatives. Arch Immunol Ther Exp 1985; 33:345–352.
14 George S, Parameswaran MK, Chakraborty AR, Ravi TK. Synthesis and
evaluation of the biological activities of some 3-{[5-(6-methyl-4-aryl-2-oxo1,2,3,4-tetrahydropyrimidin-5-yl)-1,3,
4-oxadiazol-2-yl]-imino}-1,3-dihydro2H-indol-2-one derivatives. Acta Pharm 2008; 58:119–129.
15 Takahara A, Fujita S-I, Moki K, Ono Y, Koganei H, Iwayama S, Yamamoto H.
Neuronal Ca 2 + channel blocking action of an antihypertensive drug, cilnidipine, in IMR-32 human neuroblastoma cells. Hypertension Res 2003;
26:743–747.
16 Takahara A, Konda T, Enomoto A, Kondo N. Neuroprotective effects of a dual
L/N-type Ca2 + channel blocker cilnidipine in the rat focal brain
ischemia model. Biol Pharm Bull 2004; 27:1388–1391.
17 Yamamoto T, Niwa S, Ohno S, Onishi T, Matsueda H, Koganei H, et al.
Structure-activity relationship study of 1,4-dihydropyridine derivatives
blocking N-type calcium channels. Bioorg Med Chem Lett 2006; 16:
798–802.
18 Sadanandam YS, Shety MM, Diwan PV. Synthesis and biological evaluation
of new 3,4-dihydro-6-methyl-5-N-methyl-carbamoyl-4-(substituted phenyl)2(1H)pyrimidinones and pyrimidinethiones. Eur J Med Chem 1992; 27:
87–92.
19 Kappe CO. Biologically active dihydropyrimidones of the Biginelli-type-A
literature survey. Eur J Med Chem 2000; 35:1043–1052.
20 Yadav JS, Subba Reddy BV, Reddy PT. Unprecedented synthesis of
hantzsch 1,4-dihydropyridines under biginelli reaction conditions. Synthetic
Commun 2001; 31:425–430.
21 Atwal KS, Swanson BN, Unger SE, Floyd DM, Moreland S, Hedberg A,
O’Reilly BC. Dihydropyrimidine calcium channel blockers. 3,3-Carbamoyl-4aryl-1,2,3,4-tetrahydro-6-methyl-5-pyrimidinecarboxylic acid esters as orally
effective antihypertensive agents. J Med Chem 1991; 34:806–811.
Synthesis and antihypertensive activity El-Hamouly et al. 27
22 Rovnyak GC, Atwal KS, Hedberg A, David Kimball S, Moreland S,
Gougoutas JZ, et al. Dihydropyrimidine calcium channel blockers. 4. Basic 3substituted-4-aryl-1,4-dihydropyrimidine-5-carboxylic acid esters. Potent antihypertensive agents. J Med Chem 1992; 35:3254–3263.
23 Arbačiauskiene E, Martynaitis V, Krikštolaityte S, Holzer W, Šačkus A.
Synthesis of 3-substituted 1-phenyl-1H-pyrazole-4-carbaldehydes and the corresponding ethanones by Pd-catalysed cross-coupling reactions. Arkivoc 2011;
11:1–21.
24 El-Sayed Ali T, Abdel-Aghfaar Abdel-Aziz S, Metwali El-Shaaer H, Ismail
Hanafy F, Zaky El-Fauomy A. Synthesis of some new 4-oxo-4H-chromene
derivatives bearing nitrogen heterocyclic systems as antifungal agents. Turk J
Chem 2008; 32:365–374.
25 Alok K, Pareek PE, Seth JosephDS. A convenient route for the synthesis
and characterization of novel substituted azo-coumarins and schiff’s bases.
Oriental J Chem 2009; 25:1149–1152.
26 Dehghanpour S, Heravi MM, Derikvand F. N,N0 -ethylene-bis(benzoylacetoniminato) copper (II), Cu(C 22H22N2O2), a new reagent for aromatization
of Hantzsch 1,4-dihydropyridines. Molecules 2007; 12:433–438.
27 McLeod LJ. Pharmacological experiments on intact preparations. London:
Churchill livingstone; 1970. p. 49.
28 Kerber G. Pharmacological approaches for the discovery of drugs and poisons and their mode of action analysis. In: Dr Leopold T, editor. Pharmakologische methoden zur auffinding von Arzneimittel und Gifte und Analyse
ihner Wirkungweise. Wissenschaftliche Verlag GmbH; 1941.
28 Original article
Immobilization of Mucor racemosus NRRL 3631 lipase and
characterization of silica-coated magnetite (Fe3O4) nanoparticles
Abeer A. El-Hadia,c, Hesham I. Salehb,d, Samia A. Moustafab
and Hanan M. Ahmeda
Departments of aChemistry of Natural and Microbial
Products, bInorganic Chemistry, National Research
Centre, Giza, Egypt, cDepartment of Biology, Faculty of
Science, Taif University, Taif and dDepartment of
Chemistry, Northern Border University, Arar, Saudi
Arabia
Correspondence to Abeer A. El-Hadi, Department of
Chemistry of Natural and Microbial Products, National
Research Centre, El-Behoos St.33, Dokki, Giza 12311,
Egypt
Tel: + 20 233 54974; fax: + 20 233 70931;
e-mail: [email protected]
Received 29 July 2012
Accepted 1 October 2012
Egyptian Pharmaceutical Journal
2013,12:28–35
Introduction and purpose
The uncoated magnetite (M) and silica-coated magnetite (MS) nanoparticles have
been suggested as carriers for the immobilization of enzymes to improve their activity
and stability. The objective of this study was to demonstrate the potential use of
magnetic nanoparticles in bioengineering applications, using Mucor racemosus NRRL
3631 lipase as the model enzyme.
Materials and methods
The magnetite (Fe3O4) particles were synthesized by the chemical coprecipitation
technique, that is, Massart’s process with minor modifications, using stable ferrous and
ferric salts with ammonium hydroxide as the precipitating agent. The uncoated and
coated magnetite nanoparticles for immobilizing the lipase were characterized
according to the particle sizes, as measured from the transmission electron
microscope images. The infrared and X-ray powder diffraction spectra can well explain
the bonding interaction and crystal structures of various samples, respectively.
Results and conclusion
Different concentrations of silica-coated magnetite (MS) nanoparticles were used as
cross-linking agents. A silica concentration of 1% was proven to be more suitable, with
an immobilization efficiency of 96%. The transmission electron microscope images
revealed the diameters of the uncoated magnetite particles to be 10–16 nm and those
of the coated particles to be about 11 nm. The optimal pH and temperature of the
immobilized lipase were 5–6 and 401C, respectively. There was a slight decrease in
the residual activity of the immobilized lipase at 601C for 1 h. The kinetic constants
Vmax and Km were determined to be 250 U/mg protein and 20 mmol/l, respectively, for
the immobilized lipase. The residual activity of the immobilized lipase remained over
51% despite being used repeatedly seven times. It can be concluded that Fe3O4
magnetic nanoparticles and silica-coated magnetite (MS) nanoparticles have been
successfully prepared with excellent properties using the chemical coprecipitation
technique with some modifications. The silica coating appeared to be effective in
protecting the magnetite from being converted to other oxide species. The results of
the X-ray powder diffraction indicate that the composites were in the nanoscopic
phase. The resulting immobilized lipase had better resistance to pH and temperature
inactivation compared with free lipase and exhibited good reusability.
Keywords:
Fe3O4/SiO2, immobilization efficiency, magnetic nanoparticles, Mucor racemosus
NRRL 3631 lipase, stability
Egypt Pharm J 12:28–35
& 2013 Division of Pharmaceutical and Drug Industries Research,
National Research Centre
1687-4315
Introduction
In recent years, the use of nanophase materials offers
many advantages because of their unique size and
physical properties. Magnetic nanoparticles have become
very popular when used in conjunction with biological
materials such as proteins, peptides, enzymes, antibodies,
and nucleic acids because of their unique properties [1];
this application is mainly based on the magnetic feature
of the solid phase that helps in achieving a rapid and
easy separation from the reaction medium in a magnetic
field. Previous studies have reported that magnetic
nanoparticles tend to lose their magnetizability when
biopolymer-coated nanoparticles are circulated in the
body [2]. Consequently, inorganic carrier materials
including magnetite and silica gels were being focused
on because of their thermal and mechanical stability,
nontoxicity, and high resistance against microbial attacks
and solutions of organic solvents [3]. Silica and its
derivatives when coated onto the surface of magnetic
nanoparticles may help to change their surface properties.
With the appropriate coating, the magnetic dipolar
attraction between the magnetic nanoparticles may be
screened, thus minimizing or even preventing aggregation. The coating film could also provide a chemically
inert layer against the nanoparticles, which is particularly
1687-4315 & 2013 Division of Pharmaceutical and Drug Industries Research,
National Research Centre
DOI: 10.7123/01.EPJ.0000427102.64865.32
Lipase immobilization on Fe3O4 nanoparticles El-Hadi et al. 29
useful in biological systems [1,4]. The larger specific
surface area and surface reactive groups that are
introduced by further modification of silica materials are
favorable during the preparation of silica carriers for
immobilized enzymes, and these carriers are very suitable
for adsorption and immobilization of the adsorbed protein
abundantly and steadily [5]. Lipases from different
sources are currently used in enzymatic organic synthesis [6,7]. The expanding interest in lipases mainly lies
on their wide industrial applications, including detergent
formulation, oil/fat degradation, pharmaceutical synthesis, cosmetics, paper manufacture, and oleochemistry [8]. To use lipases more economically and efficiently
in aqueous as well as in nonaqueous solvents, their
activity and operational stability needs to be improved by
immobilization. In addition, the enzyme immobilization
onto magnetic supports such as nanosized magnetite
particles allows an additional merit, namely, the selective
and easy enzyme recovery from the medium under a
magnetic force, compared with other conventional support materials. Hence, there is no need for expensive
liquid chromatography systems, centrifuges, filters, or
other equipment. In contrast, lipases obtain the highest
activity when their molecules are immobilized onto
nanoparticles because of their relatively high specific
area; this promises results on immobilizing lipases onto
surface-modified nano-sized magnetite particles [9].
The objective of this study was to demonstrate the
potential use of magnetic nanoparticles in bioengineering
applications. Mucor racemosus NRRL 3631 lipase was used
as the model enzyme in this study. The uncoated and
silica-coated magnetite nanoparticles were characterized
by X-ray powder diffraction (XRD), transmission electron
microscopy, and Fourier transform-infrared (IR) spectroscopy. The properties of the immobilized lipase such as
activity, recovery, protein analysis, and thermal stability
were investigated.
in an orbital shaker operating at 200 rpm at 301C. For
lipase production, the composition of the basal medium
(9% w/v) was: glucose, 1; olive oil, 1; peptone, 30;
KH2PO4, 0.2; KCl, 0.05; and MgSO4 7H2O, 0.05%, with
an initial pH of 6.5 [10]. The medium was heat sterilized
at 1211C for 15 min.
Standard method for enzyme activity assay
The lipase assay was performed using an olive oil
emulsion according to the procedure described by
Starr [11]. The olive oil emulsion was prepared as
follows: 10 ml of olive oil and 90 ml of 10% arabic gum
were emulsified using a homogenizer for 6 min at
20 000 rev/min. The reaction mixture composed of 3 ml
of olive oil emulsion, 1 ml of 0.2 mol/l Tris-buffer (pH
7.5), 2.5 ml of distilled water, and 1 ml of enzyme solution
was incubated at 371C for 2 h with shaking. The emulsion
was destroyed by the addition of 10 ml of acetone (95% v/v)
immediately after incubation, and the liberated free fatty
acids were titrated with 0.05 N.
Analytical procedure of protein determination
Protein measurements were carried out according to the
method of Lowry et al. [12], using BSA as the standard.
The amount of bound protein was determined indirectly
from the difference between the amount of protein
present in the filtrate and that in washing solutions after
immobilization.
Partial purification of M. racemosus lipase using
ammonium sulfate
Ammonium sulfate (60% saturation) was added to 900 ml
of the culture supernatant at 41C. The precipitate was
collected by centrifugation at 12 000g at 41C for 20 min
and dissolved in a constant amount of distilled water. The
lipase activity and protein concentrations were determined [13].
Synthesis of magnetite nanoparticles
Materials and methods
Commercial lipase enzyme was prepared from M.
racemosus NRRL 3631. All other materials were of
analytical grade and used without further purification;
these materials included tetraethyl orthosilicate
(TEOSZ98%), ammonia solution (NH3, 28 wt%), ferrous
dichloride tetrahydrate (FeCl2 4H2O), ferric trichloride
hexahydrate (FeCl3 6H2O), glucose (C6H6O6), potassium chloride (KCl), potassium dihydrogen phosphate
(KH2PO4), magnesium sulfate (MgSO4 7H2O), disodium
hydrogen phosphate (Na2HPO4), sodium dihydrogen
phosphate (NaH2PO4), peptone from animal protein, olive
oil, gum arabic, and acetone.
The nanoparticles were prepared according to the
method described by Massart [14] but without the use
of hydrochloric acid. A total of 4.05 g of FeCl3 6H2O and
1.98 g of FeCl2 4H2O was dissolved in 100 ml of distilled
water; the solution was purged with nitrogen to agitate
the mixture and prevent the oxidation of Fe2 + ions. After
30 min of purging, 143 ml of 0.7 mol/l NH4OH was added
dropwise into the solution and the now basified solution
was purged for an additional 10 min. During the addition
of NH4OH, it was noticed that the solution changed color
from the original brown to dark brown and then to black.
The precipitate was magnetically separated using a
permanent magnet and then washed with distilled water
several times and allowed to dry in air. The resulting
product was defined as M.
Microorganisms, medium, and growth conditions
Synthesis of silica-coated magnetite nanoparticles
M. racemosus NRRL 3631 was maintained on potato
dextrose agar (PDA) slants. The microorganism was
grown in 250 ml Erlenmeyer flasks containing 100 ml of
the medium. The medium was inoculated with 4 ml of
spore suspension, and the flasks were incubated for 72 h
The above-mentioned experiment was repeated until the
step in which the solution was purged with nitrogen to
agitate the mixture. After this step, the precursor
TEOS (3 ml) was carefully dropped into the reaction
mixture of iron using a syringe, with mechanical stirring.
30
Egyptian Pharmaceutical Journal
The homogenization was performed for 15 min. After
sonication for 15 min, 143 ml of 0.7 mol/l NH4OH was
added dropwise into the mixture with continuous
mechanical stirring for 30 min. The coated particles were
finally separated from the liquid using a permanent
magnet, washed with distilled water several times, and
allowed to dry in air. Finally, we also determined the
effect of silica coating by varying the amount of TEOS
added to the reaction mixture. In this regard, we studied
the effect of five different amounts of TEOS, 1.04, 2.08,
4.22, 8.33, and 12.5 ml, which are equivalent to 0.5, 1, 2,
4, and 6% molar ratios, respectively. The determine
parameter of silica-coated magnetite nanoparticles is
labeled as MS1, MS2, MS3, MS4 and MS5.
Characterization
XRD was used to investigate the crystal structure of the
magnetic nanoparticles. The size and shape of the
nanoparticles were examined using a transmission electron microscope (TEM) (Model JEOL-1230, Japan). The
IR spectra were recorded using a Fourier transforminfrared spectrophotometer (FT-IR). The sample and
KBr were pressed to form a tablet.
Immobilization of lipase
Because of the epoxy groups of the magnetite silica
nanoparticles, lipase immobilization was carried out by
treatment of the lipase solution with the nanoparticles
directly. The particles (200 mg of Fe3O4 coated with 1%
silica nanoparticles) were added to 40 ml of phosphate
buffer (0.1 mol/l, pH 6.5) containing lipase (1 ml). The
mixture was placed in a shaking incubator at 301C with
continuous shaking at 150 rpm for 6 h to finish the
immobilization of lipase. The immobilized lipase was
recovered by magnetic separation and washed with
phosphate buffer (0.1 mol/l, pH 6.5) three times to
remove excess enzyme. The resulting immobilized lipase
was held at 41C before use. The enzymatic activities of
the free and immobilized lipases were measured by
titrating the fatty acids that were obtained from the
hydrolysis of olive oil. One unit of lipase activity (U) is
defined as the amount of enzyme that hydrolyzes olive
oil, liberating 1.0 mmol of fatty acid per minute under the
assay conditions. The relative recovery (%) was the ratio
between the activity of the immobilized lipase and that of
free lipase [15].
in which K is the deactivation rate constant = slope of the
straight line [16].
The kinetic parameters Vmax and Km were determined for
the immobilized lipase. In addition, the reusability of the
immobilized lipase was determined by hydrolysis of olive
oil by the immobilized lipase recovered using magnetic
separation and compared with the first run (activity
defined as 100%).
Results and discussion
Structure and shape of the support for nanoparticles
The XRD pattern (Fig. 1) of the Fe3O4 (M) nanoparticles
prepared under standard conditions revealed diffraction
peaks at 111, 220, 311, 400, 422, 511, 440, etc., which
were the characteristic peaks of Fe3O4 crystals with a
cubic spinel structure [17]. It was clear that only the
phases of Fe3O4 were detectable and there were no other
undesired diffraction maximas of the impurities that
could be observed in the spectra. From the relatively wide
half-peak breadth, it could be estimated that the particle
size is quite small. From the XRD patterns, the average
diameter that was calculated to be 13.8 nm using the
Scherrer equation (D = Kl/b cosy, in which K is constant,
l is X-ray wavelength, and b is the peak width of halfmaximum) [18,19]. Interestingly, it was observed that
the diffraction patterns for the samples MS1 and MS2
Figure 1
Biochemical characterization of the free and
immobilized lipases and their reusability
Thermal stability of the free and immobilized lipase was
studied by incubating the biocatalyst at 30–801C for 15,
30, and 60 min in a water bath. Similarly, to determine the
stability at varying pH values, the immobilized enzyme
was reinsulated separately in 0.2 mol/l of citrate buffer at
pH 3–7 and in tris-HCl buffer at pH 7.6–9 for 1 h, and the
residual activities were determined under standard assay
conditions. The residual activity in the samples without
incubation was considered to be 100%. The inactivation
rate constant (K) and the half-life time (t1/2) were
calculated using the following formula: Half-life = 0.693/K,
X-ray powder diffraction patterns of (a) pure Fe3O4 nanoparticles and
(b) MS1 and MS2.
Lipase immobilization on Fe3O4 nanoparticles El-Hadi et al. 31
consisted of an amorphous structure, which was attributed to the amorphous silica matrix, as clearly indicated
in Fig. 1 [20]. The XRD patterns of the remaining
samples MS3, MS4, and MS5 (not presented here) also
showed an amorphous structure. The relatively low
intensity reflections and absence of significant sharp
diffraction peaks for the MS1 and MS2 patterns are
probably due to the presence of SiO2 on the surface of
the magnetic nanoparticles. Xu et al. [21] also suggested
that the low intensity of the reflection peaks could be
attributed to the ultrafine crystalline structure of the
magnetite particles used for the generation of silicacoated nanoparticles. The particle size and morphology of
Fe3O4, Fe3O4/SiO2, and Fe3O4/SiO2/enzyme were evaluated from the TEM micrographs. It is noteworthy that
the size distribution is 10–16 nm, which matched the
value calculated using the Scherrer equation, and that the
nanoparticles are spherical in shape (Fig. 2a) and their
aggregation can be discerned clearly. In Fig. 2b and c, the
coated silica layer can be observed as a typical core–shell
structure of the Fe3O4/SiO2 nanoparticles. The dispersity
of the Fe3O4/SiO2 nanoparticles was also improved, and
the average size increased to about 32 nm. After lipase
adsorption, the degree of particle aggregation increased;
however, a change in the particle size was not observed
(Fig. 2d).
FT-IR spectra of the magnetite nanoparticles
The FT-IR spectra of magnetite are shown in Fig. 3. A
factor group analysis, reported in a classic IR study on
spinels, suggested that there were four IR-active bands;
however, in most cases, including magnetite, only two of
them are observed between 400 and 800 cm – 1 [22]. In
this study, Fe3O4 showed a broad band that consisted of
two slightly split peaks identified at 573 and 621 cm – 1;
these peaks were attributed to the stretching vibration of
Figure 2
Transmission electron microscope images of (a) Fe3O4 nanoparticles, (b) MS1 nanoparticles, (c) MS1 nanoparticles with immobilized lipase, and
(d) MS1 nanoparticles without lipase.
32 Egyptian Pharmaceutical Journal
the Fe–O bond and confirmed the occupancy of Fe3 +
ions at tetrahedral sites in a manner consistent with that
reported in the literature [23–25]. On the low-frequency
side of the broad band, we observed that the weak peaks
appearing at 432 and 453 cm – 1 corresponded to the
presence of the Fe3 + –O2 – bond at octahedral sites [26].
In contrast, we found a broad peak near 3380 cm – 1 and a
sharp peak near 1635 cm – 1, which were attributed to the
stretching and binding vibrations of the hydroxyl groups.
These peaks confirm the presence of adsorbed water on
the surface of magnetite [27]. However, the peaks at
1383 and 1453 cm – 1 resulted from the stretching
vibration of the C–O bonds in CO2, which might come
from air.
FT-IR spectra of the silica-coated magnetite
nanoparticles
Figure 3 shows the IR spectrum of the silica-coated
magnetite nanoparticles. It was clear that the characteristic adsorption bands of the Fe–O bond (Fe3 + –O2 – ) of
the silica-coated magnetite nanoparticles shift to higher
wave numbers of 585, 637, 441, and 483 cm-1, respectively, compared with that of uncoated nanoparticles (in
573, 621, 432, and 453 cm – 1). The absorption bands at
around 1030, 800, and 470 cm – 1 reflect the Si–O–Si
asymmetry, Si–O–Si symmetric stretching vibrations, and
deformation mode of Si–O–Si, respectively [28]. The
bands at 569 and 965 cm – 1 are possibly because of
the Fe–O–Si and Si–O–Si stretching vibrations caused by
the perturbation of the metallic ion in the SiO4 tetrahedra [29], respectively. The FT-IR spectra of the lipase
on the silica-coated magnetite nanoparticles (Fig. 3)
showed a spectra similar to the IR spectra of the silicacoated magnetite nanoparticles with immobilized lipase.
It was observed that the characteristic bands of lipase at
1655 and 1535 cm – 1 [30] revealed that it was immobilized on the silica-coated magnetite nanoparticles.
The amount of enzyme added and the corresponding
immobilization efficiency
The relationship between the amounts of enzyme
(0.5–3 ml) and immobilization efficiency has been shown
in Fig. 4. When the enzyme amount added was 1 and
1.5 ml, with 300 mg of the magnetite coated with 1%
silica, the maximal immobilization efficiency was 87 and
96%, respectively. The curve in Fig. 4 illustrates that the
immobilization efficiency gradually decreases when the
amount of enzyme added is more than 1.5 ml. This could
be explained by an overall amount of the added enzyme
formed an intermolecular space hindrance of the immobilized enzyme, which will not only the active site of the
enzymes but also restrain the dispersion of the substrate
and product [3].
Effect of different concentrations of SiO2 coating the
magnetite nanoparticles on the immobilization
efficiency of lipase
To solve the leaching problems of the adsorbed lipase and
improve the conventional way for lipase immobilization,
different concentrations of MS nanoparticles ranging
from 0.5–4% were used as cross-linking agents for
immobilization in 300 mg of magnetite nanoparticles.
The experimental results have been given in Fig. 5. It was
shown that the immobilization efficiency decreased
slightly from 96 to 84% with an increase in the SiO2
concentration from 1 to 4% and then decreased sharply
with a further increase in the concentration of SiO2 to
6% (76%). Although a higher amount of lipase binding
occurred when a low concentration of SiO2 was used for
silica coating the magnetic nanoparticles, there was a
substantial loss of enzyme activity.
Figure 3
Biochemical properties of the free and immobilized
lipase
The effect of pH on the specific enzyme activity of lipase
immobilized by silica was studied by varying the pH of
the reaction medium from 3–9 using a 0.1 mol/l citrate
phosphate buffer (3–7) and a 0.1 mol/l Tris (hydroxy
Figure 4
(a) Fourier transform-infrared spectrophotometer spectra of Fe3O4,
(b) MSI without immobilized lipase, and (c) MSI with immobilized lipase.
Effect of different amounts of Mucor racemosus NRRL 3631 lipase on
the immobilization efficiency.
Lipase immobilization on Fe3O4 nanoparticles El-Hadi et al. 33
methyl) amino methane buffer (7.5–9), and the pH
profile has been shown in Fig. 6a. Generally, the binding
of enzymes to polycationic supports would result in an
acidic shift in the optimum pH [31,30]; similarly, after
silica immobilization, the optimum pH of lipase exhibited
an acidic shift (5–6). The variation in the residual activity
of the free and immobilized lipase with pH is shown
in Fig. 6b. The immobilized lipase was stable in the pH
range of 3–5 as compared with the free enzyme; this
indicated that immobilization appreciably improved the
stability of lipase in the acidic region.
The thermal stabilities of the free and immobilized lipase
in terms of the residual activities have been compared
in Fig. 7. Lipase immobilized on MS nanoparticles
remained fully active up to 401C. These results are
similar to those obtained by Huang et al. [30], who found
that binary immobilized lipase from Candida rugosa was
fully active at 401C; however, inactivation of the enzyme
occurred on treatment at higher temperatures. About
40% of the residual activity of free lipase was preserved at
601C for 1 h; however, about 72.9% residual activity was
Figure 5
Effect of different concentration of SiO2-coated magnetic nanoparticles
on the immobilization efficiency of Mucor racemosus NRRL 3631
lipase.
preserved in case of the immobilized enzyme. At 801C,
the free enzyme was fully inactivated, whereas the
immobilized form preserved about 37.8% of its residual
activity for 15 min. Hiol et al. [32] studied the thermostability of the free enzyme of Rhizopus oryzae and found
that it was highly inactivated at 451C and almost all
activity was lost at 501C after a 40 min incubation. This
thermal stabilization could be explained by the location
of the lipase inside the micropores of the support,
wherein the enzyme is protected against alterations of
the microenvironment. The Michaelis–Menten kinetics
of the hydrolytic activity of the free and immobilized
lipases have been represented in Table 1, using varying
initial concentrations of olive oil as the substrate. The
Michaelis constant (Km) and the maximum reaction
velocity (Vmax) were evaluated from the double reciprocal
plot. The Vmax value of 250 U/mg protein exhibited by
the immobilized lipase was found to be higher than that
of free lipase (50 U/mg protein). The Km value (20 mmol/l)
determined for the immobilized lipase was about threefolds higher than that of free lipase (6.66 mmol/l), which
indicated a lower affinity toward the substrate. This
increase in Km might be either due to the structural
changes induced in the enzymes by the immobilization or
the lower accessibility of the substrate to the active
sites [33,30]. The inactivation temperature of the soluble
and immobilized lipase was observed to be between 50 and
701C. In general, the immobilization processes protected
the enzymes against heat inactivation, for example, the
calculated half-life values at 50, 60, and 701C for the
immobilized enzyme were 630, 533, and 391.5 min,
respectively, which are higher than those (231, 198, 187,
and 3 min, respectively) of the free enzyme as shown
in Table 2, that is, the free enzyme showed a half-life of
10.5 h at 501C, 8.88 h at 601, and 6.5 h at 701C. Our results
are nearly similar to those obtained by Kumar et al. [34],
who reported the half-life of Bacillus coagulans BTS3 lipase at
55 and 601C to be 2 h and 30 min, respectively; moreover,
they reported the half-life of lipase from another mesophilic bacteria (Bacillus spp.) to be 2 h at 601C. They
reported that the deactivation rate constants of 1.1 10–3,
1.3 10–3, and 1.7 10 – 3 for the experimental immobilized enzyme at temperatures of 50,60, and 701C,
respectively were lower than those (3 10–3, 3.77 10–3,
Figure 6
Effect of pH values on the activity (a) and stability (b) of free and immobilized Mucor racemosus lipases.
34
Egyptian Pharmaceutical Journal
Figure 7
Thermal stability of free and immobilized Mucor racemosus NRRL 3631 lipases.
Table 1 Kinetic parameters (Vmax and Km) for the free and
immobilized enzymes
Types
Vmax (U/mg protein)
Km (mmol/l)
250
50
20
6.66
Immobilized lipase
Free lipase
Table 2 Kinetic parameters (half-life and the deactivation rate
constant) for the free and immobilized enzymes
Half-life (min)
Types
Immobilized
lipase
Free lipase
501C 601C 701C
630
Deactivation rate constant
501C
533 391.5 1.8 10
601C
–3
1.3 10
701C
–3
1.7 10–3
231 198 187.3 3 10–3 3.77 10–3 4.8 10–3
Figure 8
wherein the hydrophobic microenvironment makes the
enzymatic activity suffer a single experimental decay
during storage conditions [35].
Variations in the enzyme activity with repeated batch
enzyme reactions
Operational stability was the most important parameter
in the immobilization of enzymes because inactivation is
inevitable when the free enzyme is exposed to inadequate ambient conditions. The recycling efficiency of the
immobilized lipase has been presented in Fig. 8. It was
observed that the immobilized lipase retained 51% of its
original activity even after the seventh reuse; this
indicated that the resultant bound lipase had a better
reusability, which was desirable for applications in
biotechnology. The loss of activity may be ascribed to
conformational changes in the enzyme, blocking of the
lipase active sites, or the gradual loss of the bound lipase
during the reaction procedures.
Conclusion
Operational stability of the immobilized Mucor racemosus NRRL lipase
on the hydrolysis process.
and 4.8 10–3, respectively) of the free enzyme at the
same temperatures. These results could be related to a
hydrophilic or hydrophobic environment. A hydrophilic
microenvironment allowed the immobilized derivatives to
follow a double experimental decay in their activities,
From these results it can be concluded that Fe3O4
magnetic nanoparticles and silica-coated magnetite (MS)
nanoparticles with excellent properties have been successfully prepared using the chemical coprecipitation
technique with some modifications. The XRD results
indicate that the composites were in the nanoscopic
phase. Based on the TEM images, the diameters of the
uncoated magnetite particles were determined to be
around 10–16 nm and those of the coated particles to be
about 11 nm. The silica coating appeared to be effective
in protecting the magnetite from being converted to
other oxide species. The thermal and pH stabilities of the
immobilized lipase increased on immobilization. The
optimal pH and temperature of the immobilized lipase
were 5–6 and 401C, respectively. There was a slight
decrease in the residual activity of the immobilized
lipase. The operational stability of the immobilized lipase
Lipase immobilization on Fe3O4 nanoparticles El-Hadi et al. 35
over repeated cycles could substantially save on the cost
of the enzyme. The residual activity of the enzyme even
after seven repeated uses was over 51%. Conclusively,
magnetic nanoparticles provide an economically efficient
and selective system for enzyme immobilization.
Acknowledgements
Conflicts of interest
There are no conflicts of interest.
References
1 Lei L, Liu X, Li Y, Cui Y, Yang Y, Qin G. Study on synthesis of poly(GMA)grafted Fe3O4/SiO X magnetic nanoparticles using atom transfer radical
polymerization and their application for lipase immobilization. Mater Chem
Phys 2011; 125:866–871.
2 Duguet E, Vasseur S, Mornet S, Goglio G, Demourgues A, Portier J, et al.
Towards a versatile platform based on magnetic nanoparticles for in vivo
applications. Bull Mater Sci 2006; 29:581–586.
3 Bai Y-X, Li Y-F, Yang Y, Yi L-X. Covalent immobilization of triacylglycerol lipase
onto functionalized novel mesoporous silica supports. J Biotechnol 2006;
125:574–582.
4 Deng Y-H, Wang C-C, Hu J-H, Yang W-L, Fu S-K. Investigation of formation
of silica-coated magnetite nanoparticles via sol-gel approach. Colloids and
Surfaces A: Physicochem Eng Aspects 2005; 262 (1–3):87–93.
5 Park D, Haam S, Jang K, Ahn IS, Kim WS. Immobilization of starchconverting enzymes on surface-modified carriers using single and coimmobilized systems: properties and application to starch hydrolysis. Process Biochem 2005; 40:53–61.
6 Yu H, Ching CB. Theoretical analysis of the adsorption effect on kinetic
resolution of racemates catalyzed by immobilized enzymes in a batch reactor.
Ind Eng Chem Res 2008; 47:4251–4255.
7 Villeneuve P, Muderhaw JM, Graille J, Haas MJJ. Lipase-catalyzed esterification of Betulinic acid using phthalic anhydrid in organic solvent media:
study of reaction parameters. Mol Catal B Enzyme 2000; 9:113–148.
8 Mostafa H, El-Hadi AA. Immobilization of Mucor racemosus NRRL 3631
lipase with different polymer carriers produced by radiation polymerization.
Mal J Microb 2010; 6:149–155.
9 Lee SH, Doan TTN, Won K, Ha SH, Koo Y-M. Immobilization of lipase within
carbon nanotube-silica composites for non-aqueous reaction systems. J Mol
Catal B: Enzymatic 2010; 62:169–172.
10 Akhtar MW, Mirza AQ, Chughtai MID. Lipase induction in Mucor hiemalis.
Appl Environ Microbiol 1980; 40:257–263.
11 Starr MP. Spirit blue agar: a medium for the detection of lipolytic microorganisms. Science 1941; 93:333–334.
12 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with
the Folin phenol reagent. J Biol Chem 1951; 193:265–275.
13 Abbas H, Hiol A, Deyris V, Comeau L. Isolation and characterization of an
extracellular lipase from Mucor sp. strain isolated from palm fruit. Enzyme
Microb Technol 2002; 31:968–975.
14 Massart R. Preparation of aqueous magnetic liquids in alkaline and
acidic media. IEEE Trans Magnetics 1981; 17:1247–1248.
15 Lee DH, Park CH, Yeo JM, Kim SW. Lipase immobilization on silica gel using
a cross-linking method. J Ind Eng Chem 2006; 12:777–782.
16 Bailey JE, Ollis DF. Biochemical engineering fundamentals. 2nd ed. USA:
Mc Graw-Hill Book Company; 1986.
17 Nedkov I, Kolev S, Zadro K, Krezhov K, Merodiiska T. Crystalline anisotropy
and cation distribution in nanosized quasi-spherical ferroxide particles. J
Magn Magn Mater 2004; 272–276 (Suppl 1):e1175–e1176.
18 Warren BE. X-ray diffraction. New York: Dover publications; 1990.
19 Hong YR, Li HJ, Zhang ZS, Li ZH, Zheng Y, Ding MJ, Wei GD. Preparation
and characterization of silica-coated Fe3O4 nanoparticles used as precursor
of ferrofluids. App Surf Sci 2006; 255:3485–3492.
20 Poulsen HF, Neuefeind J, Neumann H-B, Schneider JR, Zeidler MD. Amorphous silica studied by high energy X-ray diffraction. J Non-Cryst Sol 1995;
188 (1–2):63–74.
21 Xu H, Tong N, Cui L, Lu Y, Gu H. Preparation of hydrophilic magnetic nanospheres with high saturation magnetization. J Magn Magn Mater 2007;
311 (Special Issue):125–130.
22 White WB, DeAngelis BA. Interpretation of the vibrational spectra of spinels.
Spectrochim Acta A Mol Spectrosc 1967; 23:985–995.
23 Ma M, Zhang Y, Yu W, Shen H-Y, Zhang H, Gu N. Preparation and characterization of magnetite nanoparticles coated by amino silane. Colloids and
Surfaces A: Physicochem Eng Aspects 2003; 212:219–226.
24 Guang-She L, Li-Ping L, Smith RL Jr, Inomata H. Characterization of the
dispersion process for NiFe2O4 nanocrystals in a silica matrix with infrared
spectroscopy and electron paramagnetic resonance. J Mol Struct 2001;
560 (1–3):87–93.
25 Li Y-S, Church JS, Woodhead AL, Moussa F. Preparation and characterization of silica coated iron oxide magnetic nano-particles. Spectrochim Acta A
Mol Biomol Spectrosc 2010; 76:484–489.
26 Maity D, Agrawal DC. Synthesis of iron oxide nanoparticles under oxidizing
environment and their stabilization in aqueous and non-aqueous media. J
Magn Magn Mater 2007; 308:46–55.
27 Tie S-L, Lin Y-Q, Lee H-C, Bae Y-S, Lee C-H. Amino acid-coated nano-sized
magnetite particles prepared by two-step transformation. Colloids and Surfaces A: Physicochem Eng Aspects 2006; 273 (1–3):75–83.
28 Arruebo M, Fernández-Pacheco R, Irusta S, Arbiol J, Ibarra MR, Santamarı́a J.
Sustained release of doxorubicin from zeolite-magnetite nanocomposites prepared by mechanical activation. Nanotechnology 2006; 17:
4057–4064.
29 Chang C-F, Lin P-H, Höll W. Aluminum-type superparamagnetic adsorbents:
synthesis and application on fluoride removal. Colloids and Surfaces A:
Physicochem Eng Aspects 2006; 280 (1–3):194–202.
30 Huang S-H, Liao M-H, Chen D-H. Direct binding and characterization of
lipase onto magnetic nanoparticles. Biotechnol Prog 2003; 19:1095–1100.
31 Goldstein L, Levin Y, Katchalski E. A water-insoluble polyanionic derivative of
trypsin. II. Effect of the polyelectrolyte carrier on the kinetic behavior of the
bound trypsin. Biochemistry 1965; 3:1913–1919.
32 Hiol A, Jonzo MD, Rugani N, Druet D, Sarda L, Comeau LC. Purification and
characterization of an extracellular lipase from a thermophilic Rhizopus
oryzae strain isolated from palm fruit. Enzyme Microb Technol 2000; 26
(5–6):421–430.
33 Anita A, Sastry CA, Hashim MA. Immobilization of urease on vermiculite.
Bioprocess Eng 1997; 16:375–380.
34 Kumar S, Kikon K, Upadhyay A, Kanwar SS, Gupta R. Production, purification, and characterization of lipase from thermophilic and alkaliphilic Bacillus
coagulans BTS-3. Protein Expr Purif 2005; 41:38–44.
35 Moreno JM, Arroyo M, Hernáiz M-J, Sinisterra JV. Covalent immobilization of
pure isoenzymes from lipase of Candida rugosa. Enzyme Microb Technol
1997; 21:552–558.
36
Original article
Extracellular polysaccharides produced by the newly discovered
source Scopularis spp.
Siham A. Ismail
Department of Chemistry of Natural and Microbial
Products, Division of Pharmaceutical and Drug
Industries, National Research Centre, Cairo, Egypt
Correspondence to Siham A. Ismail, Department of
Chemistry of Natural and Microbial Products, Division
of Pharmaceutical and Drug Industries, National
Research Centre, El-Behowth St., PO Box 12311,
Dokki, 12622 Cairo, Egypt
Tel: + 20 122 357 1676; fax: + 20 233 370 931;
e-mail: [email protected]
Received 26 August 2012
Accepted 15 November 2012
Egyptian Pharmaceutical Journal
2013,12:36–39
Background
Microorganisms are better and cheaper sources for the production of polysaccharides.
Therefore, there has been an increasing interest in isolating and identifying new
microbial polysaccharides.
Objective
The aim of this study was to produce new extracellular polysaccharides, with better
rheological properties and varied applications, from the newly discovered fungal strain
Scopularis spp., using different carbon sources.
Methods
Fourier transform infrared spectroscopy, carbohydrate analysis, and thin layer
chromatography were the methods used for the preliminary characterizing of the
produced polysaccharides.
Results
Among the 10 examined carbon sources, fructose, raffinose, sucrose, and maltose
were found to produce an appreciable amount of extracellular polysaccharides (0.90,
0.87, 0.86, and 0.74 g/l, respectively), whereas arabinose, lactose, and mannitol
produced a minimal amount of extracellular polysaccharides (0.22, 0.17, 0.12 g/l,
respectively). However, all the tested sugars enhanced the growth of the fungal strain.
The analytical method proved that the polymer was a heteropolysaccharide with six
sugar moieties, all different in their relative ratios from one carbon source to another.
Glucose was found to be the most abundant monosugar in all the polymer samples.
Galactose, rhamnose, and glucuronic acid also appeared on the thin layer
chromatography plate.
Conclusion
A new extracellular heteropolysaccharide was produced from the new source,
Scopularis spp. The produced polysaccharide contained glucose, galactose,
glucuronic acid, rhamnose, and two other unidentified sugars as indicated from the
thin layer chromatography plate.
Keywords:
acid hydrolysis, carbon source, extracellular polysaccharides, Scopularis spp.
Egypt Pharm J 12:36–39
& 2013 Division of Pharmaceutical and Drug Industries Research,
National Research Centre
1687-4315
Introduction
Polysaccharides are highly valued, biologically active
polymers with many industrial applications in food, feed,
textile, cosmetic, and pharmaceutical industries and are
also used as depolluting agents [1]. Owing to their
bioactive nature, they have many medicinal applications
as anticancer, antiviral, antioxidant, antibacterial, antiinflammatory, and prebiotic agents [2–8]. However, most
of the commercial polysaccharides are produced from
plants and algae and a small proportion is produced from
microbial sources [9]. Fungi are currently an interesting
source of biologically active compounds. Most of the
mould-produced polysaccharides are obtained from
mushrooms [8,10,11].
Microorganisms are better and cheaper sources for the
production of polysaccharides compared with plants or
algae because of their high growth rate, ability to grow in
cheaper nutrient media within a few days, lower space
requirement, and ease of manipulation [12]. Therefore,
there has been an increasing interest in isolating and
identifying new microbial polysaccharides that may
compete with traditional polysaccharides.
Therefore, the aim of this study was to examine the
ability of the new fungal strain Scopularis spp. to produce
high yields of extracellular polysaccharides (EPS), with
better rheological properties and varied applications,
using different carbon sources.
Materials and methods
Microorganisms and media
The Scopularis spp. used in this study was obtained from
the culture collection of the National Research Centre
(Egypt). The strain was maintained by subculturing on
1687-4315 & 2013 Division of Pharmaceutical and Drug Industries Research,
National Research Centre
DOI: 10.7123/01.EPJ.0000427067.33748.ea
Polysaccharide new microbial source Ismail 37
potato dextrose agar slants monthly (PDA; Merck,
Darmstadt, Germany). The slants were incubated at
28–301C for 7 days before storage at 41C.
The inoculum cultures were grown in 250 ml Erlenmeyer
flasks containing 50 ml of sterilized medium comprising
(g/l): lactose, 7.5; NaN03, 1.0; yeast extract, 1.5;
MgSO4.7H2O, 0.5; KH2PO4, 1.0; KCl, 0.5; and FeSO4.7H2O, 0.01 at pH 5 [13] in a rotatory incubator shaker at
150 rpm and 28–301C for 3 days before using.
Fermentation
Fermentation was carried out in 250 ml Erlenmeyer flasks
containing 50 ml of the above mentioned medium with
different sugars as the carbon source. The tested sugar
solutions were sterilized separately and mixed aseptically
with the other components before inoculation with 5%
(v/v) of the inoculums. The flasks were incubated
at 28–301C in a rotary shaker at 150 rpm for 7 days.
Mycelial dry weight
The mycelial pellets were separated from the viscous
liquid culture by centrifugation (6000 rpm, 20 min). After
the removal of the supernatant, the mycelia was washed
thoroughly with distilled water and dried to a constant
weight to attain the mycelial dry weight.
Isolation of the extracellular polysaccharides
The viscous supernatant obtained from the above mentioned step were collected and dialyzed against tap water
for 2 days using a 10 000–12 000 MWCO membrane (VWR
Scientific, Spectrum Companies, Goshen Parkway, West
Chester, USA), changing it three times daily; it was then
dialyzed against distilled water in the same way, after which
the solution was centrifuged again as indicated above. The
dialyzed cultures were mixed with three volumes of chilled
absolute ethanol (v/v) with stirring. The precipitated
polysaccharide was collected together as viscous filaments
and could easily be separated from the liquid and the other
compact particles that settled quickly to the bottom. The
collected EPS were washed with a water : ethanol mixture
(1 : 1, v/v) to remove the residue of the liquid culture; it
was then dried and weighted as crude EPS.
standard monosugars. The plates were developed at room
temperature in a saturated chamber containing n-propanol :
water (85 : 15, v/v). The sugars were detected by spraying
the dried plates with 3% phenol reagent, followed by
incubation at 1001C in an oven for 10 min [15].
Fourier transform infrared analysis
The crude polysaccharide was mixed with KBr powder,
ground, and pressed into 1 mm pellets for Fourier transform
infrared (FTIR 6100; Jasco, HoChi Minh City, Japan)
spectroscopy in the frequency range of 4000–400 cm – 1.
Results and discussion
Carbohydrates are very important nutritional requirements for the growth and development of all fungi.
However, different fungal species vary in their ability to
utilize different carbon sources. The results shown
in Fig. 1 indicate that the tested fungus had the ability
to grow in all the used carbon sources, but the production
of the EPS was quite distinct for each sugar used. Among
the 10 sources examined, fructose, raffinose, sucrose, and
maltose enhanced the production of EPS (0.90, 0.87,
0.86, and 0.74 g/l, respectively), whereas arabinose,
lactose, and mannitol produced minimal amounts of
EPS (0.22, 0.17, 0.12 g/l, respectively). The effect of the
different carbon sources on the amount of polysaccharides produced was recorded by all the researchers and was
found to be related to the microorganism used. The
mycelial growth did not parallel with the production of
EPS; this has also been reported by other researchers [16–18]. It has been observed that the production of
EPS increased with an increase in the concentration of
the sugars used and when the morphology of the fungal
growth was in the form of pellets rather than fibers
(unpublished data). The amount of EPS produced during
Figure 1
All the experiments were conducted in triplicate and the
results are the averages of these three independent trials.
Monosaccharide composition analysis
Acid hydrolysis of the crude polysaccharides was carried
out according to the procedure described by Fischer and
Dorfel [14]. In brief, 0.05 g of the crude EPS was mixed
with 0.5 ml of 80% sulfuric acid and left overnight at room
temperature; it was then diluted with 6.5 ml of distilled
water and boiled in a water bath for almost 6 h. The
mixtures were cold neutralized with excess BaCO3 and
subjected to thin layer chromatography for primary
investigation.
Thin layer chromatography
Silica gel plates (Merck) were used to identify the
composition of the hydrolyzed polysaccharides. The samples were spotted onto the plates along with different
The effect of different sugars on the production of extracellular
polysaccharides (EPS) and cell growth [expressed as constant dry
weight (CDW)].
38
Egyptian Pharmaceutical Journal
Figure 2
(a)
(b)
100
Transmittance (%)
90
80
70
60
50
4000 3600 3200 2800 2400 2000 1600 1200
800
100
80
70
60
100
90
80
70
800
400
800
400
Wavenumber (cm-1)
(d)
FT-IR of crude EPS from raffinose
FT-IR of crude EPS from Surose
90
80
70
60
60
50
4000 3600 3200 2800 2400 2000 1600 1200
Wavenumber (cm-1)
90
50
4000 3600 3200 2800 2400 2000 1600 1200
400
Wavenumber (cm-1)
(c)
Transmittance (%)
FT-IR of crude EPS from glucose
FT-IR of crude EPS from fructose
Transmittance (%)
Transmittance (%)
100
800
400
50
4000 3600 3200 2800 2400 2000 1600 1200
Wavenumber (cm-1)
(a) Fourier transform infrared (FTIR) of the crude extracellular polysaccharides (EPS) produced from fructose as the carbon source in the culture
medium. (b) FTIR of the crude EPS produced from glucose as the carbon source in the culture medium. (c) FTIR of the crude EPS produced from
raffinose as the carbon source in the culture medium. (d) FTIR of the crude EPS produced from sucrose as the carbon source in the culture medium.
our study is within the range that has been published by
other authors [17–19].
Figure 3
Fourier transform infrared spectroscopic analysis
The configuration of the crude EPS produced from four
different sugars has been shown in Fig. 2a–d.The spectra
clearly indicate that all the samples had a broad band
around 3400 cm – 1 representing a large number of
hydroxyl groups and a sharp band at 2922 cm – 1 for the
C–H bending vibration of the CH2 groups, and the two
bands are characteristic of carbohydrate polymers. The
bands near 1736 cm – 1 and those around 1250 cm – 1 may
be attributed to the stretching vibration of the C = O and
C–O–C of the acyl groups. The bands at 1420 cm – 1 and
those around 1606–1621 cm – 1 have been suggested to
represent the carboxyl groups of acids, whereas the bands
in the range of 820–955 cm – 1 represent the linkages
between the mono sugars. All the data were within the
range that has been reported by other authors [5,6,20–23].
Effect of different carbon sources on the composition of
the extracellular polysaccharides
The monosugar composition of the crude EPS produced
from the different carbon sources was identified using
thin layer chromatography as shown in Fig. 3. The plates
indicate the presence of more than six distinguishable
spots in most of the samples. However, the relative ratio
Thin layer chromatography plate for the different samples: 1, 7, 8, 9, 12,
and 13 for glucuronic acid, fucose, glucose, N-acetyl glucosamine,
galactose, and rhamnose standards, respectively, and 2, 3, 4, 5, 6, 10,
and 11 for fructose, raffinose, maltose, sucrose, glucose, lactose, and
arabinose samples, respectively.
of the monosugars was entirely different. All the samples
mainly contain glucose, galactose, glucuronic acid, and
rhamnose. Although glucose is the main monosugar
component of the produced EPS, neither glucose nor
Polysaccharide new microbial source Ismail 39
its isomer galactose gave the highest yield of the
produced EPS, when used in the culture medium as
the carbon source. However, both glucose and galactose
gave an appreciable amount of EPS (0.6 and 0.56 g/l,
respectively).The influence of the carbon source on the
production and composition of the EPS has been
reported by other authors as well [5,17,24]. The presence
of different sugar moieties suggests that the produced
EPS was a heteropolysaccharide.
Conclusion
A new extracellular heteropolysaccharide was produced from
a newly discovered source, Scopularis spp. The strain has the
ability to grow and produce EPS in the presence of all the
tested sugars. The produced polysaccharide contains
glucose, galactose, glucuronic acid, rhamnose, and two other
unidentified sugars. This study will open doors for further
studies on attaining a greater production of EPS from this
newly discovered source and also for clarifying their exact
composition, structures, and biological activities. Moreover,
the oligosaccharides and low-molecular-weight polysaccharides that come out of the dialysis bag have to be identified.
5 Kanmani P, Satish kumar R, Yuvaraj N, Paari KA, Pattukumar V, Arul V. Production and purification of a novel exopolysaccharide from lactic acid bacterium Streptococcus phocae PI80 and its functional characteristics activity
in vitro. Bioresour Technol 2011; 102:4827–4833.
6 Wang Z-M, Peng X, Lee K-LD, Tang JC-O, Cheung PC-K, Wu J-Y. Structural
characterisation and immunomodulatory property of an acidic polysaccharide
from mycelial culture of Cordyceps sinensis fungus Cs-HK1. Food Chem
2011; 125:637–643.
7 Cimini D, de Rosa M, Schiraldi C. Production of glucuronic acid-based
polysaccharides by microbial fermentation for biomedical applications.
Biotechnol J 2012; 7:237–250.
8 Zong A, Cao H, Wang F. Anticancer polysaccharides from natural resources:
a review of recent research. Carbohydr Polym 2012; 90:1395–1410.
9 Donot F, Fontana A, Baccou JC, Schorr-Galindo S. Microbial exopolysaccharides: main examples of synthesis, excretion, genetics and extraction.
Carbohydr Polym 2012; 87:951–962.
10 Ikekawa T. Beneficial effect of edible and medicinal mushroom on
health care. Int J Med Mushroom 2001; 3:291–298.
11 Wasser S. Medicinal mushrooms as a source of antitumor and immunomodulating polysaccharides. Appl Microbiol Biotechnol 2003;
60:258–274.
12 Raza W, Makeen K, Wang Y, Xu Y, Qirong S. Optimization, purification,
characterization and antioxidant activity of an extracellular polysaccharide
produced by Paenibacillus polymyxa SQR-21. Bioresour Technol 2011;
102:6095–6103.
13 Ismail SA-A, Hashem AM. Nutrition requirement for the production of
Penicillium chrysogenum a-galactosidase and its potential for hydrolysis of
raffinose family oligosaccharides. J Appl Sci Res 2012; 8:945–952.
14 Fischer FG, Dorfel H. Polyuronic acids in brown algae. Hoppe Seylers
Z Physiol Chem 1955; 302 (4–6):186–203.
15 Adachi S. A rapid method for the assay of lactulose. Anal Biochem 1965;
12:137–142.
1 Raza W, Yang W, Jun Y, Shakoor F, Huang Q, Shen Q. Optimization and
characterization of a polysaccharide produced by Pseudomonas fluorescens
WR-1 and its antioxidant activity. Carbohydr Polym 2012; 90:921–929.
2 Li R, Jiang X-L, Guan H-S. Optimization of mycelium biomass and exopolysaccharides production by Hirsutella sp. in submerged fermentation and
evaluation of exopolysaccharides antibacterial activity. Afr J Biotechnol
2010; 9:195–202.
16 Pokhrel CP, Ohga S. Submerged culture conditions, for mycelia yield and
polysaccharides production by Lyophyllum decastes. Food Chem 2007;
105:641–646.
17 Chang TT, Chao CH, Lu MK. Enhanced biomass production of Pycnoporus
sanguineus and alterations in the physiochemical properties of its polysaccharides. Carbohydr Polym 2011; 83:796–801.
18 Smiderle FR, Olsen LM, Ruthes AC, Czelusniak PA, Santana-Filho AP,
Sassaki GL, et al. Exopolysaccharides, proteins and lipids in Pleurotus
pulmonarius submerged culture using different carbon sources. Carbohydr
Polym 2012; 87:368–376.
19 Yun Xiang W, Zhao-Xin L. Optimization of processing parameters for the
mycelial growth and extracellular polysaccharide production by Boletus spp.
ACCC 50328. Process Biochem 2005; 40 (3–4):1043–1051.
20 Na YS, Kim WJ, Kim SM, Park JK, Lee SM, Kim SO, et al. Purification,
characterization and immunostimulating activity of water-soluble polysaccharide isolated from Capsosiphon fulvescens. Int Immunopharmacol
2010; 10:364–370.
21 Zhao L, Dong Y, Chen G, Hu Q. Extraction, purification, characterization and
antitumor activity of polysaccharides from Ganoderma lucidum. Carbohydr
Polym 2010; 80:783–789.
22 Freitas F, Alves VD, Torres CAV, Cruz M, Sousa I, Melo MJ, et al. Fucosecontaining exopolysaccharide produced by the newly isolated Enterobacter
strain A47 DSM 23139. Carbohydr Polym 2011; 83:159–165.
3 Zhao L, Chen Y, Ren S, Han Y, Cheng H. Studies on the chemical structure
and antitumor activity of an exopolysaccharide from Rhizobium sp. N613.
Carbohydr Res 2010; 345:637–643.
4 Deng P, Zhang G, Zhou B, Lin R, Jia L, Fan K, et al. Extraction and in vitro
antioxidant activity of intracellular polysaccharide by Pholiota adiposa SX-02.
J Biosci Bioeng 2011; 111:50–54.
23 Ye S, Liu F, Wang J, Wang H, Zhang M. Antioxidant activities of an exopolysaccharide isolated and purified from marine Pseudomonas PF-6. Carbohydr Polym 2012; 87:764–770.
24 Jin Y, Zhang L, Chen L, Chen Y, Cheung PCK, Chen L. Effect of culture
media on the chemical and physical characteristics of polysaccharides isolated from Poria cocos mycelia. Carbohydr Res 2003; 338:1507–1515.
Acknowledgements
This work was supported by National Research Centre, Cairo, Egypt;
as part of the project of the Department of Chemistry of Natural and
Microbial Products. Thank’s for the responsible professors of the
financial department.
Conflicts of interest
There are no conflicts of interest.
References
40
Original article
Biotransformation of soybean saponin to soyasapogenol B by
Aspergillus parasiticus
Hala A. Amina, Yousseria M. Hassanb and Soad M. Yehiaa
a
Department of Chemistry of Natural and Microbial
Products, National Research Center, Dokki and
b
Department of Microbiology, Faculty of Science,
Ain shams University, Cairo, Egypt
Correspondence to Hala A. Amin, PhD, Department of
Chemistry of Natural and Microbial Products, National
Research Center, Dokki, 12311 Cairo, Egypt
Tel: + 20 233 464 472; fax: + 20 237 622 603;
e-mail: [email protected]
Received 7 November 2012
Accepted 15 January 2013
Egyptian Pharmaceutical Journal
2013,12:40–45
Objectives
The aim of this study was to select of the most potent fungus that is able to hydrolyze
soybean saponin (SS) to soyasapogenol B (SB). The selected fungus was cultivated
under different physiological conditions to evaluate its ability to transform SS to
achieve the maximal conversion output.
Materials and methods
Within 72 h, the biotransformation of SS by Aspergillus parasiticus, followed by
isolation and purification of SB as a main product were carried out. The identity of SB
was established by determination of its RF value and IR, mass spectra, and NMR
spectra. Furthermore, different sets of experiments were carried out to enhance the
activity of the tested organism and consequently, SB production.
Results and conclusion
Screening of different fungal isolates for transformation of SS to SB revealed that
A. parasiticus produced the highest yield of SB. The maximum SB yield was obtained
using a production medium composed of (%, w/v): malt extract, 4; yeast extract, 2;
KH2PO4, 0.2; (NH4)2SO4, 0.2; MgSO4 7H2O, 0.03; CaCl2 2H2O, 0.03; galactose,
0.5; and SS, 3 (pH 8). The medium was inoculated with 6% (v/v) inoculum of a 72 h
old culture and incubated on a rotary shaker (150 rpm) at 301C for 72 h. Under these
optimal conditions, the cell biotransformation efficiency was increased from 13.44 to
65%.
Keywords:
Aspergillus parasiticus, biotransformation, soyasapogenol B, soybean saponin
Egypt Pharm J 12:40–45
& 2013 Division of Pharmaceutical and Drug Industries Research,
National Research Centre
1687-4315
Introduction
Saponins are structurally diverse molecules that are
chemically referred to as triterpenes and steroid glycosides.
They consist of nonpolar aglycones coupled with one or
more monosaccharide moieties [1]. This combination of
polar and nonpolar structural elements in their molecules
explains their soap-like behavior in aqueous solutions.
Soyasaponins are a group of oleanane triterpenoids found
in soy and other legumes. They are divided into three
groups, based on the structure of the aglycone moiety, the
A, B, and E saponins [2]. Soyasapogenols A, B, and E are
conjugated as glycosides in soy [3,4]. The current
consensus is that soyasapogenols A, B, and E are true
aglycons, whereas soyasapogenols C, D, and E are artifacts
of hydrolysis that occur during the isolation process of
A, B, and E soyasapogenols .
Soyasaponins have various physiological effects including
hepatoprotective [5], anticarcinogenic [6], antiviral [7],
and anti-inflammatory [8] activities. Soyasapogenol B (SB),
obtained from soybean saponin (SS), is known to have
hepatoprotective [9], antiviral [10], antimutagenic [11],
anti-inflammatory [8], and growth suppressing effects on
cells derived from human colon and ovarian cancer [11,12].
Results from in-vitro fermentation suggest that colonic
microflora readily hydrolyzed SS to aglycones [2]. These
observations suggested that the dietary chemopreventive
effects of SS against colon cancer may involve alteration
by the microflora [12]. There is some evidence, as with
many other saponins, that bioactivity of SS is increased as
sugar moieties are eliminated from the saponin structure,
thereby reducing the polarity.
Aglycones, soyasapogenols, are produced by acid hydrolysis
of saponins, but there have been reports of aglycone
production by microorganisms. Kudou et al. [13] cultured
158 strains of the genus Aspergillus in a medium containing
SS and reported that 26 of them had a marked SS hydrolase
activity. Watanabe et al. [14] isolated a SS hydrolase from
Neocosmospora vasinfecta var. vasinfecta PF1225, a filamentous
fungus that can degrade SS and generate SB. Recently,
Amin and Mohamed [15] reported the production of SB
(86.3%) from SS using immobilized Aspergillus terreus on a
loofah sponge. The aim of this study was to select the most
potent fungus that is able to hydrolyze SS to SB. The
selected isolate was cultivated under different physiological
conditions to evaluate its ability to transform SS to achieve
the maximal conversion output.
Materials and methods
Cultivation of fungal isolates
The different fungal isolates used in this work (Table 1)
were donated by the Center of Cultures of Chemistry of
1687-4315 & 2013 Division of Pharmaceutical and Drug Industries Research,
National Research Centre
DOI: 10.7123/01.EPJ.0000427332.31862.10
SS biotransformation to SB Amin et al.
Table 1 Bioconversion of soybean saponin to soyasapogenol B
by different fungal strains
Soyasapogenol B
Fungal isolates
Aspergillus fumigatus
Aspergillus flavus
Aspergillus niger
Aspergillus parasiticus
Aspergillus ruber
Rhizopus riori
Penicillium aurantiacum
Penicillium waksmanii
Penicillium frequentans
Penicillium cyclopium
Trichoderma harzianum
Trichoderma viride
mg/100 ml
Molar yield (%)
6.8
22.34
18.58
32
4.92
14.6
–
1.86
9.6
8.12
3.9
3.82
2.85
9.38
7.8
13.44
2.06
6.13
–
0.78
4.03
3.41
1.63
1.6
Strains were cultivated on a transformation culture medium consisting
of (%, w/v): malt extract, 4; yeast extract, 2; KH2PO4, 0.2; (NH4)2SO4,
0.2; MgSO4 7H2O, 0.03; CaCl2 2H2O, 0.03; and SS, 1 (pH 5.7) at
150 rpm and 30 ± 21C for 72 h.
SS, soybean saponin
Natural and Microbial Products Department, National
Research Center (Cairo, Egypt). They were maintained
on potato dextrose agar slants at 41C and subcultured
at intervals of 1–2 months. Unless otherwise stated, the
fermentations were carried out in 250 ml Erlenmeyer
flasks containing 100 ml of the fermentation medium
composed of (%, w/v): malt extract, 4; yeast extract, 2;
KH2PO4, 0.2; (NH4)2SO4, 0.2; MgSO4 7H2O, 0.03;
CaCl2 2H2O, 0.030; and SS, 1 (pH 5.7) [16]. The flasks
were inoculated with 6% inoculum and agitated on a
rotary shaker at 150 rpm at 30 ± 21C for 72 h.
General assessment of the chemicals and instruments
used
SS (50%) was purchased from Organic Technologies Co.
(Coshocton, Ohio, USA). Potato dextrose agar and yeast
extract were products of Biolife Italiana (Milano, Italy).
Bacto malt extract and bacto peptone were purchased
from Difco Laboratories (New Jersey, USA). 1H NMR
and 13C NMR spectra were measured using a Bruker
AMX 500 instrument (Weizmann Institute of Science
Chemical, Rehovot, Israel) operating at 500 MHz for 1H
NMR and at 125 MHz for 13C NMR. Samples were dissolved in fully deuterated dimethyl sulfoxide (DMSO-d5).
The chemical shifts (d) are reported in ppm and the
coupling constants (J) in Hz. Mass spectra were measured
using a Finnigan mat. SSQ 7000 instrument at an
ionization voltage of 70 eV and EI mode.
Quantitative analysis of soyasapogenol B
At the end of the biotransformation period, the reaction
mixture was extracted twice with double the volume of
ethyl acetate. Thereafter, the organic layer was dried over
anhydrous sodium sulfate and concentrated under
reduced pressure. The residue was dissolved in a
chloroform–methanol mixture (1 : 1) and mounted on
thin-layer chromatography (TLC) plates. The plate was
first chromatographed for soyasapogenols using the
above-mentioned solvent system and then for SS using
a solvent system comprising chloroform–methanol–acetic
41
acid (10 : 20 : 1, v/v). SS and SB were detected on TLC
plates by spraying with 10% H2SO4 and then heating for
10 min at 1101C; they were then quantitatively analyzed
using a TLC-scanner (Shimadzu CS-9000 dual wavelength flying spot, thin layer chromato-scanner, Tokyo,
Japan) at l equal to 530 nm [16]. The obtained weight of
SB was calculated by calibration of the line obtained from
the standard sample using the area under the curve for
the biotransformation products in each chromatogram.
SB molar yield ð % Þ¼
Weight of soyasapogenol B/MW of soyasapogenol B
100;
Weight of soyasaponin I/MW of soyasaponin I
where MW is the molecular weight and soyasaponin I
represents SS.
Separation and identification of the biotransformation
products
After cultivation of Aspergillus parasiticus on the biotransformation culture medium containing 1% SS, the resulting filtrate (500 ml) was extracted twice with ethyl
acetate, and the organic layer was concentrated under
reduced pressure to obtain an oily sample (415 mg). A
preparative silica gel plate (silica gel 60 F-254 aluminum
plates; Merck, Darmstadt, Germany) was spotted and
developed using the same solvent system (benzene :
ethyl acetate : acetic acid; 24 : 8 : 1, v/v). The areas
containing soyasapogenols were detected by a slight
discoloration on the plates, and these sections were
scraped, extracted with chloroform : methanol (1 : 1), and
evaporated to dryness. This led to isolation of compound
I (56 mg) as the main product.
Compound I
Compound I was identified as SB, with a melting point of
2301C. The H1 NMR (DMSO-d5) results were as follows:
d at 5.18 (t, 1H, J12,11a = J12,11b = 4 Hz, H-12), 4.85 (d, 1H,
J24a-24b = 4.6 Hz, H-24a), 4.14 (dd, 2H, H-3, and H-21), 4.05
(d, 1H, J24b-24a = 4.6 Hz, H-24b), 3.82 (d, 1H, J22a-21b = 8.4
Hz, J22a,21a = 2.4 Hz, H-22a), 1.2 (s, 3H, H-23), 1.18 (s, 3H,
H-27), 0.95 (s, 3H, H-28), 0.90 (s, 3H, H-26), 0.84 (s, 6H,
H-25), 0.82 (s, 3H, H-29), and 0.80 (s, 3H, 30) and the 13C
NMR (CD3Cl) results were shown in Table 2.
Optimization of soybean saponin biotransformation
using Aspergillus parasiticus
Optimization of the environmental conditions for microbial biotransformation processes on a laboratory scale
is important to obtain information for the scaled-up
production of the target product in a large-scale
fermentor. The parameters assessed were pH (4, 5, 5.7,
6, 7, 8, and 9) of the medium, inoculum size (1, 2, 3, 4, 5,
6, 8, and 10%, v/v) and age (24, 48, 72, and 96 h), duration
of the biotransformation process (24, 48, 72, 96, 120, and
144 h), SS concentration (0.5, 1, 2, 3, and 4%, w/v),
incubation temperature (20, 25, 30, 35, and 401C), and
shaking incubator speed (static,100, 150, 200, and
250 rpm). For examining the effect of the cultivation
medium composition on the biotransformation process,
different levels of either malt extract (2, 3, 4, 5, and 6%,
42 Egyptian Pharmaceutical Journal
Table 2
13
C NMR assignments of soyasapogenol B
Carbon number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Soyasapogenol B
38.13
27.14
78.57
42.05
55.34
18.54
32.77
41.14
47.11
36.24
23.13
121.46
144.03
41.56
25.45
27.83
36.87
44.51
46.01
30.11
41.56
73.98
22.80
62.94
15.57
16.50
24.99
28.23
32.59
20.23
w/v) or yeast extract (0.5, 1, 2, 2.5, and 3%, w/v), different
carbon sources (glucose, galactose, mannose, sucrose,
arabinose, and starch), and different concentrations of
galactose (0.5, 1, 2, 3, 4, and 5% w/v) were individually
used.
Results and discussion
Screening experiments
Twelve fungal isolates were screened for their saponinhydrolyzing abilities to produce SB from the SS that was
added to the culture medium. Results in Table 1 indicate
different capacities of the tested cultures to produce SB.
P. aurantiacum failed to perform the desired reaction,
whereas the other fungal isolates (Aspergillus flavus,
Aspergillus fumigates, Aspergillus niger, Aspergillus parasiticus,
Aspergillus ruber, Penicillium cyclopium, Penicillium frequentans,
Penicillium waksmannii, Rhizopus riori, Trichoderma harzianum,
and Trichoderma viride) could. Among the 12 examined
fungal cultures, A. parasiticus produced the highest yield
of SB; it could transform about 13.44% of the added SS,
with the formation of 32 mg/100 ml SB. In this connection, Kudou et al. [13] reported that 26 of 158 strains of
the genus Aspergillus had a marked SS hydrolase activity
when cultured in a medium containing SS. Moreover,
Watanabe et al. [17] purified a SS hydrolase from
Aspergillus oryzae PF1224.
Identification of the biotransformation products
As A. parasiticus was cultivated for 72 h on a medium
containing 1% SS; compound I was isolated as a major
product (about 80%) in addition to some other minor
by-products. Physicochemical characteristics and various
spectral data of the obtained compound I were identical
to those of standard SB. Compound I produced red color
with sulfuric acid alone or with Liebermann–Burchard
reagent for the triterpenes. The molecular formula was
assigned to be C30H50O3 from the EI-mass spectra
(458 m/z). The presence of seven tertiary methyl singlets
(d 0.8–1.2) and a triplet olefinic proton at d 5.18 (t, 1H,
J12,11a = J12,11b = 5 Hz, H-12) in the NMR spectra
suggested a olean-12-en structure with three hydroxyl
groups. The hydroxyl groups were identified as being
attached at C-3, C-22, and C-24 from the H1 and 13C
NMR spectral data. The downfield shift of both C-3 and
C-22 (d 78.57 and 73.98, respectively) in the 13C NMR
spectrum suggested that two hydroxyl groups were
attached at these positions. The third hydroxyl group
was supported at C-24 by the presence of two signals at
d 4.85 (d, 1H, J24a-24b = 4.6 Hz, H-24a) and d (d, 1H,
J24b-24a = 4.6 Hz, H-24b), in addition to a methylene carbon
signal at 62.94 ppm in the 13C NMR spectrum. The signal
at d 3.82 (d, 1H, J22a-21b = 8.4 Hz, J22a,21a = 2.4 Hz, H-22a)
was assigned as the H-22a proton, which suggested a
b-orientation of the oxygen atom. Therefore, compound I
was identified as 3 b, 22 b, 24-trihydroxyolean-12 (13)-ene
(SB). All spectral data were in agreement with those
published by Kitagawa and colleagues [18,19].
Optimization of soybean saponin biotransformation by
Aspergillu sparasiticus
Effect of pH
Results presented in Table 3 show that the highest SS
conversion activities were maintained within the pH
range of 7–9; however, the biotransformation process was
markedly impedd at pH values below 5.7. In addition, the
initial pH values of the medium (4–9) were found to be
shifted toward more acidic values (3.39–6.83) at the end
of the bioconversion process. A maximum concentration
of SB (89.39 mg/100 ml) corresponding to a molar yield of
37.59% was obtained at pH 8. These findings supported
the data reported by Amin et al. [19] for the bioconversion
of SS to SB by A. terreus. Kudou et al. [20] found that
saponin hydrolase enzyme from A. oryzae KO-2 was stable
at pH values ranging from 5.0 to 8.0.
Effect of inoculum size
Results illustrated in Fig. 1 indicate that the yield of SB
was positively correlated to the increase in the inoculum
size up to 6% inoculum (v/v), corresponding to 0.0568 mg
cell dry weight, which led to the highest yield of SB
(37.59%). In contrast, an increase or decrease in the
inoculum size led to a gradual decrease in the SB yield.
Effect of the incubation period
The capacity of A. parasiticus to transform SS proved to be
markedly affected by the duration of the transformation
process. As shown in Fig. 2, biotransformation of SS to SB
increased gradually with increase of the incubation period
until the maximum value of 37.59% after 72 h was
reached, giving an SB yield of 89.5 mg/100 ml. However,
this yield sharply decreased upon increasing the time
more than 72 h, probably due to a further metabolism of
SS biotransformation to SB Amin et al.
43
trends of SB production and cell growth were roughly
equivalent.
Table 3 Effect of different initial pH values on production of
soyasapogenol B from soybean saponin by Aspergillus
parasiticus
Soyasapogenol B
Initial pH
Final pH
mg/100 ml
Molar yield (%)
4.10
4.96
5.03
5.71
7.22
6.83
11.38
32
64.33
83.16
89.5
83.39
4.78
13.44
27.01
34.92
37.59
35.02
5
5.7
6
7
8
9
Initial medium pH was adjusted using 1N HCl and 1N KOH at different
pH values. Aspergillus parasiticus was cultivated on a transformation
culture medium at 150 rpm and 30 ± 21C for 72 h.
Figure 1
40
100
"SB"
Molar Yield
30
60
20
40
Molar Yeild (%)
SB (mg/100ml)
80
10
20
0
0
1
2
3
6
7
4
5
Inoculum size (ml)
8
9
10
11
Effect of inoculum size on production of soyasapogenol B (SB) from
soybean saponin by Aspergillus parasiticus. Biotransformation was
performed on a transformation culture medium (pH 8) inoculated
separately with different inoculum sizes. Flasks were incubated at
150 rpm and 30 ± 21C for 72 h.
Effect of the culture medium composition
Results given in Figs 3 and 4 indicate that A. Parasiticus
acts optimally at malt extract concentrations of 40 g/l and
yeast extract concentrations of 20 g/l, producing an SB
yield of 37.59%. Lower or higher levels of malt or yeast
extract gave lower yields of SB. Watanabe et al. [14] used
the same concentrations of malt and yeast extracts to
isolate a SS hydrolase from Neocosmospora vasinfecta var.
vasinfecta PF1225.
As regards the additional carbon sources, results illustrated in Fig. 5 clearly indicate that the maximum yield
of SB (41.6%) was achieved when galactose was added to
the transformation medium; this is may be due to the
enhanced growth of the fungus by using lactose as the
carbon source. In contrast, the other tested carbon
sources supported comparatively lower conversion estimates and were thus excluded.
Moreover, the effect of different levels of galactose on SB
production was studied. Data given in Fig. 6 reveal that a
low level of galactose (0.5%) supported maximum SB
production (49%), whereas increasing galactose levels
over 1% resulted in a dramatic decrease in SB production,
possibly because the cells preferred the easily oxidizable
galactose as an exclusive carbon source and repressed the
induction of saponin-hydrolyzing activity [19].
Effect of soybean saponin levels
100
"SB"
MolarYeild
Cell Biomass
3.0
50
2.5
40
60
30
40
(Molar Yeild)
SB (mg/100ml)
80
60
2.0
1.5
20
1.0
10
0.5
0
0.0
20
cell biomass (gm/50ml)
Figure 2
0
20
40
60
80
Time (hour)
100
120
Duration of soyasapogenol B (SB) accumulation during hydrolysis of
soybean saponin by Aspergillus parasiticus. A. parasiticus was
cultivated on a transformation culture medium at pH 8, 150 rpm, and
30 ± 21C. Molar yield of soyasapogenol B and cell dry weight were
determined at different time intervals.
Kudou et al. [20] reported that saponin hydrolase was an
enzyme induced by the existence of SS as it has high
substrate specificity for the glucuronide bonds of glycosides. Thus, to enhance the productivity, different
substrate (SS) concentrations ranging from 0.5 to 4%
(w/v) were supplemented to the transformation culture
medium at the inoculation time. Results given in Fig. 7
indicate that molar yields of SB increased on increasing
the amounts of SS supplemented to the culture medium
up to the 3% level. Above the latter concentration, the
yield of SB decreased gradually; this is may be due to
inhibition of the SS hydrolase on increasing the substrate
concentration to more than 3%. Kudou et al. [20]
indicated that SS hydrolase from A. oryzae KO-2 is
inhibited by increasing the substrate level above the
optimum concentration (2.5 mmol/l).
Effect of incubation temperature
the product. Watanabe et al. [14] isolated a SS hydrolase
from Neocosmospora vasinfecta var. vasinfecta PF1225 after a
72 h incubation period. Moreover, the cell biomass yields
were determined at different time intervals (24, 48, 72,
96, 120, and 144 h) and were found to be 2.118, 3.04,
4.558, 4.566, and 5.386 g/100, respectively. Therefore, the
Results in Fig. 8 show that relatively high SB yields were
maintained at temperatures ranging from 25 to 351C.
Maximum SS conversion (65%) was achieved at 301C,
leading to a production of 464.24 mg/100 ml SB. Watanabe
et al. [14] cultivated Neocosmospora vasinfecta var. vasinfecta
PF1225 on an MY medium at 251C to isolate a SS
hydrolase; this means that the optimal incubation
temperature depends on the type of organism used.
Egyptian Pharmaceutical Journal
Figure 3
Figure 6
90
44
"SB"
Molar Yield
120
70
42
85
80
38
36
75
60
100
50
80
40
Molar yield (%)
40
Molar yield (%)
SB (mg/100ml)
SB
Molar yield
SB (mg/100ml)
44
34
70
32
2
3
4
Malt extract (%)
5
0
Effect of malt extract concentration on production of soyasapogenol B
(SB) from soybean saponin by Aspergillus parasiticus. A. parasiticus
was cultivated on a transformation culture medium supplemented with
varying amounts of malt extract (2–6%, w/v) at pH 8, 150 rpm, and
30 ± 21C for 72 h. Control treatment: using 4% malt extract.
1
2
3
Galactose (%)
4
5
Effect of galactose concentration on soyasapogenol B (SB) production.
Aspergillus parasiticus was cultivated on a transformation culture
medium supplemented with different concentrations of galactose
(0.5–5%, w/v) at pH 8, 150 rpm, and 301C for 72 h. Control treatment:
using 1% galactose.
Figure 7
Figure 4
500
100
40
20
20
10
70
65
300
60
200
55
100
50
0
0
0.0
0.5
1.0
1.5
2.0
Yeast extract (%)
2.5
3.0
0.0
3.5
Molar yield (%)
40
Molar Yeild (%)
30
60
75
" SB"
Molar Yield
400
SB (mg/100ml)
"SB"
Molar Yield
80
SB (mg/100ml)
30
60
6
45
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
Soybean saponin (SS, %)
Effect of yeast extract concentration on production of soyasapogenol B
(SB) from soybean saponin by Aspergillus parasiticus. A. parasiticus
was cultivated on a transformation culture medium supplemented with
varying amounts of yeast extract (0.5–3%,w/v) at pH 8, 150 rpm, and
30 ± 21C for 72 h. Control treatment: using 2% yeast extract.
Effect of substrate concentration on production of soyasapogenol B
(SB) from soybean saponin (SS) by Aspergillus parasiticus. A.
parasiticus was cultivated on a transformation culture medium
supplemented with different levels of SS (0.5–4%, w/v) at pH 8,
150 rpm, and 30 ± 21C for 72 h. Control treatment: using 1% SS.
Figure 8
Figure 5
110
34
32
SB (mg/100ml)
36
Molar yield (%)
SB (mg/100ml)
40
38
80
400
42
90
"SB"
Molar Yeild
60
300
40
200
Molar yeild(%)
"SB"
Molar Yeild
100
80
500
20
100
70
30
60
28
Glucose Galactose Mannose Arabinose Sucrose
Carbon sources
Starch
control
Effect of adding different carbon sources to the fermentation medium
on soyasapogenol B (SB) production. Aspergillus parasiticus was
cultivated on a transformation culture medium supplemented with 1%
(w/v) of one of these carbon sources at pH 8, 150 rpm, and 301C for
72 h. Control treatment: without addition of the carbon source.
0
0
20
25
30
35
Temperature (°C)
40
Effect of different temperature values on production of soyasapogenol B
(SB) from soybean saponin (SS) by Aspergillus parasiticus. A.
parasiticus was cultivated on a transformation culture medium composed
of (%, w/v): malt extract 4; yeast extract, 2; KH2PO4, 0.2; (NH4)2SO4,
0.2; MgSO4 7H2O, 0.03; CaCl2 2H2O, 0.03; and SS, 3 (pH 8). Flasks
were incubated at different temperatures and 150 rpm for 72 h.
SS biotransformation to SB Amin et al.
Conclusion
A. parasiticus was screened and selected on the basis of
its ability to hydrolyze SS, producing a high yield of SB.
A maximum conversion value of 65% was obtained using
a production medium composed of (%, w/v): malt extract,
4; yeast extract, 2; galactose, 0.5; and SS, 3 (pH 8). The
medium was inoculated with 6% (v/v) inoculum and
incubated at 301C for 72 h. Under these optimal conditions, the SB molar yield increased from 13.44 to 65%.
Acknowledgements
Conflicts of interest
There are no conflicts of interest.
References
1 Oleszek WA. Chromatographic determination of plant saponins. J Chromatogr 2002; A967:147–162.
2 Hu J, Reddy MB, Hendrich S, Murphy PA. Soyasaponin I and sapongenol B
have limited absorption by Caco-2 intestinal cells and limited bioavailability
in women. J Nutr 2004; 134:1867–1873.
3 Kudou S, Tonomura M, Tsukamoto C, Shimoyamada M, Uchida T, Okubo K.
Isolation and structural elucidation of the major genuine soybean saponin.
Biosci Biotechnol Biochem 1992; 56:142–143.
4 Shiraiwa M, Harada K, Okubo K. Composition and structure of ‘group B
saponin’ in soybean seed. Agric Biol Chem 1991; 55:911–917.
5 Kinjo J, Hirakawa T, Tsuchihashi R, Nagao T, Okawa M, Nohara T, Okabe H.
Hepatoprotective constituents in plants 14. Effects of soyasapogenol B,
sophoradiol, and their glucuronides on the cytotoxicity of tert-butyl hydroperoxide to HepG2 cells. Biol Pharm Bull 2003; 26:1357–1360.
6 Zhang W, Popovich DG. Effect of soyasapogenol A and soyasapogenol B
concentrated extracts on Hep-G2 cell proliferation and apoptosis. J Agric
Food Chem 2008; 56:2603–2608.
45
7 Hayashi K, Hayashi H, Hiraoka N, Ikeshiro Y. Inhibitory activity of soyasaponin
II on virus replication in vitro. Planta Med 1997; 63:102–105.
8 Ahn K-S, Kim J-H, Oh S-R, Min B-S, Kinjo J, Lee H-K. Effects of oleananetype triterpenoids from fabaceous plants on the expression of ICAM-1.
Biol Pharm Bull 2002; 25:1105–1107.
9 Sasaki K, Minowa N, Kuzuhara H, Nishiyama S, Omoto S. Synthesis and
hepatoprotective effects of soyasapogenol B derivatives. Bioorg Med Chem
Lett 1997; 7:85–88.
10 Kinjo J, Yokomizo K, Hirakawa T, Shii Y, Nohara T, Uyeda M. Anti-herpes virus
activity of fabaceous triterpenoidal saponins. Biol Pharm Bull 2000; 23:
887–889.
11 Berhow MA, Wagner ED, Vaughn SF, Plewa MJ. Characterization
and antimutagenic activity of soybean saponins. Mutat Res 2000; 448:
11–22.
12 Gurfinkel DM, Rao AV. Soyasaponins: the relationship between chemical structure and colon anticarcinogenic activity. Nutr Cancer 2003; 47:
24–33.
13 Kudou S, Tsuizaki I, Shimoyamada M, Uchida T, Okubo K. Screening for
microorganisms producing soybean saponin hydrolase. Agric Biol Chem
1990; 54:3035–3037.
14 Watanabe M, Sumida N, Yanai K, Murakami T. A Novel saponin hydrolase
from Neocosmospora vasinfecta var. vasinfecta. Appl Environ Microbiol
2004; 70:865–872.
15 Amin HA, Mohamed SS. Immobilization of Aspergillus terreus on loofa
sponge for soyasapogenol B production from soybean saponin. J Mol Catal
B: Enzymatic 2012; 78:85–90.
16 Sullivan C, Sherma J. Development and validation of an HPTLCdensitometry method for assay of glucosamine of different forms in dietary
supplement tablets and capsules. Acta Chromatographica 2005; 15:
119–130.
17 Watanabe M, Sumida N, Yanai K, Murakami T. Cloning and characterization
of saponin hydrolases from Aspergillus oryzae and Eupenicillium brefeldianum. Biosci Biotechnol Biochem 2005; 69:2178–2185.
18 Kitagawa I, Yoshikawa M, Wang HK, Saito M, Tosirisuk U, Fujiwara T, Tomita K.
Revisions of the structure of the sapogenols. Chem Pharm Bull 1982;
30:2294.
19 Amin HAS, Hanna AG, Mohamed SS. Comparative studies of acidic and
enzymatic hydrolysis for production of soyasapogenols from soybean saponin. Biocatalysis Biotransformation 2011; 29:311–319.
20 Kudou S, Tsuizaki I, Uchida T, Okubo K. Purification and some properties of
soybean saponin hydrolase from Aspergillus oryzae KO-2. Agric Biol Chem
1991; 55:31–36.
46
Original article
Characterization of ternary solid dispersions of nimesulide
with Inutec SP1 and b-cyclodextrin and evaluation
of anti-inflammatory efficiency in rats
Rawia M. Khalila, Mamdouh M. Ghorabb, Noha Abd El Rahmana
and Silvia Kocova El-Arinia
a
Department of Pharmaceutical Technology, National
Research Centre, Cairo and bDepartment of
Pharmaceutics, Faculty of Pharmacy, Suez Canal
University, Ismailiya, Egypt
Correspondence to Rawia M. Khalil, PhD, Department
of Pharmaceutical Technology, National Research
Centre, El-Bohowth St., PO Box 12622, Dokki,
11371 Cairo, Egypt
Tel: + 20 233 335 456; fax: + 20 233 370 931;
e-mail: [email protected]
Received 5 November 2012
Accepted 26 January 2013
Egyptian Pharmaceutical Journal
2013, 12:46–56
Objective
The objective of this investigation is to enhance the physicochemical properties
of nimesulide (NS) and the stability of NS solid dispersions in order to improve
the anti-inflammatory activity of the drug.
Background
NS – a NSAID – is sparingly soluble in water and this low aqueous solubility in addition
to its poor wettability leads to variability in the bioavailability of the drug.
Materials and methods
In the present study, ternary dispersions of NS were investigated using a new
polymeric carrier, Inutec SP1 (Inutec), in combination with b cyclodextrin (b-CD).
The ternary dispersions were prepared using different ratios of NS and b-CD (2 : 1;
1 : 1; 1 : 2), to which a fixed amount of Inutec (20% w/w of total formula) was added
using different methods of incorporation of the drug. Physical mixtures of equivalent
compositions were prepared by physically mixing the ingredients. The optimal
formulation obtained with a full factorial experimental design was used for the
evaluation of anti-inflammatory activity.
Results
In the ternary dispersions, the dissolution behavior improved in comparison with the
physical mixtures and was found to be dependent on the technique of incorporation
of the drug, the method of preparation, and the molar ratio of drug to b-CD. Physical
characterization of the ternary dispersions by infrared spectroscopy (FTIR), differential
scanning calorimetry, and X-ray powder diffraction indicated a decrease in crystallinity
because of partial inclusion in b-CD and the effect of Inutec, which promoted the
formation of microcrystals or partial amorphization of the drug during the processing
of the dispersions by kneading. Differential scanning calorimetry and X-ray powder
diffraction curves of the dispersions prepared by the solvent method indicated the
presence of a polymorphic form of NS with a lower melting point. The optimized ternary
dispersion predicted by the full factorial design showed good physical stability
following an accelerated stability test. The ternary dispersion of NS, Inutec, and
b-CD was found to show better anti-inflammatory efficiency in rats compared with
a commercial tablet of NS.
Conclusion
It can be concluded that the dissolution properties and the anti-inflammatory efficacy of
the ternary dispersions of NS with b-CD and Inutec were enhanced because of a
secondary solubilization of the inclusion by the polymeric surfactant.
Keywords:
accelerated stability, b-cyclodextrin, Inutec SP1, in-vivo evaluation in rats, nimesulide,
ternary solid dispersion
Egypt Pharm J 12:46–56
& 2013 Division of Pharmaceutical and Drug Industries Research,
National Research Centre
1687-4315
Introduction
Nimesulide (NS) is an important anti-inflammatory drug
and shows selective COX-2 inhibition, which contributes
toward its good gastrointestinal tolerability. Moreover,
despite concerns over its potential hepatotoxicity, it
remains approved for the market because of the
beneficial action overweighing the risks associated with
the drug [1]. However, the very poor aqueous solubility of
NS is a huge hurdle in formulation development.
Therefore, enhancement of water solubility has been an
ongoing challenge for pharmaceutical researchers as it can
lead to more efficient and safer formulations of this
important medicament.
1687-4315 & 2013 Division of Pharmaceutical and Drug Industries Research,
National Research Centre
DOI: 10.7123/01.EPJ.0000427333.85411.b9
Ternary solid dispersions of nimesulide Khalil et al. 47
Numerous studies have dealt with the use of different
carriers for the preparation of solid dispersions (SD) of
NS [2–8]. Considerable amount of research has been
published on complexation of NS with b-cyclodextrin
(b-CD). Nalluri and colleagues [9,10] studied binary
systems in 1 : 1 and 1 : 2 molar ratios of drug and carrier.
They reported that the increase in dissolution properties
was because of the formation of a 1 : 1 complex in
solution. Further increase in b-CD led to the formation of
a 1 : 2 complex in the solid state. However, despite the
true inclusion formed, the dissolution rate and efficiency
values obtained were not as anticipated. The reason for
this is the formation of crystalline inclusion complexes
(IC) [9,10].
To overcome the drawback of the limited aqueous
solubility of b-CD, Dutet et al. [6] examined the effect
of double hydrophilization in ternary systems of NS and
b-CD and PEG 6000, but found no improvement in the
bioavailability of NS in rats.
Generally, these and other attempts have failed to
produce a stable marketable NS product, and thus there
is a need for novel, more efficient carriers for NS. Recent
advances in excipient technology have resulted in new
surfactants such as Inutec SP1 (Inutec) of Orafti NonFood. It is derived frominulin by grafting alkyl groups on
a polyfructose backbone (Fig. 1).
In this way, a structure is obtained, with polyfructose loops
providing steric stability. The polymeric nature of this
surfactant has made Inutec very useful as an emulsifier in
cosmetic and food industries [11]. Its application in
Figure 1
pharmaceutical formulations has been reported by Van den
Mooter et al. [12] as a carrier for SD of itraconazole, a drug
with very low aqueous solubility. A 20/80 w/w SD of
itraconazole and Inutec led to an improved dissolution rate.
The dissolution efficiency (DE) depended more on the
method of preparation than on the degree of amorphization.
In a recent study, Janssens et al. [13] investigated further the
effect of Inutec on itraconazole in ternary dispersions with
polyvidone-vinylacetate 64 (PVPVA 64) and found that the
improvement vis-à-vis the binary systems depended on the
incorporation of a sufficient amount of PVPVA 64 required
for the molecular dispersion of itraconazole. Ibrahim
et al. [14] used Inutec and hydroxypropyl-b-CD for the
preparation of chewable tablets of etodolac. The authors
reported that the dissolution rate of etodolac at pH 1.2 and
6.8 was improved compared with a pure drug and physical
mixture (PM) as a result of loss of crystallinity.
We reported in a previous publication [15] on the effect
of Inutec on the dissolution behavior of NS in binary
dispersions of NS with increasing amounts of Inutec. The
dissolution rate was enhanced proportionally with the
increase in the Inutec concentration and a ratio of drug to
Inutec of 1 : 3 led to a maximum of 87% DE after 180 min.
The aim of the present study is to evaluate the effect of
Inutec at a low concentration (20% w/w) to act as a second
hydrophilization factor in ternary dispersions based on
NS – b-CD complexes. Another aim is to use an experimental design for optimization of the formula to conduct
accelerated stability tests and consequently for its use in
the evaluation of anti-inflammatory efficiency in rats.
Materials and methods
Materials
NS was obtained as a gift sample from Sigma (Monofia,
Egypt). Inutec SP1 was generously provided by Orafti
Non-Food (Tienen, Belgium). b-CD (MW 1135) was
purchased from Sigma Chemical Company (St Louis,
Missouri, USA). All other materials were of analytical
grade.
Preparation of ternary systems from inclusion complex
and Inutec (ICSD)
First, the IC of NS and b-CD were prepared in 2 : 1, 1 : 1,
and 1 : 2 molar ratios using two methods: solvent and
kneading methods.
Solvent method (IC/S)
The alcoholic solution of NS was added to an aqueous
solution of b-CD. The resulting mixture was stirred for
30 min and evaporated under reduced pressure at a
temperature of 601C until dry. The dried mass was
ground in a mortar and passed through a sieve (250 mm).
Kneading method (IC/K)
Chemical structure of Inutec SP1.
A mixture of NS and b-CD was wetted with water and
kneaded thoroughly for 30 min in a glass mortar.
The resulting paste was dried under vacuum for 24 h.
48
Egyptian Pharmaceutical Journal
The dried mass was ground in a mortar and passed
through a sieve (250 mm).
Second, the IC of NS–b-CD was mixed with a fixed
amount of Inutec (20% w/w of the total formula), and
then wetted together with water and kneaded as
discussed in the kneading method.
These systems are distinguished by the preparation
method of the binary IC.
It should be noted that by maintaining the amount of
Inutec added to the ternary systems constant, the ratio of
Inutec to NS increases with an increase in the molar ratio
of b-CD to drug.
Preparation of ternary solid dispersions
NS, b-CD, and Inutec were dispersed together using
either the solvent or the kneading method.
Solvent method (SD/S)
The aqueous solution of b-CD was added to an alcoholic
solution of NS and Inutec. The solvents were evaporated
using the rotavapor as discussed previously.
using 0.45 mm millipore filters. An aliquot portion of the
filtrate was diluted with phosphate buffer (pH = 7.4) and
analyzed for drug content by measuring its absorbance
spectrophotometrically at 392 nm against a blank solution
containing the same concentrations of the carrier. Each
experiment was conducted in triplicate.
Solid-state characterization
Fourier transform infrared spectroscopy
Fourier transform infrared (FTIR) spectra were recorded
using an FTIR-6100 type A spectrophotometer (Jasco,
Tokyo, Japan) equipped with a deuterated triglycine
sulfate detector. Samples were prepared in KBr disks
using a hydrostatic press. The scanning range was
between 4000 and 400 cm–1 at 4 cm–1 resolution.
Differential scanning calorimetry
Differential scanning calorimetry (DSC) was performed
using a Pyris5 instrument (Perkin Elmer, Waltham,
Massachusetts, USA) equipped with an intercooler. A
dry purge of nitrogen gas was used at 20 ml/min. The
instrument was calibrated with pure indium. Samples
(2–3 mg) were analyzed in closed Al pans from 50 to
2201C at a heating rate of 101C/min.
Kneading method (SD/K)
A mixture of NS, b-CD, and Inutec was wetted with
water and kneaded as discussed previously.
Preparation of physical mixtures
The corresponding PM were obtained by mixing the
various components together in a mortar by trituration for
5 min, followed by sieving (250 mm).
Determination of NS content in the prepared
formulations
An accurately weighed amount of NS formulation was
dissolved in phosphate buffer (pH = 7.4) and sonicated
for 30 min to ensure complete extraction of the drug from
the dispersion. The content of NS was determined
spectrophotometrically at 392 nm using a UV spectrophotometer. Each preparation was tested in triplicate.
Wettability study
A powder sample (3 g) was placed in a sintered glass
funnel (33 mm internal diameter). The funnel was
plunged into a beaker containing water such that the
surface of water in the beaker remained at the same level
as the powder in the funnel. Methylene blue powder
(100 mg) was layered uniformly on the surface of the
powder in the funnel. The time required for wetting of
the methylene blue powder was measured. The average
of three observations was used for drawing the conclusions [3].
Phase solubility study
Solubility studies were carried out as described by
Higuchi and Connors [16]. An excess amount of NS
was added to screw-capped vials containing different
concentrations of the carrier solution. The vials were
shaken mechanically at 37 ± 0.51C for 72 h until reaching
equilibrium. Filtration of the suspension was carried out
X-ray powder diffraction
X-ray powder diffraction (XRPD) patterns of the pure
ingredients and all of the SD containing varying proportions of NS in the matrix were recorded using an X-ray
diffractometer (Scintag Inc., Cupertino, California, USA)
equipped with CuKa as the source of radiation.
Measurements were carried out using 45 kV voltage and
9 mA current. The 2y values and the intensities of the
peaks were compared for pure ingredients, the PM, and
the SD systems.
Solubility study
The solubility was determined in distilled water at 371C.
A sample equivalent to 25 mg of NS (excess amount of
NS) was added to 10 ml of distilled water in a vial with a
teflon-lined screw cap. The vials were shaken mechanically at 37 ± 0.51C for 72 h until reaching equilibrium.
Filtration of the suspension was carried out using 0.45 mm
millipore filters. An aliquot portion of the filtrate was
diluted with phosphate buffer (pH = 7.4) and analyzed
for drug content by measuring its absorbance spectrophotometrically at 392 nm. Each experiment was conducted in triplicate.
In-vitro dissolution study
The dissolution rate was determined in the USP
Dissolution Tester, Apparatus I, at 37 ± 0.51C. The
dissolution medium was 900 ml of phosphate buffer
(pH = 7.4) at a rotation speed of 50 rpm. Powder samples
containing 25 mg of NS or its equivalent of PM or SD
were filled in transparent zero-sized hard gelatin
capsules. Aliquots, each of 5 ml, from the dissolution
medium were withdrawn at time intervals 15, 30, 45, 60,
90, 120, 150, and 180 min and replaced by an equal
volume of fresh dissolution medium. The samples were
filtered through a 0.45 mm millipore filter and assayed
Ternary solid dispersions of nimesulide Khalil et al. 49
spectrophotometrically for NS at 392 nm using fresh
dissolution medium as a blank.
The DE was calculated according to Khan [17] and is
defined as the area under the dissolution curve up to the
time, t, expressed as a percentage of the area of the
rectangle described by 100% dissolution at the same
time. The DE can have a range of values depending on
the time interval chosen. The DEs at 30, 60, and 180 min.
were calculated from the dissolution profiles. The
experiments were conducted in triplicate.
Statistical analysis of data
All data were analyzed statistically using the analysis of
variance test for a P value of 0.05 using the social package
for statistical study Software (SPSS Company, IBM
Corporation, New York, USA). Differences were considered statistically significant at a value of P less than 0.05.
Results and discussion
The composition and method of preparation of all the
systems studied are listed in Table 1.
Experimental design
An experimental design was generated to estimate the
effects on the dissolution properties of the following
experimental variables: method of preparation at two
levels (solvent, kneading) and the NS : b-CD : Inutec
ratio at three levels (2 : 1 : 20%, 1 : 1 : 20%, and 1 : 2 : 20%).
Nimesulide content in the prepared formulations
The drug content and the percentage recovery were
determined in all prepared formulations in order to
confirm that there was no drug loss during preparation
and that the SD showed good content uniformity.
Accelerated stability study
An accelerated stability study was carried out by
subjecting the SD to stressed conditions at 401C and
75% relative humidity (maintained using a saturated
solution of NaCl) for a period of 3 months. The effect of
the stressed conditions was determined by measuring
in-vitro dissolution and by DSC and XRPD studies.
Evaluation of anti-inflammatory activity of selected
nimesulide solid dispersions
Twenty-four adult female albino rats 150 ± 20 g were used.
The rats were randomly allocated into four groups, each
including six animals. Carrageenan was used to induce rat’s
paw edema. This effect was determined according to the
method described previously in the literature [18,19]. The
animals were kept on a standard laboratory diet. The rats
were kept fasted for 16 h before the experiment, but were
allowed free access to water. The samples were administered orally as a suspension to the respective animal groups
at a dose of 50 mg/kg [20,21].
One hour after administration, edema was induced by an
injection of 0.1 ml of 1% (w/v) carrageenan solution in
distilled water into the planter aponeurosis of rats’ right
hind paws. The volume of the injected paw was measured
immediately after carrageenan injection and after 1, 2, 3,
4, and 5 h using a plethsymometer. The percentage
increase in paw volume was calculated according to the
equation given by Delporte et al. [22].
% increase in paw volume¼ðVf Vi Þ/Vi 100;
where Vf and Vi are the final and the initial paw volume
of an animal, respectively.
In addition, the percentage inhibition of edema volume for
each time was calculated from the mean effect in control and
in treated animals according to the following equation [23].
% inhibition of edema volume¼ð1Vt /Vc Þ100;
where Vt and Vc are the mean increase in the volume of the
carrageenan-injected paw of the treated group and the
control group, respectively. The one-way analysis
of variance test was carried out on the area under percentage
increase in edema volume versus time curve.
Wettability study
The mean wetting times of representative PM and
dispersions are shown in Fig. 2. It can be seen that the
wetting time for pure NS (8 h) was significantly reduced
in the PM. The wettability was further improved in the
dispersions and the best results were obtained for the
dispersions prepared using the SD technique [SD/S
(1 : 2 : 20%) and SD/K (1 : 2 : 20%) in Fig. 2]. It is also
evident from Fig. 2 that the wetting times decreased
significantly (Po0.05) on adding Inutec to the binary
complexes (compare ternary ICSD/S; ICSD/K to binary
IC/S; IC/K in Fig. 2). This confirms the secondary
hydrophilization effect of Inutec in the ternary systems.
Phase solubility study
The phase solubility diagrams of NS and b-CD with and
without 0.5% Inutec can be classified as AL-type according
to Higuchi and Connors [16] as shown in Fig. 3. The
aqueous solubility of NS increased linearly (R2 = 0.9952
and 0.9971 in the absence and in the presence of 0.5% of
Inutec, respectively) as a function of the b-CD concentration. The phase solubility of NS in aqueous solutions of
b-CD increased in the presence of 0.5% of Inutec as
reflected by the small increase in the stability constant
from Kc = 354 to Kc = 430 mol/l. This is in agreement with
the increased wettability of the PM (Fig. 2).
Solid-state characterization
Studies were carried out to determine the nature of the
products obtained.
Fourier transform infrared spectroscopy
FTIR data were obtained to determine whether chemical
interactions occurred during the preparation of the SD.
Figure 4 shows the FTIR spectra of the individual
components, their PM, and the different dispersions. The
FTIR spectra of the PM showed the patterns of each
component. In the FTIR spectra of the ICSDs and the SDs,
the peak of the N–H function at 3290 cm–1 was slightly
pronounced or invisible. Otherwise, no other new bonds
Egyptian Pharmaceutical Journal
Table 1 Composition of physical mixtures and solid dispersions
prepared using different methods
NS formulation
NS : b-CD
(mol/mol)
Inutec %
(w/w)
2:1
1:1
1:2
2:1
1:1
1:2
2:1
1:1
1:2
2:1
1:1
1:2
2:1
1:1
1:2
2:1
1:1
1:2
2:1
1:1
1:2
2:1
1:1
1:2
0
0
0
20
20
20
0
0
0
0
0
0
20
20
20
20
20
20
20
20
20
20
20
20
PM (2 : 1)
PM (1 : 1)
PM (1 : 2)
PM (2 : 1 : 20%)
PM (1 : 1 : 20%)
PM (1 : 2 : 20%)
IC/S (2 : 1)
IC/S (1 : 1)
IC/S (1 : 2)
IC/K (2 : 1)
IC/K (1 : 1)
IC/K (1 : 2)
ICSD/S (2 : 1 : 20%)
ICSD/S (1 : 1 : 20%)
ICSD/S (1 : 2 : 20%)
ICSD/K (2 : 1 : 20%)
ICSD/K (1 : 1 : 20%)
ICSD/K (1 : 2 : 20%)
SD/S (2 : 1 : 20%)
SD/S (1 : 1 : 20%)
SD/S (1 : 2 : 20%)
SD/K (2 : 1 : 20%)
SD/K (1 : 1 : 20%)
SD/K (1 : 2 : 20%)
0.003
Method of preparationa
Physical mixing
Physical mixing
Physical mixing
Physical mixing
Physical mixing
Physical mixing
IC/solvent (50 ml)
IC/solvent (50 ml)
IC/solvent (50 ml)
IC/kneading
IC/kneading
IC/kneading
ICSD/solvent (50 ml)
ICSD/solvent (50 ml)
ICSD/solvent (50 ml)
ICSD/kneading
ICSD/kneading
ICSD/kneading
SD/solvent (70 ml)
SD/solvent (100 ml)
SD/solvent (150 ml)
SD/kneading
SD/kneading
SD/kneading
b-CD, b cyclodextrin; IC/K, inclusion complex prepared using the
kneading method; IC/S, inclusion complex prepared using the solvent
method; ICSD/K, inclusion complex in solid dispersion prepared using
the kneading method; ICSD/S, inclusion complex in solid dispersion
prepared using the solvent method; NS, nimesulide; PM, physical
mixture; SD/K, solid dispersion prepared using the kneading method;
SD/S, solid dispersion prepared using the solvent method.
a
Values in parentheses represent the amount of methanol used in the
preparation (for 1 mol NS).
20.22
20.59
SD/S (1:2:20%)
SD/K (1:2:20%)
22.33 23.52
ICSD/S (1:2:20%)
24.19
ICSD/K (1:2:20%)
25.49
IC/K (1:2)
PM (1:2:20%)
PM (1:2)
49.22 50.13
IC/S (1:2)
480.00
NS
Time (minutes)
Figure 2
500
450
400
350
300
250
200
150
100
50
0
Figure 3
NS Formulation
Wettability of selected NS–b-CD and NS–b-CD–Inutec SP1 formulations: comparison of different methods of preparation.
were observed, which indicates that there was no interaction
between NS and the carriers at the molecular level.
0.0025
NS Concentration (M)
50
0.002
0.0015
0.001
0.0005
0
0
0.05
0.1
0.15
0.2
β-CD Concentration (M)
Phase solubility diagrams of NS in different concentrations of b-CD
with and without 0.5% Inutec SP1 at 371C: K, NS–b-CD:
y = 0.0112x + 2E – 05, R 2 = 0.9952; ’, NS–b-CD–Inutec: y = 0.0136x +
8E – 05, R 2 = 0.9971. b-CD, b cyclodextrin; NS, nimesulide.
The thermogram of NS showed a single endothermic
peak with onset at 148.81C and a peak at 1501C
corresponding to its melting point. These results were
also reported by Chowdary and Nalluri [24] and by
Abdelkader et al. [5]. The DSC thermogram of b-CD
showed a broad endothermic effect with a peak at 971C.
Inutec showed a small endothermic peak at 103.51C at
the tail of the water evaporation endotherm at 601C and a
glass transition signal at 1431C as also reported by Van
den Mooter et al. [12].
The thermogram of the PM is a combination of the DSC
curves of the individual components without changes in
the melting peaks. The ternary ICSDs prepared by both
methods (S, K) and the ternary SD prepared by the
kneading method (K) showed a marked reduction in the
intensity of the NS endotherm when compared with that
of the PM, indicating progressive partial inclusion of NS
within the b-CD cavity. The thermogram of the ternary
SD prepared by the solvent method was characterized by
a split endotherm indicating that the NS showed
polymorphism because of the use of an organic solvent
(methanol) in the preparation of the dispersions. As we
reported earlier, the use of a solvent induced a different
polymorphic form of NS that melts at a slightly lower
temperature [15]. Bergese et al. [25] also reported such a
polymorphic form of the drug and Di Martino et al. [26]
obtained a split endotherm because of the use of an
organic solvent in their study.
The ICSD (solvent) system, in contrast, did not show the
double-peaked endotherm. This might be attributed to
the use of a smaller amount of alcoholic solvent used in
its preparation (Table 1).
Differential scanning calorimetry
DSC thermograms were generated to test for the
possibility of the inclusion of NS in b-CD.
Figure 5 shows the DSC thermograms of the individual
components and their ternary systems at a 1 : 2 molar ratio
prepared by the solvent (S) and kneading (K) methods.
X-ray powder diffraction
XRPD patterns were obtained to determine the crystallinity of the products obtained and to confirm the results
of the DCS study.
Ternary solid dispersions of nimesulide Khalil et al.
Figure 4
51
Figure 5
SD/K (1:2:20%)
SD/K (1:2:20%)
SD/S (1:2:20%)
SD/S (1:2:20%)
ICSD/K (1:2:20%)
ICSD/K (1:2:20%)
ICSD/S (1:2:20%)
Heat Flow (mW)
%T
ICSD/S (1:2:20%)
PM (1:2:20%)
PM (1:2:20%)
β-CD
β-CD
Inutec
Inutec
NS
4000
3500
3000
2500
2000
1500
1000
NS
500
Wavenumber [cm-1]
FTIR spectra of NS, Inutec SP1, b-CD, their PM and their ternary ICSD,
and SD at 1 : 2 molar ratio prepared by solvent (S) and kneading (K)
methods. b-CD, b cyclodextrin; FTIR, Fourier transform infrared
spectroscopy; ICSD, inclusion complex in solid dispersion;
NS, nimesulide; PM, physical mixture; SD, solid dispersions.
Figure 6 shows the diffractograms of NS, b-CD, Inutec,
their ternary PM, and the different SD (ICSD and SD) at
1 : 2 molar ratios with b-CD, prepared using the solvent
and the kneading methods.
NS showed the characteristic diffraction pattern with
numerous distinctive peaks, indicating the highly crystalline nature of the drug. The most abundant peaks were
observed at 2y values of 19.3 and 23.11. The diffraction
pattern of b-CD showed numerous peaks, with a major
peak at 2y = 12.481, whereas the diffraction pattern of
Inutec was characterized by very small peaks protruding
from the halo around 2y = 16–201.
The XRPD pattern of the PM represents a combination
of the individual patterns of the drug and the carriers and
the intensities of the peaks reflect the fraction of the
drug in the mixture. The diffractograms of ICSD
prepared using the solvent and kneading methods and
SD prepared using the kneading method showed a
notable reduction in the intensity of the characteristic
peaks of the drug in comparison with the PM. This
reduction in peak intensity is a result of loss of
crystallinity of the drug in the preparation, indicating
partial inclusion of NS within the b-CD cavity. The
XRPD patterns of the ternary SD prepared using the
solvent method showed a diffraction peak at 2y = 18.91,
which was not observed in the XRPD pattern of the pure
NS. This indicates the presence of polymorphism
because of the use of an organic solvent (methanol).
Temperature °C
DSC thermograms of NS, Inutec SP1, b-CD, their PM, and their ternary
ICSD and SD at a 1 : 2 molar ratio prepared by solvent (S) and kneading
(K) methods at a heating rate of 101C/min. b-CD, b cyclodextrin; DSC,
differential scanning calorimetry; ICSD, inclusion complex in solid
dispersion; NS, nimesulide; PM, physical mixture; SD, solid dispersions.
The diffractograms of the binary and ternary systems
were compared quantitatively with the diffractogram of
the PM. For this purpose, the values of the relative
intensity (I/Io) were used, which were calculated from
the intensity (I) of a selected peak (2y = 23.11) and the
intensity (Io) of the major peak (2y = 19.31). The relative
intensity values decreased to I/Io = 85 and 88% in the ICs
and further to 80 and 82% in the ternary ICSDs
depending on the method of preparation (solvent vs.
kneading). The highest decrease in the relative intensity
was observed in the ternary systems prepared using the
SD technique, using the kneading method (I/Io =
69.8%), indicating the highest degree of amorphization
compared with the PM and the other binary and ternary
formulations. However, the I/Io value of ternary dispersion prepared using the SD technique with methanol was
not calculated because of the appearance of a new peak
indicative of a different polymorphic form of NS. Janssens
et al. [13] investigated the diffractograms of ternary
systems of itraconazole with PVPVA 64 and Inutec SP1 in
different ratios of polymer to Inutec. They reported that
all the systems showed XRPD amorphous behavior,
except for the one with the lowest ratio. They therefore
concluded that itraconazole was molecularly dispersed
in the PVPVA, whereas Inutec did not interact with any
52 Egyptian Pharmaceutical Journal
In the dispersions, the solubility increased compared with
the PMs, indicating further interaction between NS and
the carriers in the solid state at all drug to b-CD molar
ratios. It can be noted that the addition of Inutec to the
preformed IC of NS and b-CD did not induce a change in
saturation solubility in any significant way (compare ICs
with ICSDs). However, direct dispersion of the components enhanced the solubility significantly (Po0.05). Of
the two methods of dispersion, the use of solvent yielded
better results (compare ternary SD/S with ternary SD/K
in Fig. 7).
Figure 6
SD/K (1:2:20%)
SD/S (1:2:20%)
ICSD/K (1:2:20%)
ICSD/S (1:2:20%)
Intensity
In-vitro dissolution study
The dissolution rate (%D) and the DEs at 30, 60, and
180 min of all the systems studied are summarized
in Table 2. Maximum values were obtained after
180 min of dissolution testing. It should be noted that
although the maximum values obtained did not reach
100% after 3 h of dissolution testing, conducting the tests
for a longer period was not considered practical.
PM (1:2:20%)
β-CD
The dissolution enhancement in the PM was 2.2-fold
compared with pure drug, which is in agreement with the
improved wettability and complexation of the drug with
b-CD in solution. The difference between the dissolution rates of the ternary PM and the binary PM was not
statistically significant (P40.05).
Inutec
NS
0
5
10
15
20
25
30
35
40
45
50
2
X-ray powder diffractograms of NS, Inutec SP1, b-CD, their PM, and
their ternary ICSD and SD at a 1 : 2 molar ratio prepared by solvent (S)
and kneading (K) methods. b-CD, b cyclodextrin; ICSD, inclusion
complex in solid dispersion; NS, nimesulide; PM, physical mixture;
SD, solid dispersions.
of the components on a molecular level. This is in
agreement with the present study that is NS interacts
with the b-CD by means of partial inclusion. Further
decrease in crystallinity in ternary systems compared with
binary IC may be attributed to Inutec, which promoted
the formation of microcrystals in the ternary systems.
Consequently, the addition of this polymeric surfactant
increased the saturation solubility and this could lead to
better dissolution rates of the ternary dispersions vis-à-vis
the binary systems. The XRPD findings are in full
agreement with the DSC results (Fig. 5).
Solubility study
The aqueous solubility in distilled water of pure NS and
the ternary ICSDs and SDs is shown in Fig. 7. The
solubilities obtained for the PMs and the binary ICs are
also shown for comparison. The solubility obtained for
pure drug was 14.3 mg/ml. In the PM, the solubility
increased as a result of the IC formed in the solution. The
addition of Inutec led to a slight increase in solubility
(compare ternary PMs with binary PMs in Fig. 7). This is
in agreement with the results from the phase solubility
study.
The dissolution profiles of all the dispersions showed a
significant improvement (Po0.05) compared with the
PM because of the progressive inclusion in the b-CD and/
or the secondary hydrophilization action of Inutec. It can
be seen from Table 2 that the increase in dissolution
could be related to both the method of preparation and
the presence of Inutec in the systems.
The dissolution profiles of the IC also increased
compared with the PM but to a lesser degree than when
compared with the dispersions. Similar to the saturation
solubility, the dissolution rate is also the highest for
solvent SD.
The improvement in the dissolution rate and the DE of
the PM in comparison with the pure drug can be
attributed to the formation of a soluble 1 : 1 complex of
NS with b-CD as confirmed by the phase solubility
studies and the results of the solubility tests. The
addition of Inutec did not affect the dissolution of the
ternary PM significantly (P40.05), although this would
have been anticipated from the improved wettability
because of the solubilizing action of this polymeric
surfactant. In the dispersion systems, however, the
improved wettability resulting from the addition of
Inutec significantly increased the dissolution of the
ternary dispersions in comparison with the binary
dispersions (Po0.05). This is because by physical
mixing, the polymer is only deposited on the drug,
whereas during the kneading process, there is deeper
entrapment of the drug in the polymer network. Because
of the unique action of Inutec to adsorb onto hydrophobic
substrates with its alkyl chains, more hydrophobic
particles become occupied by Inutec with its hydrophilic
fructose loops in the solution. This leads to an increase in
Ternary solid dispersions of nimesulide Khalil et al.
53
Figure 7
100
90
SD
Solubility of NS (µg/ml)
80
ICSD
IC
70
60
50
40
PM
30
20
10
SD/K (1:2:20%)
SD/K (1:1:20%)
SD/K (2:1:20%)
SD/S (1:2:20%)
SD/S (1:1:20%)
SD/S (2:1:20%)
ICSD/K (1:2:20%)
ICSD/K (1:1:20%)
ICSD/K (2:1:20%)
ICSD/S (1:2:20%)
ICSD/S (1:1:20%)
ICSD/S (2:1:20%)
IC/K (1:2)
IC/K (1:1)
IC/K (2:1)
IC/S (1:2)
IC/S (1:1)
IC/S (2:1)
PM (1:2:20%)
PM (1:1:20%)
PM (2:1:20%)
PM (1:2)
PM (1:1)
PM (2:1)
NS
0
Nimesulide Formulations
Solubility (mg/ml) of physical mixtures and dispersions of NS with b-CD and Inutec SP1.
wettability and consequently to better dissolution properties of the ternary systems.
Generally, all dispersions showed better solubility and
dissolution rate when compared with the PM. This can be
attributed to the changes in the solid states as shown by
the results of the physical characterization of the SD. In
the kneaded dispersions, the predominant factor is the
reduced crystallinity of the drug because of partial inclusion
in the b-CD or the formation of microcrystals dispersed in
the polymer network or the formation of some amorphous
drug during the processing of the formulations. This was
evidenced by the decrease in the melting endotherm on
the DSC curves and by the reduced relative intensity of
the characteristic peaks on the X-ray diffractograms. In
addition to this, the dispersions prepared using the solvent
method showed polymorphism as indicated by the split
endotherm in the DSC curves and by the presence of a
new peak on the X-ray diffractograms. Polymorphism of NS
has been reported in the literature [25, 26] and the
enhanced dissolution of the solvent dispersions found in
the present study could be because of the polymorphic
form with a lower melting point.
On the basis of the data obtained, it can be concluded
that several factors contributed toward the enhanced
dissolution rate and DE of the ternary dispersions of NS,
b-CD, and Inutec, that is increased wettability because of
a second solubilization of the inclusion by the polymeric
surfactant Inutec, decreased crystallinity because of
partial inclusion of NS in b-CD, and increased solubility
because of polymorphism.
The use of Inutec may offer advantages not found with the
more commercial carriers used for the preparation of SD.
Evaluation of the results of an experimental design
Three characteristic points on the dissolution curves
(responses) were used for the evaluation of the effects of
the experimental variables (factors). They are D30, D60,
and DE180, representing the percentage drug dissolved at
30, 60 min and the DE at 180 min, respectively (Table 2).
A 2 2 3 experimental design was generated for the
three factors (technique, method, and ratio) at the
selected levels.
The least square model was used in order to predict the
optimal values of the responses within the ranges of the
factors used in the experimental design. The main effects
represent the values of the estimates of the parameters
calculated from the model that was used to fit the data.
The largest effect on the DE180 (7.4) was because of the
ratio. The second highest effect (3.2) was because of the
method of preparation of the ternary system, whereas
the effect of the technique (i.e. SD or ICSD) exerted the
smallest effect, with a value of the estimate of 2.8.
The optimal formulation was predicted to be at the
following levels of the experimental variables: technique = SD; method = solvent; ratio = 1 : 2 : 20%.
This formula was used for further studies (accelerated
stability and in-vivo evaluation of anti-inflammatory
activity).
Accelerated stability study
The physical stability was investigated by comparing the
dissolution profiles as well as the solid-state characteristics of the freshly prepared samples and of samples aged
1, 2, and 3 months. For this study, the formula optimized
by the factorial design was used, that is the ternary SD
54
Egyptian Pharmaceutical Journal
Table 2 Dissolution rate (%D) and dissolution efficiency of different formulations
%D (min)
NS formulation
PM (2 : 1)
PM (1 : 1)
PM (1 : 2)
PM (2 : 1 : 20%)
PM (1 : 1 : 20%)
PM (1 : 2 : 20%)
IC/S (2 : 1)
IC/S (1 : 1)
IC/S (1 : 2)
IC/K (2 : 1)
IC/K (1 : 1)
IC/K (1 : 2)
ICSD/S (2 : 1 : 20%)
ICSD/S (1 : 1 : 20%)
ICSD/S (1 : 2 : 20%)
ICSD/K (2 : 1 : 20%)
ICSD/K (1 : 1 : 20%)
ICSD/K (1 : 2 : 20%)
SD/S (2 : 1 : 20%)
SD/S (1 : 1 : 20%)
SD/S (1 : 2 : 20%)
SD/K (2 : 1 : 20%)
SD/K (1 : 1 : 20%)
SD/K (1 : 2 : 20%)
DE (%) (min)
30
60
180
30
60
180
22.35 ± 2.1
27.25 ± 1.6
33.78 ± 2.5
23.74 ± 2.2
28.62 ± 1.7
36.51 ± 2.7
43.62 ± 2.2
51.66 ± 2.8
64.71 ± 3.8
39.90 ± 3.6
44.77 ± 3.4
50.71 ± 2.5
47.81 ± 4.5
48.94 ± 2.6
59.18 ± 2.1
47.05 ± 3.3
47.68 ± 3.3
53.88 ± 3.3
56.08 ± 2.9
65.51 ± 3.8
75.68 ± 3.2
46.34 ± 2.7
49.39 ± 4.4
61.68 ± 2.3
30.6 ± 1.5
35.0 ± 3.4
42.1 ± 2.6
31.7 ± 1.5
35.6 ± 3.4
43.9 ± 2.8
62.4 ± 2.3
68.9 ± 2.6
78.3 ± 2.5
54.9 ± 3.5
61.1 ± 2.9
72.4 ± 3.0
66.0 ± 3.4
65.6 ± 4.3
79.4 ± 3.2
58.6 ± 4.8
65.8 ± 3.3
75.2 ± 3.5
69.4 ± 2.9
77.6 ± 2.3
86.7 ± 4.3
59.9 ± 2.3
69.3 ± 2.9
77.5 ± 3.7
40.6 ± 3.9
47.3 ± 2.2
54.6 ± 1.9
41.5 ± 4.0
49.6 ± 2.3
55.2 ± 1.9
77.2 ± 1.0
80.8 ± 1.8
84.9 ± 2.5
75.1 ± 4.2
78.3 ± 4.2
83.6 ± 2.9
80.0 ± 3.7
81.9 ± 3.1
88.9 ± 2.1
70.5 ± 3.2
81.7 ± 3.8
85.2 ± 3.3
82.3 ± 3.6
86.5 ± 3.3
98.6 ± 2.8
73.8 ± 2.3
83.7 ± 1.2
89.1 ± 3.9
12.7 ± 0.1
16.3 ± 1.1
19.0 ± 1.9
13.7 ± 0.2
17.1 ± 1.1
21.7 ± 0.5
25.4 ± 1.0
28.0 ± 2.4
36.4 ± 2.1
20.5 ± 2.3
24.7 ± 2.4
26.2 ± 2.4
26.0 ± 1.7
23.0 ± 2.2
32.5 ± 1.6
26.0 ± 2.4
25.0 ± 2.6
31.7 ± 2.3
35.9 ± 2.0
40.7 ± 2.5
48.5 ± 1.9
25.3 ± 2.2
28.7 ± 2.4
33.2 ± 2.4
19.7 ± 0.2
23.9 ± 1.6
27.0 ± 2.4
20.3 ± 0.2
24.9 ± 1.7
31.3 ± 1.5
40.2 ± 1.2
45.3 ± 2.2
54.5 ± 2.5
34.5 ± 2.3
39.8 ± 3.1
45.6 ± 2.7
41.7 ± 1.0
41.3 ± 2.1
51.8 ± 2.1
39.5 ± 2.3
41.2 ± 2.9
48.3 ± 2.6
50.4 ± 0.8
56.7 ± 2.5
65.1 ± 2.8
39.3 ± 2.7
44.6 ± 2.4
52.1 ± 1.6
29.9 ± 1.4
36.3 ± 0.3
40.1 ± 3.3
30.9 ± 1.5
37.5 ± 0.3
44.6 ± 1.8
61.3 ± 0.9
65.5 ± 2.2
73.0 ± 2.8
56.5 ± 2.9
60.9 ± 3.5
66.8 ± 2.4
62.5 ± 0.6
64.9 ± 2.5
73.8 ± 2.1
57.4 ± 1.6
63.1 ± 0.7
69.6 ± 2.5
67.5 ± 1.7
73.6 ± 2.7
84.5 ± 3.1
59.0 ± 2.7
66.5 ± 1.9
74. 0 ± 2.5
DE, dissolution efficiency; IC/K, inclusion complex prepared using the kneading method; IC/S, inclusion complex prepared using the solvent method;
ICSD/K, inclusion complex in solid dispersion prepared using the kneading method; ICSD/S, inclusion complex in solid dispersion prepared using
the solvent method; NS, nimesulide; PM, physical mixture; SD/K, solid dispersion prepared using the kneading method; SD/S, solid dispersion
prepared using the solvent method.
obtained using the solvent method, SD/S (1 : 2 : 20%). In
addition, the ternary SD obtained using the kneading
method, SD/K (1 : 2 : 20%), was also investigated.
The dissolution rates of fresh and aged samples using the
solvent method and those using the kneading method are
shown in Fig. 8a and b, respectively. Statistical analysis
showed that under the conditions of the accelerated test,
no significant changes occurred in the dissolution
behavior of the ternary dispersions of NS, b-CD, and
Inutec (P40.05).
The thermal (by DSC) and crystalline (by XRPD)
characteristics of the aged samples after 3 months of
accelerated stability testing did not show any significant
changes compared with those of the fresh samples,
indicating good stability in the solid state.
Evaluation of the anti-inflammatory activity in rats
The presence of edema is one of the prime signs of
inflammation [27]. It has been documented that carrageenan-induced rat paw edema is a suitable in-vivo model to
predict the efficacy of anti-inflammatory agents, which act
by inhibiting the mediators of acute inflammation [28]. The
efficiency of NS in the inhibition of the edema volume was
determined using the method described in the experimental part.
The samples used in the study included the optimized
ternary SD obtained using the solvent method [SD/S
(1 : 2 : 20%)] and the commercially available NS tablet
(designated as market tablet) as well as the control. The
anti-inflammatory effect was monitored during 5 h
following carrageenan injection. The results are presented in Fig. 9.
The results shown in Fig. 9 indicate that there is a
marked increase (Po0.05) in the mean percentage
inhibition of edema volume with the SD when compared
with the commercially available tablet.
With respect to the pharmacodynamic parameters, it can be
seen that the maximum percentage inhibition of edema
volume for all samples occurred 1 h after dosing. Also, it can
be seen in Fig. 9 that the dispersion inhibited the increase
in paw volume during the early phase of inflammation
(1–3 h after carrageenan injection) and also showed a good
inhibitory effect at a later phase (up to 5 h). This is in
agreement with the studies of Garcia-Pastor et al. [29], who
suggested a biphasic model in carrageenan-induced edema.
The first phase begins immediately after injection and
decreases within 1–1.5 h. The second phase remains
through 3 h. The delayed phase is considered to result
from the effect of prostaglandins on mediator release.
Conclusion
The effects of the carriers investigated in this study that
resulted in the enhancement of the dissolution properties
and the anti-inflammatory activity of the water-insoluble
drug NS represent potential incentive toward the
development of a stable formulation that can lead to a
reduction in the dose without the need to modify the
basic molecule of the drug. The addition of a hydrophilic
polymeric surfactant (Inutec) in a small concentration
markedly enhanced the dissolution rate of NS compared
with the binary IC with b-CD.
Ternary solid dispersions of nimesulide Khalil et al.
The polymeric surface-active agent Inutec, which was
shown to improve the anti-inflammatory activity of NS
but that has not as yet been fully investigated or reported
in the literature, might have huge potential as a carrier for
other water-insoluble drugs.
Figure 8
(a)
100
90
80
% Drug dissolved
55
70
60
Acknowledgements
50
Conflicts of interest
40
There are no conflicts of interest.
30
20
References
10
1 European Medicines Agency: Doc. Ref. EMEA/432604, London; 2007.
0
0
50
100
150
200
Time (minutes)
(b)
3 Gohel MC, Patel LD. Processing of nimesulide-PEG 400-PG-PVP solid
dispersions: preparation, characterization, and in vitro dissolution. Drug
Dev Ind Pharm 2003; 29:299–310.
4 Babu GV, Kumar NR, Himasankar K, Seshasayana A, Murthy KV.
Nimesulide-modified gum karaya solid mixtures: preparation, characterization, and formulation development. Drug Dev Ind Pharm 2003; 29:855–864.
100
90
80
70
% Drug dissolved
2 Gohel MC, Patel LD. Improvement of nimesulide dissolution from solid
dispersions containing croscarmellose sodium and Aerosil 200. Acta
Pharm 2002; 52:227–241.
5 Abdelkader H, Abdallah OY, Salem HS. Comparison of the effect of
tromethamine and polyvinylpyrrolidone on dissolution properties and
analgesic effect of nimesulide. AAPS PharmSciTech 2007; 8:E1–E8.
60
50
6 Dutet J, Lahiani-Skiba M, Didier L, Jezequel S, Bounoure F, Barbot C, et al.
Nimesulide/cyclodextrin/PEG 6000 ternary complexes: physico-chemical
characterization, dissolution studies and bioavailability in rats. J Incl Phenom
Macrocycl Chem 2007; 57 (1–4):203–209.
40
30
20
7 Dhanaraju MD, Thirumurugan G. Dissolution profiling of nimesulide solid
dispersions with polyethylene glycol, Talc and their combinations as dispersion carriers. Int J Pharm Tech Res 2010; 2:480–484.
10
0
0
50
100
Time (minutes)
150
200
Dissolution profiles of fresh and aged solid dispersions prepared using
the solvent method [(a) SD/S (1 : 2 : 20%)] and the kneading method
[(b) SD/K (1 : 2 : 20%)]:m, fresh sample; ’, after 1 month; ~, after 2
months; K, after 3 months. Data represent mean ± SD (n = 3).
8 Chandrashekhar PB, Basawaraj P, Srinivas SR, Amaregouda BRC
Rajesh. Formulation and evaluation of fast dissolving nimesulide tablets by
solid dispersion technique. Int Res J Pharm 2011; 2:145–148.
9 Nalluri BN, Chowdary KPR, Murthy KVR, Hayman AR, Becket G. Physicochemical characterization and dissolution properties of nimesulide-cyclodextrin binary systems. AAPS PharmSciTech 2003; 4:1–12.
10 Nalluri BN, Chowdary KP, Murthy KV, Becket G, Crooks PA. Tablet formulation studies on nimesulide and meloxicam-cyclodextrin binary systems.
AAPS PharmSciTech 2007; 8:E1–E7.
11 Booten K, Levecke B. Inutec surfactants: the properties of a new type of
surfactants. Household Pers. Care Today 2003; 1:26–28.
12 Van den Mooter G, Weuts I, De Ridder T, Blaton N. Evaluation of Inutec SP1
as a new carrier in the formulation of solid dispersions for poorly
soluble drugs. Int J Pharm 2006; 316 (1–2):1–6.
13 Janssens S, Humbeeck JV, Mooter GVd. Evaluation of the formulation of solid
dispersions by co-spray drying itraconazole with Inutec SP1, a polymeric
surfactant, in combination with PVPVA 64. Eur J Pharmaceut Biopharmaceut
2008; 70:500–505.
Figure 9
14 Ibrahim MM, El-Nabarawi M, El-Setouhy DA, Fadlalla MA. Polymeric
surfactant based etodolac chewable tablets: formulation and in vivo evaluation. AAPS PharmSciTech 2010; 11:1730–1737.
15 Khalil RM, Ghorab MM, Abd El-Rahman N, Kocova El-Arini S. Enhancement
of dissolution of nimesulide using a novel surface-active polymeric carrier,
Inutec SP1. Egypt Pharm J (NRC) 2010; 9:181–201.
16 Higuchi T, Connors KA. Phase solubility techniques. Adv Anal Chem Instrum
1965; 4:117–212.
17 Khan KA. The concept of dissolution efficiency. J Pharm Pharmacol 1975;
27:48–49.
18 Winter CA, Porter CC. Effect of alterations in side chain upon antiinflammatory and liver glycogen activities of hydrocortisone esters. J Am
Pharm Assoc Am Pharm Assoc (Baltim) 1957; 46:515–519.
80
Inhibition of oedema (%)
70
60
50
40
30
19 Winter CA, Risley EA, Nuss GW. Carrageenin-induced edema in hind paw of
the rat as an assay for antiiflammatory drugs. Proc Soc Exp Biol Med 1962;
111:544–547.
MT
SD/S (1:2:20%)
20
10
20 Suleyman H, Salamci E, Cadirci E, Halici Z. Beneficial interaction of nimesulide with NSAIDs. Med Chem Res 2007; 16:78–87.
0
0
1
2
3
Time (h)
4
5
6
Mean percentage inhibition of edema volume after administration of the
selected NS formulations in Carrageenan-induced paw edema in rats:
K, market tablet; ’, solid dispersion prepared using the solvent
method [SD/S (1 : 2 : 20%)]. NS, nimesulide.
21 Achar KCS, Hosamani KM, Seetharamareddy HR. In-vivo analgesic and antiinflammatory activities of newly synthesized benzimidazole derivatives. Eur J
Med Chem 2010; 45:2048–2054.
22 Delporte C, Backhouse N, Negrete R, Salinas P, Rivas P, Cassels BK, San
Feliciano A. Antipyretic, hypothermic and antiinflammatory activities and
metabolites from Solanum ligustrinum Lood. Phytother Res 1998; 12:
118–122.
23 Abdel-Allah DH. Formulation and quality improvement of tablets of
antirheumatic activity [PhD thesis]. Egypt: Cairo University; 2008.
56
Egyptian Pharmaceutical Journal
24 Chowdary KPR, Nalluri BN. Nimesulide and b-cyclodextrin inclusion complexes: physicochemical characterization and dissolution rate studies. Drug
Dev Ind Pharm 2000; 26:1217–1220.
27 Sur TK, Pandit S, Battacharyya D, Kumar CKA, Lakshmi SM, Chatttopadhyay D,
Mandal SC. Studies on the antiinflammatory activity of Betula alnoides bark.
Phytother Res 2002; 16:669–671.
25 Bergese P, Bontempi E, Colombo I, Gervasoni D, Depero LE. Microstructural
investigation of nimesulide-crospovidone composites by X-ray diffraction and
thermal analysis. Composites Sci Tech 2003; 63:1197–1201.
26 Di Martino P, Censi R, Barthélémy C, Gobetto R, Joiris E, Masic A, et al.
Characterization and compaction behaviour of nimesulide crystal forms. Int J
Pharm 2007; 342 (1–2):137–144.
28 Morebise O, Fafunso MA, Makinde JM, Olajide OA, Awe EO. Antiinflammatory and analgesic property of leaves of Gongronema latifolium.
Phytother Res 2002; 16:S75–S77.
29 Garcia-Pastor P, Randazzo A, Gomez-Paloma L, Alcaraz MJ, Paya M. Effects
of petrosaspongiolide M, a novel phospholipase A2 inhibitor, on acute and
chronic inflammation. J Pharmacol Exp Ther 1999; 289:166–172.
Original article 57
DNA fingerprinting and profile of phenolics in root and root calli
of Arctium lappa L. grown in Egypt
Elsayed A. Aboutabla, Mona El-Tantawyb, Nadia Sokkara and Manal M. Shamsb
a
Department of Pharmacognosy, Faculty of Pharmacy,
Cairo University and bDepartment of Pharmacognosy,
National Organization for Drug Control and Research
(NODCAR), Cairo, Egypt
Correspondence to Elsayed A. Aboutabl, Department
of Pharmacognosy, Faculty of Pharmacy, Cairo
University, Kasr-el-Aini Str., 11562 Cairo, Egypt
Tel: + 20 100 242 8817; fax: + 20 223 628 426;
e-mail: eaboutabl@ yahoo.com
Received 17 December 2012
Accepted 19 February 2013
Egyptian Pharmaceutical Journal
2013,12:57–62
Aim
The aim of this study was the establishment of an efficient and promising protocol for
callus production from Arctium lappa L. roots (family Asteraceae) and comparison of
the metabolic profile of their phenolic and flavonoid content. DNA fingerprinting of
A. lappa L. was carried out using the molecular generic marker technique (random
amplification of polymorphic DNA-PCR), which was newly introduced in Egypt, for
identification and authentication of the plant.
Methods
The effect of different concentrations of benzyladenine and naphthalene acetic acid
added to MS media on initiation of root callus production and mass of callus produced
was investigated. The presence or absence of various secondary metabolites of the
root and calli was also determined using colorimetric methods and high performance
liquid chromatography.
Results and conclusion
The growth parameters of the callus were determined. Each callus differs from the root
in the profile of phenolic and flavonoid content. The calli have a higher phenolic content
than the root and differ in the flavonoid profile.
Keywords:
Arctium lappa L, DNA fingerprinting, flavonoids, phenolics, root callus
Egypt Pharm J 12:57–62
& 2013 Division of Pharmaceutical and Drug Industries Research,
National Research Centre
1687-4315
Introduction
Subjects and methods
Arctium lappa L. or burdock (Asteraceae) is native to
Europe and north Asia. Traditionally, it has been used as a
safe and edible food product [1,2] and for the treatment
different ailments [3–5]. Phytochemical investigation of
different organs of the plant revealed the presence of
fixed oil, phenolic acids, flavonoids, lignans [2,6], resin,
mucilage, essential oil [7], polyacetylenes [7], and
caffeoylquinic acid derivatives [8]. In a previous study [9],
bioactive lignans and phenolics and the biological
activities of extracts from different organs of A. lappa L.
cultivated in Egypt were studied. PCR sequencing was
carried out for six A. lappa L. breeds from southern
Taiwan using two primers, ITS1-5.8S and rRNA-ITS2,
which revealed that they all had an amplified fragment
that was 358 bp in length [10]. Automatic sequence
analysis showed that the DNA sequences for different
breeds of Arctium can differ [10]. Hypocotyls and cotyls of
the plant were induced to produce callus for high
frequency plant regeneration [11]. In the current
literature, few studies on tissue culture and DNA
fingerprinting of the plant were found, but no reports
dealing with the phenolic profile of the callus were found.
Accordingly, the aim of the present work was to carry out
PCR sequencing for the identification and authentication
of A. lappa L., a plant grown in Egypt, and to study the
root callus metabolites, as the accumulation of secondary
products in plant cell cultures depends on the composition of the culture medium.
Plant material
Authentic seeds of A. lappa L. were kindly provided to
Prof. Dr E.A. Aboutabl by the Botanic Garden, Bonn,
Germany and were cultivated in the Experimental
Station of Medicinal and Aromatic Plants, Faculty of
Pharmacy, Cairo University. For tissue culture, seeds were
collected from the cultivated plant during the fruiting
stage.
Plant material for DNA fingerprinting
Freeze-dried leaves of A. lappa (10 g) were powdered in
liquid nitrogen, and genomic DNA was extracted by a
modification of the cetyltrimethylammonium bromide
method [12].
Reference standards
Rutin, daidzein, genistein, isorhamnetin, luteolin, biochanin A, hyperoside, gallic acid, chlorogenic acid, caffeic
acid, ferulic acid, and coumarin were obtained from the
Department of National Organization of Drug Control
and Research Standards.
Primers were obtained from Operon Technologies Inc.
(Almeda, California, USA).
Methods
DNA amplification was carried out using the random
amplification of polymorphic DNA (RAPD) technique
1687-4315 & 2013 Division of Pharmaceutical and Drug Industries Research,
National Research Centre
DOI: 10.7123/01.EPJ.0000428269.66909.9a
58
Egyptian Pharmaceutical Journal
Table 1 Sequence of 15 primers assayed using the RAPD-PCR
technique
Primer
Sequences (50 –30 )
A-01
A-11
B-06
B-08
B-15
B-18
P-01
O-02
O-09
E-08
E-05
E-11
G-06
Z-13
G-17
50 -CAGGCCCTTC-30
50 -CAATCGCCGT-30
50 -TGCTCTGCCC-30
50 -GTCCACACGG-30
50 -GGAGGGTGTT-30
50 -CCACAGCAGT-30
50 -GTAGCACTCC-30
50 -ACGTAGCGTC-30
50 -TCCCACGCAA-30
50 -TCACCACGGT-30
50 -TCACCACGGT-30
50 -GAGTCTCAGG-30
50 -GTGCCTAACC-30
50 -GACTAAGCCC-30
5’- ACGACCGACA-3’
RAPD, random amplification of polymorphic DNA.
with 15 primers (the sequences are shown in Table 1).
The GeneAmp PCR system 9700 (Perkin Elmer,
Cambridge, UK) and a gel documentation system
(Bio-Rad Gel Doc-2000, Bio-Rad Laboratories, GmbH,
Munich, Germany) were used for photographing of PCR
products.
PCR reactions[13,14] were carried out in a total volume
of 25 ml with 10 ng/ml of genomic DNA as a template, 3 ml
of random primer, 2.5 ml of 2 mmol/l dNTP mix (Abgene,
Surrey, UK), 2.5 ml of 10 PCR buffer, 2 ml of 25 mmol/l
MgCl2, and 0.3–5 U/ml of Taq DNA polymerase. An
aliquot of 22 ml of master mix solution was dispensed in
each PCR tube (0.2 ml Eppendorf tube) containing 3 ml
of the appropriate template DNA. The reaction involved
initial denaturation by heating for 4 min at 941C.
Complete denaturation of DNA indicated efficient
utilization of the template in the first amplification cycle
and a good yield of the PCR product. The reaction
mixture was then subjected to 40 cycles of the following
program: a denaturation step at 941C for 45 s, an
annealing step at 361C for 1 min, and an elongation or
extension step at 721C for 2 min. After the last cycle, the
mixture was subjected to a final extension step for 7 min
at 721C, followed by soaking at 41C until removal of the
reaction mixture from the PCR machine. The amplification products were resolved by electrophoresis on a 1.4%
agarose gel containing ethidium bromide (0.5 mg/ml) in
1 tris-borate-EDTA buffer. A total of 15 ml of each PCR
product was mixed with 3 ml of loading buffer (tracking
dye) and loaded into the wells of the gel. The gel
was run at 85 V for about 3 h or until the tracking dye
reached the gel. An ultraviolet (UV) Polaroid camera
was used for visualization of RAPD. Polaroid camera was
used for 6 visualization of RAPD; markers being scored
as DNA fragments present in some lanes and absent
in others.
Ramadan, Sharkiah, Egypt) (an antiseptic solution
containing 0.3% w/v chlorhexidine gluconate and 3% w/v
cetrimide) for about 5 min with shaking. The seeds were
then washed three times with sterile distilled water and
immersed in 30% commercial Clorox solution (10th of
Ramadan) (a disinfectant containing 1.5% sodium hypochlorite) with 1–2 drops of wetting agent (Tween 80)
while shaking on a shaker for 10 min. Thereafter, the
seeds were washed three times with sterile distilled
water. They were then cultured in a jar containing sterile
solid MS control media without a plant growth regulator
and incubated at 22–281C with a photoperiod of 16 h/day
(200–2500 lx). After 6–8 weeks, the plantlets grown were
used to obtain the explants used for callus cultures. The
6–8-week-old seedlings grown in vitro on sterile MS
medium (Fig. 1) were used as a sources of explants [15].
Dissection of uniformly-sized explants (about 0.5 cm in
length) from different organs – that is, shoot tips, leaves,
roots, and stems – was performed under aseptic conditions using a sterile scalpel and forceps [16]. The
different explants were cultured in jars containing sterile
medium supplemented with different concentrations of
various plant growth regulators such as benzyladenine
(BA), kinetin, naphthalene acetic acid (NAA), indolebutyric acid, 2,4-dichlorophenoxy acetic acid, and indole
acetic acid. For each condition, 30 jars were prepared,
each jar containing five explants. The cultures were
incubated at 211C ( ± 21C) with a photoperiod of
16 h/day (1500–2000 lx) for a period of 6 weeks.
Determination of total phenolics
Air-dried plant root calli 1 and 2 (1 g each) were defatted
with petroleum ether and extracted with 70% methanol
by sonication at room temperature. Stock solution
(concentration: 1 mg/ml) was prepared from the concentrated residue by dissolving in distilled water. The
phenolic compound in the root was found to be gallic
acid on Folin–Ciocalteau colorimetry [17] using a
Shimadzu 1601 spectrophotometer at 730 nm, and the
total phenolic content of the root was compared with that
Figure 1
Tissue culture
The seeds were washed thoroughly with running tap
water for about 15 min and surface-sterilized by immersion in 10% commercial Savlon solution (10th of
The random amplification of polymorphic DNA electrophoretic profile
of Arctium lappa L., cultivated in Egypt, generated by 15 primers
(M: 100 bp plus fermentas).
Phenolics of Arctium lappa root and calli Aboutabl et al. 59
of the calli. Determination of total the phenolic content
of the cultivated roots, leaves, and seeds was carried out
in our previous work [9].
Colorimetric determination of total flavonoid content
Powdered, air-dried (2 g) plant root calli 1 and 2 were
defatted with petroleum ether, extracted with 70%
methanol till exhaustion, and evaporated to dryness.
The combined methanolic extract was adjusted to 50 ml.
A 5 ml aliquot of each extract was treated with a 5 ml
aliquot of 0.1 mol/l AlCl3 reagent [18]. The absorbance of
the color developed was measured at lmax 422 nm against
a blank, and the corresponding amount of rutin was
recorded.
HPLC determination of isoflavones
Dried root (1 g) and root calli (1 and 2, 0.25 g) were
separately defatted, filtered, and extracted with 50%
ethanol. The ethanol was evaporated under vacuum at
351C, and the phenolics in the remaining aqueous
solution were extracted with ethyl acetate (1 : 1). The
phenolic fractions were stored in the dark at 41C until
analysis by high performance liquid chromatography
(HPLC). An Aglient 1100 system (Agilent Technologies
Deutschland GmbH, Germany) equipped with a column
compartment, quaternary pump, degasser, auto sampler,
and UV detector was used for HPLC analysis. Elution was
performed at a flow rate of 1 ml/min with a mobile phase
of water/acetic acid (98 : 2 v/v, solvent A) and methanol/
acetonitrile (50 : 50 v/v, solvent B), starting with 5% B and
increasing the level of B to 30% at 25 min, 40% at 35 min,
52% at 40 min, 70% at 50 min, and 100% at 55 min; the
UV detector was set at 254 nm [19]. Retention times
were compared with those of certain standard isoflavones.
Before injection into the HPLC system, each sample was
filtered through a 0.4 mm membrane filter into the sample
vial for injection.
The number of banding patterns generated by each
primer was recorded to obtain the DNA profile of A. lappa
under investigation, in order to compare it with
previously reported phenotypic characters as well as for
chemical investigations. Molecular size, in base pairs, of
amplified DNA fragments produced by 15 decamer
primers in A. lappa L. is listed in Table 1, and their
reproducible RAPD profiles generated are shown
in Fig. 1. The total number of bands generated by the
15 primers was 93, the smallest size of amplified product
being 245 bp, whereas the largest size of the amplified
product being 3030 bp. Primer P1 produced nine bands,
with 245 bp being the smallest size and 3030 bp being the
largest size; primer A1 was the least reproducible and
generated three bands with molecular sizes 1739, 724,
and 276 bp.
Callus production
Figure 2 shows 6–8-week-old seedlings of A. lappa grown
in vitro on sterile MS media; these were used as a source
of explants for callus production. Trials using different
explants (shoot tips, leaves, stems, and roots) and
different growth regulators were carried out for initiation
of callus. Calli were obtained successfully on MS media
supplemented with plant growth regulators for roots:
MS + 0.5 mg/l BA + 1 mg/l NAA (callus 1) and MS + 0.5
mg/l BA + 0.1 mg/l NAA (callus 2). The different callus
Figure 2
HPLC determination of phenolics
Extraction and HPLC analysis of phenolics were carried
out under the same conditions as those for isoflavones,
but measurements were made with a detector set at
330 nm. Retention times were compared with those of
available phenolic standards.
Results
Total genomic DNA profiling of A. lappa L., grown
in Egypt, was performed using 15 random primers.
Six- to eight-week-old seedling of Arctium lappa L. grown in vitro in MS
medium.
Table 2 The effect of plant growth regulators on callus growth parameters of Arctium lappa L. root and total phenolic and flavonoid
content of Arctium lappa L. root and two calli
Root calli (greenish brown compact undifferentiated callus)
Characteristics
Root
Callus fresh weight (g)
–
Callus dry weight (g)
–
Total phenolic content (%; calculated as gallic acid content in dried material) 5.33
Total flavonoid content (%; calculated as rutin content in dried material)
0.05
Callus 1
Callus 2
5.01 + 0.3
0.48 + 0.05
6.53
0.003
4.22 + 0.2
0.36 + 0.03
7.98
0.002
60
Egyptian Pharmaceutical Journal
Figure 3
(a) Callus 1 ( 0.76; MS + 0.5 mg/l BA + 1 mg/l NAA). (b) Callus 2 ( 1; MS + 0.5 mg/l BA + 0.1 mg/l NAA).
growth parameters are listed in Table 2, and callus types
are presented in Fig. 3a and b.
Table 3 Phenolics identified by high performance liquid
chromatography in Arctium lappa L. root and calli
Concentration (mg/g)
Determination of total phenolic content of the root calli
compared with that of the root
Colorimetric determination showed that there was a
variation in the phenolic content of the root compared
with that of the calli (Table 2). Callus 2 showed a higher
phenolic content than callus 1 and the root because of the
effect of plant growth regulators (BA and NAA) on the
biosynthesis of polyphenols [15].
Determination of the flavonoid compounds of the root
calli compared with those of the A. lappa L. root
Compounds
Gallic acid
Daidzein
Genistein
Isorhamnetin
Chlorogenic acid
Caffeic acid
Biochanin A
Hyperoside
Rutin
Ferulic acid
Coumarin
Luteolin
Rt (min)
Root
Callus 1
Callus 2
2.51
3.03
3.57
4.29
5.54
6.15
7.17
7.95
8.11
9.17
9.70
11.78
0.49
–
0.005
–
0.62
–
–
0.31
0.22
0.01
0.02
0.01
0.36
–
–
0.080
0.06
0.06
0.018
–
–
0.06
0.22
–
0.78
0.054
0.014
–
0.58
0.70
–
–
–
–
0.66
0.62
The flavonoid content in each of the two calli was less
than that in the root (Table 2).
HPLC determination of isoflavones in the root callus
compared with those in the main plant parts
The concentration of isoflavones (in mg/g; Table 3)
indicates that the root contains only genistein and differs
in metabolic profile compared with root callus 1 (MS +
0.5 mg/l BA + 1 mg/l NAA), which contains isorhamnetin
and biochanin A, and root callus 2 (MS + 0.5 mg/l
BA + 0.1 mg/l NAA), which contains daidzein and genistein. The flavonoid content in callus culture differs
qualitatively and quantitatively from that in the parent
plant [20].
HPLC determination of the phenolic content of the root
callus compared with that of the root
The root differs in its phenolic metabolic profile
compared with the two calli. The phenolic compounds
present in the root were identified as gallic acid, ferulic
acid, chlorogenic acid, hyperoside, rutin, coumarin, and
luteolin. Callus 1 was found to contain gallic acid,
chlorogenic acid, caffeic acid, ferulic acid, and coumarin,
whereas callus 2 was found to contain gallic acid,
chlorogenic acid, caffeic acid, coumarin, and luteolin.
The corresponding concentrations are listed in Table 3
(in mg/g; Table 4).
Phenolics of Arctium lappa root and calli Aboutabl et al.
61
Table 4 Molecular size, in base pairs, of amplified DNA fragments produced by 15 decamer primers in Arctium lappa L.
Molecular size
of DNA marker (bp)
245
268
276
310
359
370
440
453
467
525
556
573
590
644
683
703
724
745
813
838
862
888
914
941
969
998
1028
1058
1090
1122
1155
1189
1225
1298
1337
1377
1417
1503
1547
1593
1640
1989
1739
1791
1844
1899
2013
2073
2134
2197
2330
2399
2776
2858
3030
Sum
A1
A11
O2
O9
E5
E8
E11
B6
B8
B15
B18
G6
G17
Z13
+
p1
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
3
7
5
+
7
4
9
Conclusion
From the present study, it was deduced that using BA and
NAA for the induction of root callus production caused an
increase in the phenolic content compared with that
of the main root. Decreasing the amount of NAA in callus
2 (MS + 0.5 mg/l BA + 0.1 mg/l NAA) resulted in a higher
phenolic content than that in callus 1 (MS + 0.5 mg/l
BA + 1 mg/l NAA). In addition, HPLC results for callus 2
show a marked increase in caffeic acid, coumarin, and
luteolin content; however, the flavonoid content in the two
calli decreased, and the metabolic profile of isoflavones
8
7
5
+
4
7
6
5
6
+
9
showed great variation. DNA fingerprinting helps in the
authentication and identification of A. lappa L., which is
grown in Egypt. This is the first report on tissue culture
and molecular biological study of this plant. The current
literature, our previous work [9], and also the results of the
present work prove the importance of the plant; hence,
the authors recommended that the study on the effects of
plant growth regulators, precursors, and other factors that
increase the main active constituents of the plant, which
can be used as a source of natural raw material for
phytopharmaceuticals, be continued.
62 Egyptian Pharmaceutical Journal
Acknowledgements
Conflicts of interest
There are no conflicts of interest.
References
1 Bown D. Encyclopedia of herbs and their uses. London: Dorling Kindersley;
1995. pp. 240–241.
2 Leung A. Encyclopedia of common natural ingredients. 2nd ed. New York:
John Wiley and Sons; 1996.
3 Foster S, Duke JA. A field guide to medicinal plants. New York: Houghton
Mifflin Company; 1990. p. p166.
4 Kenner D, Requena Y. Botanical Medicine. Massachuetts, USA: Pardicm
Publications Brookline; 2001. p. 137.
5 Tamayo C, Richardson MA, Diamond S, Skoda I. The chemistry and
biological activity of herbs used in Flor-Essence herbal tonic and Essiac.
Phytother Res 2000; 14:1–14.
6 Ferracane R, Graziani G, Gallo M, Fogliano V, Ritieni A. Metabolic profile of
the bioactive compounds of burdock (Arctium lappa) seeds, roots and
leaves. J Pharm Biomed Anal 2010; 51:399–404.
7 Penelope O. The complete medicinal herbal. NewYork: Dorling Kindersley;
1993. p. p58.
8 Maruta Y, Kawabata J, Niki R. Antioxidative caffeoylquinic acid derivatives in
the roots of burdock (Arctium lappa L.). J Agric Food Chem 1995; 43:
2592–2595.
9 Aboutabl EA, El-Tantawy M, Sokar N, Shams MM, Selim A. Bioactive lignans
and other phenolics from roots, leaves and seeds of Arctium lappa L. grown
in Egypt. Egypt Pharmaceutical J 2012; 11:59–65.
10 Chang H-J, Huang W-T, Tsao D-A, Huang K-M, Lee S-C, Lin S-R, et al.
Identification and authentication of Burdock (Arctium lappa Linn) using PCR
sequencing. Fooyin J Health Sci 2009; 1:28–32.
11 Hou HEWT, Wang SW. CY. Callus induction and high frequency plant
regeneration from hypocotyl and cotyledon explants of Arctium lappa L. In
Vitro Cell Dev Biol Plant 2006; 42:411–414.
12 Doyle JJ, Doyle JL. A rapid DNA isolation procedure for small quantities of
fresh leaf tissue. Phytochem Bull 1987; 19:11–15.
13 Welsh J, McClelland M. Fingerprinting genomes using PCR with arbitrary
primers. Nucleic Acids Res 1990; 18:7213–7218.
14 Abd El Samad EH, El-Gizawy AM, El Kishin DA, Lashine ZA. Estimation of
genetic diversity in wild and cultivated form of beet using RAPD and AFLP
Markers. Res J Agric Biol Sci 2009; 5:207–217.
15 Murashige T, Skoog F. A revised medium for rapid growth and bioassays with
tobacco tissue cultures. Physiol Plant 1962; 15:473–497.
16 Evans DA, Sharp WR, Ammirato PV, Yamada Y. Handbook of plant cell
culture, techniques for propagation and breeding. 1 New York, USA: Macmillan Publishing Company; 1983. pp. 2–3.
17 Singleton VL, Orthofer R, Lamuela-Raventós RM. Analysis of total phenols
and other oxidation substrates and antioxidants by means of folin-ciocalteu
reagent. Methods Enzymol 1998; 299:152–178.
18 Karawya MS, Aboutabl EA. Phytoconstituents of Tabernaemontana coronaria Jacq. Willd and Tabernaemontana dichotoma Roxb. growing in Egypt
Part IV. The flavonoids. Bull Fac Pharm Cairo Univ 1982; 21:41–49.
19 Campos MG, Webby RF, Markham KR, Mitchell KA, Da Cunha AP. Age-induced
diminution of free radical scavenging capacity in bee pollens and the contribution
of constituent flavonoids. J Agric Food Chem 2003; 51:742–745.
20 Delle Monache G, De Rosa MC, Scurria R, Vitali A, Cuteri A, Monacelli B,
et al. Comparison between metabolite productions in cell culture and in
whole plant of Maclura pomifera. Phytochemistry 1995; 39:575–580.
Original article 63
Influence of formulation parameters on the physicochemical
properties of meloxicam-loaded solid lipid nanoparticles
Rawia M. Khalila, Ahmed Abd El-Baryb, Mahfoz A. Kassema,
Mamdouh M. Ghorabc and Mona Bashaa
a
Department of Pharmaceutical Technology, National
Research Centre, bDepartment of Pharmaceutics,
Faculty of Pharmacy, Cairo University, Cairo and
c
Department of Pharmaceutics, Faculty of Pharmacy,
Suez Canal University, Ismailia, Egypt
Correspondence to Rawia M. Khalil, Department
of Pharmaceutical Technology, National Research
Centre, El-Bohowth St., PO Box 12622, Dokki,
12311 Cairo, Egypt
Tel: + 20 1006935895/ + 1006550825;
fax: + 20 233370931;
e-mail: [email protected]
Received 26 November 2012
Accepted 4 February 2013
Egyptian Pharmaceutical Journal
2013, 12:63–72
Objective
The aim of this research was to investigate novel particulate carrier systems such as
solid lipid nanoparticles (SLNs) for topical delivery of a lipophilic drug, meloxicam
(MLX).
Methods
MLX-loaded SLNs were prepared using a modified high-shear homogenization and
ultrasonication technique using different types of lipids and surfactants. Lipid
nanoparticles were characterized in terms of entrapment efficiency, particle size,
Zeta potential, differential scanning calorimetry, transmission electron microscopy,
and in-vitro release studies.
Results
The lipid nanoparticles showed mean diameters of 210–730 nm, whereas the
entrapment efficiency ranged from 50 to 84% depending on emulsifier and lipid
concentration or type. MLX-loaded SLNs showed spherical particles with Zeta
potentials varying from – 15.7 to – 30.5 mV. A differential scanning calorimetry study
revealed that MLX encapsulated in SLNs was in the amorphous form. All nanoparticle
formulations exhibited sustained release characteristics, and the release pattern
followed the Higuchi’s equation. The analysis of results revealed that the type and
concentration of the emulsifier or lipid used had a significant effect on the
physicochemical properties on the investigated SLNs formulations.
Conclusion
The present study indicates that SLNs could potentially be exploited as carrier systems
for MLX, with improved drug loading capacity and controlled drug release.
Keywords:
differential scanning calorimetry, in-vitro release study, meloxicam, solid lipid
nanoparticles, topical delivery
Egypt Pharm J 12:63–72
& 2013 Division of Pharmaceutical and Drug Industries Research,
National Research Centre
1687-4315
Introduction
In recent years, significant effort has been devoted to
develop nanotechnology for drug delivery. Solid lipid
nanoparticles (SLNs) are aqueous colloidal dispersions,
the matrix of which comprises solid biodegradable lipids.
SLNs combine the advantages and avoid the drawbacks
of several colloidal carriers of their class such as physical
stability, protection of incorporated labile drugs from
degradation, controlled release, and excellent tolerability [1]. SLNs offer a suitable means of delivering drugs
for various application routes; they attract great attention
as novel colloidal drug carriers for topical use [2]. The
advantages of these carriers include negligible skin
irritation, controlled release, and protection of active
substances [3]. Because they are composed of nonirritative and nontoxic lipids, SLNs seem to be well suited for
use on inflamed and damaged skin. Moreover, SLNs have
distinct occlusive properties because of the formation of
an intact film on the skin surface upon drying, which
decreases transepidermal water loss and favors drug
penetration through the stratum corneum [4]. Besides
having a highly specific surface area, nanometer-sized
SLNs also facilitate the contact of the encapsulated drug
with the stratum corneum [4]. The nanometer-sized
particles can make close contact with superficial junctions
of corneocyte clusters and furrows between corneocyte
islands, which may favor accumulation for several hours,
allowing for sustained drug release [5]. Other advantages
of SLNs include a high drug payload and incorporation of
lipophilic and hydrophilic drugs [2]. SLNs have been
used to improve skin/dermal uptake of several drugs [6,7],
which supports the idea that SLNs can be used as carriers
for topical delivery of meloxicam (MLX).
MLX is a potent, nonsteroidal anti-inflammatory
water-insoluble drug [8,9]. It inhibits cyclooxygenase
(COX). MLX is more selective for the COX-2 isoform
of prostaglandin synthetase compared with the COX-1
form. Therefore, MLX has been labeled a ‘preferential’
inhibitor instead of a ‘selective’ inhibitor of COX-2
[10].
1687-4315 & 2013 Division of Pharmaceutical and Drug Industries Research,
National Research Centre
DOI: 10.7123/01.EPJ.0000428643.74323.d9
64
Egyptian Pharmaceutical Journal
The intention of this study was to prepare and evaluate
MLX-loaded SLNs and to optimize the formulation
parameters in order to fabricate SLN dispersions of
desired characteristics for topical delivery of MLX, aiming
to improve skin uptake and reduce systemic absorption
and dermal irritation.
Materials and methods
using a validated UV-spectrophotometric method (model
2401/PC; Shimadzu, Kyoto, Japan) after suitable dilution.
The EE% was calculated using the following equation [13]:
EE % ¼
Winitial drug Wfree durg
100;
Winitial drug
where Winitial drug is the initial mass of the drug used and
Wfree drug is the mass of the free drug detected in the
supernatant after centrifugation of the aqueous dispersion.
Materials
MLX was supplied by Medical Union Pharmaceuticals
(Ismailia, Egypt). Geleol (glyceryl monostearate 40–55;
40–55% monoglycerides, 30–45% diglycerides, melting point
(m.p.) 54.5–58.51C), Compritol 888 ATO (glyceryl behenate; 15–23% monoglycerides, 40–60% diglycerides, 21–35%
triglycerides, m.p. 69.0–74.01C), and Precirol ATO5 (glyceryl palmitostearate; 8–22% monoglycerides, 40–60% diglycerides, 25–35% triglycerides, m.p. 50–601C) were kindly
donated by Gattefossé (Saint-Priest, France). Tween 80
(polysorbate 80), methanol Chromasolv, and dialysis tubing
cellulose membrane (molecular weight cutoff 12 000 g/mole)
were purchased from Sigma Chemical Company (St. Louis,
Missouri, USA). Cremophor RH40 (polyoxyl 40 hydrogenated
castor oil) was kindly donated by BASF (Ludwigshafen,
Germany). All other chemicals and reagents used were of
analytical grade.
Methods
Preparation of solid lipid nanoparticles
SLNs were prepared by a slight modification of the
previously reported high-shear homogenization and ultrasonication technique [11,12]. Briefly, the lipid phase
consisted of Geleol, Compritol, or Precirol as the solid
lipid was melted 51C above the melting point of the lipid
used. MLX (0.5%w/w) was dissolved therein to obtain a
drug–lipid mixture. An aqueous phase was prepared by
dissolving the surfactant in distilled water and heated up
to the same temperature of the molten lipid phase.
The hot lipid phase was poured onto the hot aqueous
phase and homogenization was carried out at 25 000 rpm
for 5 min using a Heidolph homogenizer (Heidolph
Instruments, Schwabach, Germany). The resultant hot
oil-in-water emulsion was sonicated for 30 min (Digital
Sonicator; MTI, Michigan, USA). MLX-loaded SLNs
were finally obtained by allowing the hot nanoemulsion to
cool to room temperature. Blank SLNs were prepared
using the same procedure variables.
Meloxicam entrapment efficiency
The entrapment efficiency percentage (EE%), which
corresponds to the percentage of MLX encapsulated
within the nanoparticles, was determined by measuring
the concentration of free MLX in the dispersion medium.
The unentrapped MLX percentage was determined by
adding 500 ml of MLX-loaded nanoparticles to 9.5 ml of
methanol and centrifuging this dispersion at 9000 rpm
(Union 32R; Hanil Science Industrial, Gangwondo, Korea)
for 30 min. The supernatant was filtered through a
Millipore (Sigma-Aldrich, St. Louis, USA) membrane filter
(0.2 mm) and analyzed for unencapsulated MLX at 360 nm
Particle size analysis
Particle size analysis of MLX-loaded nanoparticles was
performed using a laser diffraction (LD) particle size analyzer (Master Sizer X; Malvern Instruments, Worcestershire,
UK) at 251C. The LD data obtained were evaluated using
volume distribution as diameter values of 10, 50, and 90%
and span values. The diameter values indicate the
percentage of particles possessing a diameter equal to
or lower than the given value. The span value is a
statistical parameter used to evaluate the particle size
distribution: lower the span value, narrower is the particle
size distribution. It is calculated using the following
equation [14]:
Span¼
LD90 % LD10 %
:
LD50 %
Zeta Potential and pH measurement
The z potential was measured in folded capillary cells
using a Laser Zetameter (Malvern Instruments). Measurements were performed in distilled water adjusted with a
solution of 0.1 mmol/l NaCl at 251C. The z potential
values were calculated using the Smoluchowski equation.
The pH values of MLX lipid nanoparticles were
measured at 251C using a digital pH meter (Jenway,
Staffordshire, UK).
Transmission electron microscopy
Morphological examination of MLX-loaded SLNs was performed using transmission electron microscopy (TEM)
(model JEM-1230; Jeol, Tokyo, Japan). One drop of the
diluted sample was deposited onto the surface of a carboncoated copper grid and negatively stained with a drop of
2% (w/w) aqueous solution of phosphotungstic acid for
30 s. Excess staining solution was wiped off with filter
paper, leaving a thin aqueous film on the surface. After
staining, the samples were allowed to dry at room
temperature for 10 min for analysis [15].
Differential scanning calorimetry
Differential scanning calorimetry (DSC) analysis was
carried out using a Shimadzu Differential Scanning
Calorimeter (DSC-50; Shimadzu). About 10 mg of sample
was added into a 40 ml aluminum pan, which was sealed
and heated in the range of 30–3001C at a heating rate of
101C/min. An empty aluminum pan was used as a
reference standard. The analysis was carried out under
nitrogen purge.
Meloxicam loaded solid lipid nanoparticles Khalil et al.
Rheological study
The rheological properties of the prepared lipid nanoparticles were determined using Brookfield’s Viscometer
(Brookfield LV-DV II + ; Brookfield, Massachusetts,
USA). The sample (20 g) was placed in a beaker and
allowed to equilibrate for 5 min. The measurements were
carried out at ambient temperature using the suitable
spindle. The spindle speed rate was increased in
ascending order from 1 to 100 rpm and then decreased
in descending order from 100 to 1 rpm, with each kept
constant for 10 s before a measurement was made.
In-vitro release study
The in-vitro release of MLX was evaluated using the
dialysis bag diffusion technique described by Yang
et al. [16]. The release studies of MLX from SLNs were
performed in phosphate buffer (pH 5.5) and methanol
(75 : 25). Aqueous nanoparticulate dispersion equivalent
to 2 mg of MLX was placed in a cellulose acetate dialysis
bag and sealed at both ends. The dialysis bag was
immersed in the receptor compartment containing 50 ml
of dissolution medium, which was stirred in a water bath
shaker at 100 rpm (Memmert GmbH, Schwabach,
Germany) and maintained at 32 ± 21C. The receptor
compartment was covered to prevent evaporation of the
dissolution medium. A 2 ml sample of the receiver
medium was withdrawn at predetermined time intervals
(0.5, 1, 2, 3, 4, 5, 6, 8, 24, and 48 h) and replaced by an
equivalent volume of fresh medium to maintain constant
volume. The samples were analyzed for drug content
spectrophotometrically at 360.5 nm. The data were
analyzed using linear regression equations, and the order
of drug release from the different formulations was
determined.
Statistical analysis
All experiments were repeated three times, and data were
expressed as mean value ± SD. The statistical analysis
65
was carried out using one-way analysis of variance.
A P value of less than 0.05 was considered statistically
significant.
Results and discussion
Preparation of solid lipid nanoparticles
In the present study, MLX-loaded SLNs dispersions were
composed of Geleol, Compritol 888 ATO, or Precirol
ATO5 as core matrices used in different concentrations of
5, 7.5, and 10% (w/w). These lipid-based carrier systems
were stabilized using 0.5, 1, 2.5, and 5% (w/w) Tween 80
or Cremophor RH40. MLX was incorporated at a constant
concentration of 0.5% (w/w). The w/w percentage
composition of the investigated MLX SLNs is shown
in Tables 1 and 2.
Meloxicam entrapment efficiency
The entrapment efficiencies of all SLN formulations are
presented in Tables 1 and 2. The entrapment efficiencies
varied from 50.42 ± 2.07 to 84.38 ± 0.65%. It can be
observed that increasing the amount of surfactant from
0.5 to 1 to 2.5 to 5% (w/w) at a constant amount of lipid
(5% w/w) resulted in a gradual significant decrease
(Po0.05) in the entrapment efficiencies. However, no
change in EE% was observed (Table 1) for Compritol
(SLN7 and SLN8) and Precirol SLNs (SLN13 and
SLN14) on increasing the Tween 80 concentration from
0.5 to 1%. Moreover, for Geleol SLNs (SLN3 and SLN4),
no significant decrease in EE% was observed on increasing the Tween 80 concentration above 2.5% (w/w)
(P40.05). Table 2 shows that using Cremophor RH40
resulted in the same gradual decrease in EE% (Po0.05);
however, in case of Geleol SLNs (SLN21 and SLN22)
and Precirol SLNs (SLN33 and SLN34), a further
increase in the Cremophor RH40 concentration from
2.5 to 5% did not result in significant changes in EE%
(P40.05). This observed decrease in EE% could be
Table 1 Composition and entrapment efficiency of meloxicam solid lipid nanoparticles (%w/w) of different lipids using Tween 80
Lipid
Formulas
SLN1
SLN2
SLN3
SLN4
SLN5
SLN6
SLN7
SLN8
SLN9
SLN10
SLN11
SLN12
SLN13
SLN14
SLN15
SLN16
SLN17
SLN18
Type
Geleol
Concentration
Tween 80 (%)
Entrapment efficiency %a
5
0.5
1
2.5
5
0.5
59.78 ± 1.04
56.63 ± 0.88
51.03 ± 0.96
50.42 ± 2.07
62.30 ± 0.23
67.49 ± 1.27
62.47 ± 0.25
62.22 ± 1.03
57.31 ± 1.92
54.79 ± 0.21
65.76 ± 1.77
72.63 ± 1.66
65.68 ± 0.09
65.53 ± 0.40
62.00 ± 0.39
58.51 ± 0.71
70.02 ± 0.89
75.99 ± 3.36
Compritol
7.5
10
5
Precirol
7.5
10
5
SLN, solid lipid nanoparticle.
a
Values represent mean ± SD.
7.5
10
0.5
1
2.5
5
0.5
0.5
1
2.5
5
0.5
66
Egyptian Pharmaceutical Journal
Table 2 Composition and entrapment efficiency of meloxicam solid lipid nanoparticles (%w/w) of different lipids using
Cremophor RH40
Lipid
Formulas
SLN19
SLN20
SLN21
SLN22
SLN23
SLN24
SLN25
SLN26
SLN27
SLN28
SLN29
SLN30
SLN31
SLN32
SLN33
SLN34
SLN35
SLN36
Type
Geleol
Concentration (%)
Cremophor RH40 (%)
Entrapment efficiency %a
5
0.5
1
2.5
5
0.5
63.31 ± 1.11
58.33 ± 1.42
52.28 ± 1.89
53.34 ± 1.20
65.89 ± 0.83
69.10 ± 0.42
68.63 ± 0.34
62.16 ± 1.64
59.34 ± 0.32
56.64 ± 0.91
70.41 ± 0.58
77.47 ± 0.93
78.77 ± 0.85
73.33 ± 1.31
67.71 ± 2.76
66.79 ± 0.92
79.51 ± 0.24
84.38 ± 0.65
Compritol
7.5
10
5
Precirol
7.5
10
5
7.5
10
0.5
1
2.5
5
0.5
0.5
1
2.5
5
0.5
SLN, solid lipid nanoparticle.
a
Values represent mean ± SD.
explained by the partition phenomenon. High surfactant
levels in the external phase might increase the partition
of the drug from the internal to the external phase of the
medium. This increased partition is due to the increased
solubilization of the drug in the external aqueous phase
such that more volumes of the drug can disperse and
dissolve in it [17]. However, some cases in which further
increase of surfactant concentration did not lead to a
significant change in EE% could suggest that an optimum
concentration of the surfactant was reached, sufficient to
cover the surface of the nanoparticles effectively. The
data also clearly showed that the formulations prepared
using Cremophor RH40 as a surfactant had higher EE%
compared with those prepared using Tween 80. Similar
results were reported by Lv et al. [18] for penciclovirloaded SLNs.
The structure of the lipid used has a great influence on
the capacity for drug incorporation. Therefore, the effect
of lipid type and concentration on the entrapment
efficiency of MLX SLNs was also investigated (Tables 1
and 2). Geleol SLNs exhibited the lowest entrapment of
MLX when compared with Compritol and Precirol. This
can be attributed to the difference in composition and
chain length of the three lipids used. The higher drug
entrapment efficiency observed with Precirol and Compritol was attributed to the high hydrophobicity due to
the long chain fatty acids attached to the triglycerides,
resulting in increased accommodation of lipophilic
drugs [19].
The results also showed that increasing the lipid
concentration from 5 to 7.5 to 10% (w/w) led to a gradual
increase in the entrapment efficiency, which was
observed for lipids used at constant concentrations of
Tween 80 and Cremophor RH40 (Po0.05). However,
this increase in the entrapment efficiency is not
proportional to the increase in lipid content, which can
be observed for the three lipids. An exception was
observed for SLN31 and SLN35 wherein a significant
increase in EE% occurred only on increasing Precirol
concentrations from 7.5 to 10% (w/w). A possible
explanation for these observations is that the increase
in lipid content can afford more space to encapsulate
more drug, thus reducing drug partition in the outer
phase [18,20]. This may also be due to an increase in the
viscosity of the medium, resulting in faster solidification
of nanoparticles, which would further prevent drug
diffusion to the external phase of the medium [21].
Particle size analysis
The LD 90% of the formulated SLNs is presented
in Table 3. In case of Tween 80 and Cremophor RH40,
the nanoparticulate dispersions showed sizes ranging
from 210 ± 35.36 to 740 ± 14.14 nm and from 235 ± 21.21
to 730 ± 14.14 nm, respectively. The low span values of
different formulations indicate a narrow particle size
distribution. The results clearly showed that there was a
gradual decrease in particle size with an increase in
surfactant concentration from 0.5 to 1 to 2.5 to 5% (w/w)
(Po0.05). This was observed for all formulations except
for SLN1 and SLN2 and for SLN19 and SLN20, in which
an initial increase in surfactant concentration from 0.5 to
1% did not lead to a significant decrease in particle size
(P40.05). However, a further increase in surfactant
concentration above 2.5% for SLN33 and SLN34 did not
result in a significant change in particle size (P40.05).
The decrease in size of nanoparticles at high surfactant
concentrations might be due to an effective reduction in
the interfacial tension between the aqueous and lipid
phases, leading to the formation of emulsion droplets of
smaller sizes [22]. Higher surfactant concentrations
effectively stabilize the particles by forming a steric
barrier on the particle surface and thereby protect smaller
particles and prevent their coalescence into bigger
ones [17]. For the formulations in which further increase
of surfactant concentration above 2.5% did not reduce the
particle size significantly, the data clearly suggest that an
optimum concentration of the surfactant was reached,
Meloxicam loaded solid lipid nanoparticles Khalil et al. 67
Table 3 Particle size, f potential, and pH values of meloxicam solid lipid nanoparticles
Formulas
SLN1
SLN2
SLN3
SLN4
SLN5
SLN6
SLN7
SLN8
SLN9
SLN10
SLN11
SLN12
SLN13
SLN14
SLN15
SLN16
SLN17
SLN18
LD 90%
Span
z potential (mV)
pH
Formulas
LD 90%
Span
z potential (mV)
pH
420 ± 14.14
385 ± 7.07
250 ± 28.28
210 ± 35.36
480 ± 14.14
555 ± 7.07
580 ± 14.14
545 ± 7.07
440 ± 28.28
385 ± 7.07
680 ± 28.28
740 ± 14.14
470 ± 14.14
415 ± 21.21
310 ± 14.14
265 ± 7.07
570 ± 28.28
685 ± 7.07
0.51
0.63
0.18
0.34
0.67
1.15
0.80
1.27
1.06
0.80
1.28
1.27
0.88
0.83
0.81
0.48
1.03
1.34
– 15.9
– 16.0
– 17.9
– 20.9
– 25.5
– 25.5
– 18.8
– 21.1
– 21.0
– 22.3
– 23.0
– 27.1
– 16.9
– 15.7
– 18.6
– 22.4
– 29.8
– 30.5
6.15 ± 0.03
5.68 ± 0.04
5.61 ± 0.06
5.53 ± 0.05
5.72 ± 0.16
5.80 ± 0.04
5.77 ± 0.02
6.26 ± 0.10
5.96 ± 0.08
6.09 ± 0.01
5.56 ± 0.01
5.67 ± 0.08
5.91 ± 0.01
6.42 ± 0.04
5.70 ± 0.05
5.70 ± 0.03
5.76 ± 0.06
5.49 ± 0.08
SLN19
SLN20
SLN21
SLN22
SLN23
SLN24
SLN25
SLN26
SLN27
SLN28
SLN29
SLN30
SLN31
SLN32
SLN33
SLN34
SLN35
SLN36
425 ± 17.68
370 ± 14.14
265 ± 7.07
235 ± 21.21
490 ± 28.28
565 ± 7.07
565 ± 3.54
505 ± 7.07
465 ± 21.21
390 ± 14.14
685 ± 10.61
730 ± 14.14
490 ± 14.14
435 ± 3.54
315 ± 7.07
285 ± 21.21
580 ± 28.28
685 ± 7.07
1.39
1.23
1.10
1.21
1.61
1.64
1.64
1.84
1.72
1.30
1.90
1.95
1.36
1.55
1.29
1.27
1.30
1.71
– 15.8
– 17.5
– 19.8
– 19.1
– 20.5
– 25.2
– 15.9
– 17.8
– 21.6
– 21.7
– 19.8
– 22.8
– 20.2
– 20.0
– 21.4
– 22.6
– 20.4
– 24.3
5.97 ± 0.01
5.82 ± 0.02
5.88 ± 0.05
5.93 ± 0.08
5.85 ± 0.01
5.84 ± 0.07
5.87 ± 0.01
5.71 ± 0.03
6.08 ± 0.02
5.95 ± 0.07
5.92 ± 0.01
5.53 ± 0.02
6.24 ± 0.08
5.26 ± 0.03
5.63 ± 0.02
5.94 ± 0.03
5.56 ± 0.13
5.48 ± 0.28
LD, laser diffraction; SLN, solid lipid nanoparticle.
f Potential analysis and pH measurements
As shown in Table 3, all formulations were negatively
charged; the z potential varied from – 15.7 mV (SLN14)
to – 30.5 mV (SLN18), indicating relatively good stability
(a)
Particle size (nm)
The results also showed that increasing the lipid content
from 5 to 7.5 to 10% (w/w) led to a subsequent increase
in particle size (Table 3). Statistical analysis of the data
showed no significant increase in particle size in case of
SLN19 and SLN23 on increasing the lipid concentration
from 5 to 7.5%. A similar result was obtained on increasing
the lipid concentration from 7.5 to 10% in case of SLN11
and SLN12 and in SLN29 and SLN30. This increase in
particle size may partially be related to the viscosity of
the samples, as viscosity is a key factor affecting the
ability to create a fine dispersion. At higher lipid
contents, the efficiency of homogenization decreases
because of a higher viscosity of the sample, resulting in
larger particles. Moreover, a high particle concentration at
high lipid contents increases the probability of particle
contact and subsequent aggregation [24]. The LD 90%
values of MLX SLNs of different lipids at a constant
surfactant concentration (0.5% w/w) are shown in Fig. 1.
For both surfactants used, Compritol showed the largest
particle sizes, followed by Precirol and then Geleol.
These differences in sizes may be due to differences in
the chain lengths and viscosities of the lipids used [25].
Compritol 888 ATO (m.p. 69.0–74.01C) is a solid lipid
based on glycerol esters of behenic acid (C22), in which
the main fatty acid is behenic acid (485%) but other fatty
acids (C16–C20) are also present. Precirol ATO5 (m.p.
50.0–60.01C) and Geleol (m.p. 54.5–58.41C) are composed mainly of palmitic (C16) and stearic acids (C18)
(490%). A high melting temperature resulting in higher
viscosity and the long hydrocarbon chain length of
Compritol might result in larger particle sizes in
comparison with Precirol and Geleol.
Figure 1
800
700
600
500
400
300
200
100
0
Geleol
Compritol
Precirol
0.5
(b)
7.5
Lipid conc.(%)
10.0
800
700
Particle size (nm)
sufficient to cover the surface of nanoparticles effectively
and prevent agglomeration during the homogenization
process [23].
Geleol
Compritol
Precirol
600
500
400
300
z
200
100
0
0.5
7.5
Lipid conc.(%)
10.0
Effect of lipid concentration and type on particle size measured by laser
diffraction 90% of meloxicam solid lipid nanoparticles using (a) Tween
80 and (b) Cremophor RH40.
and dispersion quality. It was noticeable that as the
amount of surfactant increased in the formulation the
z potential became more negative. However, the influence of surfactant type is less pronounced.
Tween 80 and Cremophor RH40 being nonionic surfactants could successfully be used in the production of
relatively stable dispersions. This behavior could be a
result of the strong effect of surfactants in an emulsion
system on the adsorbed layer thickness [26]. Although
nonionic surfactants could not ionize into charged groups
like ionic ones, they still demonstrated an effect on the
z potential. This might be due to molecular polarization
and adsorption of emulsifier molecules onto the charge in
water: they were absorbed onto the emulsifier layer of the
68
Egyptian Pharmaceutical Journal
particle/water interface, and an electric double layer
similar to an ionic layer was formed. Considering the
effect of lipid type and concentration on the z potential
of the produced SLN formulations, the results showed no
direct relationship between the type of lipid used and the
measured z values. In contrast, as the lipid concentration
increased, the z potential was found to become more
negative. Rahman et al. [17] reported the same observation when studying the effect of increasing Compritol
concentrations in the final formulation.
The bulk pH values of the stratum corneum and upper
viable epidermis have been measured to be 4.0–4.5 and
5.0–7.0, respectively [27]. For a topical preparation to be
applied safely onto the skin, its pH should lie within this
range. The pH values of different MLX SLN formulations ranged from 5.26 ± 0.03 to 6.42 ± 0.04 (Table 3)
and hence were in the required range.
Transmission electron microscopy
TEM was used to investigate the morphology of MLXloaded SLNs. It was evident from the TEM images that
the nanoparticles were almost spherical with smooth
morphology, appeared as black dots, and were well
dispersed and separated on the surface (Fig. 2). This
description is in agreement with a previous observation
that the use of chemically heterogeneous lipids in
combination with heterogeneous surfactants favors the
formation of ideally spherical lipid nanoparticles [11].
The figure illustrates the presence of a very thin layer
surrounding the particles, which suggests a drug-enriched
core model. This model can be achieved if during the
lipid solidification process, the drug precipitates first,
which results in a drug-enriched core covered with a lipid
shell that has a lower drug concentration. This drug
distribution within the nanoparticles will have its impact
on the in-vitro drug release profile discussed.
Differential scanning calorimetry analysis
Figure 3 shows the DSC thermograms of pure MLX, bulk
lipids (Geleol, Compritol 888 ATO, and Precirol ATO5),
and MLX-loaded SLNs. Pure MLX showed a sharp
endothermic peak at 259.541C, corresponding to its
melting point, indicating its characteristic crystalline
nature. Bulk Geleol showed a distinctive melting peak at
66.011C, whereas Compritol 888 ATO showed a sharp
peak at 74.221C. The bulk Precirol ATO5 exhibits a sharp
endothermic event, ascribing to melting, around 63.351C,
with a small but well-defined shoulder at 57.371C, which
might be due to melting of the a-polymorphic form [28].
These sharp melting endothermic peaks of bulk lipids
indicate that the starting materials were crystalline. As
observed in Fig. 3, the thermograms of all investigated
SLN systems did not show the melting peak of MLX,
indicating the conversion of crystalline MLX to the
amorphous form, which could be attributed to complete
dissolution of the drug in the molten lipid matrix. The
melting points of Geleol, Compritol 888 ATO, and
Precirol ATO5 in the SLN form were depressed, showing
a slight shift toward the lower temperatures when
compared with the corresponding bulk lipids. This
Figure 2
SLN4
SLN22
SLN10
SLN28
Transmission electron micrographs of meloxicam solid lipid nanoparticles.
SLN16
SLN34
Meloxicam loaded solid lipid nanoparticles Khalil et al. 69
Figure 3
Heat flow (W/g)
(b)
Heat flow (W/g)
(a)
Temperature (oC)
Temperature (oC)
Differential scanning calorimetry thermograms of pure meloxicam (MLX), bulk lipids (Geleol, Compritol, and Precirol), and MLX solid lipid
nanoparticles (SLNs) using. (a) Tween 80 and (b) Cremophor RH40.
melting point depression could be due to the small
particle size (nanometer range), the high specific surface
area, and the presence of a surfactant. In other words, the
depression can be attributed to the Kelvin effect [4].
Kelvin realized that small, isolated particles would melt at
a temperature lower than the melting temperature of
bulk materials. In the same way, the melting enthalpy
values of different lipids in SLN formulations showed
drastic depression compared with those of their bulk
lipids. These lower melting enthalpy values should
suggest a less-ordered lattice arrangement of the lipid
within the nanoparticles compared with those of the bulk
materials [13]. For the less-ordered crystalline or
amorphous state, the melting of the substance requires
less energy compared with the perfectly crystalline
substance, which needs to overcome the lattice force.
Rheological study
The rheological properties of MLX SLNs were presented
by plotting the shear stress (SS) versus the shear rate
(SR) (flow curves) and the viscosity versus the shear rate
(viscosity curves) curves [29,30]. The rheograms of
selected different SLN formulations are shown
in Fig. 4. As shown from the continuous shear rheometry,
SLN dispersions revealed a non-Newtonian flow. The
viscosity of non-Newtonian fluids changes according
to the shear rate, that is, has no constant viscosity [31].
This flow was characterized by the shear-thinning
behavior, in which the viscosity of the SLN dispersions
decreased with an increase in the shear rate. At present,
shear-thinning materials are also considered thixotropic,
because it always takes time, even though limited, to
regroup the microstructural elements [32]. In addition,
the type of lipid affected the viscosity of the final
product. For both surfactants used, Geleol SLNs showed
lower viscosities compared with Precirol and Compritol
SLNs.
In-vitro release studies
To compare the drug release profile from the prepared
SLN formulations, the release efficiency (RE%) after
48 h was used. The data clearly showed that the release of
the drug from the investigated SLN formulations can
be influenced by the type and concentration of the
surfactant, in addition to the nature and concentration of
lipid matrix used. Some formulations of Tween 80 and
Cremophor RH40 SLNs were selected, representing
those of highest and lowest surfactant and lipid
concentrations. The selected formulations of Tween 80
SLNs were SLN1, SLN4, SLN6, SLN7, SLN10, SLN12,
SLN13, SLN16, and SLN18, whereas those of Cremophor RH40 SLNs were SLN19, SLN22, SLN24, SLN25,
SLN28, SLN30, SLN31, SLN34, and SLN36. The
percentage of MLX released during B48 h ranged from
70 Egyptian Pharmaceutical Journal
Figure 4
Viscosity (cP)
upward curve
downward curve
100
SLN4
SR (sec-1)
80
60
40
50
upward curve
40
downward curve
30
20
10
20
0
0
0
50
100
150
200
0
50
SS (dyne/cm2)
SR (sec-1)
SLN10
Viscosity (cP)
upward curve
downward curve
100
80
60
40
20
60
upward curve
50
downward curve
40
30
20
0
100
0
300
200
400
0
50
SS (dyne/cm2)
Viscosity (cP)
SR (sec-1)
80
60
40
20
0
0
50
100
150
200
70
60
50
40
30
20
10
0
downward curve
0
250
50
Viscosity (cP)
SR (sec-1)
50
80
60
40
0
upward curve
downward curve
40
30
20
0
200
0
400
0
600
50
SS (dyne/cm2)
120
Viscosity (cP)
SR (sec-1)
60
80
60
40
150
upward curve
downward curve
50
40
30
20
10
20
0
0
100
0
200
300
0
50
SS (dyne/cm2)
120
50
upward curve
downward curve
100
100
150
SR (sec-1)
Viscosity (cP)
SR (sec-1)
100
SR (sec-1)
upward curve
downward curve
100
SLN34
150
10
20
SLN28
100
SR (sec-1)
upward curve
downward curve
100
150
upward curve
SS (dyne/cm2)
120
100
SR (sec-1)
upward curve
downward curve
100
SLN22
150
10
0
SLN16
100
SR (sec-1)
80
60
40
20
0
upward curve
downward curve
40
30
20
10
0
0
50
100
150
200
0
SS (dyne/cm2)
Rheograms of meloxicam solid lipid nanoparticles (SLNs). SR, shear rate; SS, shear stress.
50
100
SR (sec-1)
150
Meloxicam loaded solid lipid nanoparticles Khalil et al.
29.42 (SLN18) to 76.61% (SLN4) in case of Tween 80
SLNs and from 29.33 (SLN31) to 72.72% (SLN28) in
case of Cremophor RH40 SLNs (Fig. 5). Interestingly,
the amount of surfactant used had a great influence on
the release pattern of SLNs. Increasing the surfactant
concentration from 0.5 to 5% (w/w) led to an increase in
the percentage of MLX released and the RE% (Po0.05)
(Fig. 5 and Table 4).
The fast or rapid release and higher release efficiency
observed at higher surfactant concentrations could be
explained by the partitioning of the drug between the
melted lipid phase and aqueous surfactant phase during
particle production. During particle production by the
hot homogenization technique, the drug partitions from
Figure 5
% MLX released
(a)
100
90
80
70
60
50
40
30
20
10
0
SLN1
SLN4
SLN6
SLN7
SLN10
SLN12
SLN13
SLN16
SLN18
0
10
20
30
40
50
60
Times (h)
% MLX released
(b)
SLN19
SLN22
SLN24
SLN25
SLN28
SLN30
SLN31
SLN34
SLN36
100
90
80
70
60
50
40
30
20
10
0
0
10
20
30
Times (h)
40
50
60
The release profile of meloxicam (MLX) from solid lipid nanoparticles
(SLNs) using (a) Tween 80 and (b) Cremophor RH40 as surfactants.
71
the liquid oil phase to the aqueous water phase. The
amount of drug partitioning to the water phase will
increase with the increase of drug solubility in the water
phase as a result of increasing the temperature of
the aqueous phase and surfactant concentration. Higher
the temperature and surfactant concentrations, greater
is the solubility of the drug in the water phase. During
cooling of the produced O/W nanoemulsion, the solubility
of the drug in the water phase decreases continuously
with decrease in the temperature of the water phase,
which implies a repartitioning of the drug into the lipid
phase. When reaching the recrystallization temperature
of the lipid, a solid lipid core starts forming, including the
drug that is present at this temperature in this lipid
phase. Reducing the temperature of the dispersion
further increases the pressure on the drug because of
its reduced solubility in water to further repartition into
the lipid phase. The already crystallized core is not
accessible anymore for the drug; consequently, the drug
concentrates in the still liquid outer shell of the SLN
and/or on the surface of the particles. The amount of drug
in the outer shell is released relatively rapidly, whereas
the drug incorporated into the particle core is released
gradually [33].
As regards the type of lipid matrix, the results clearly
showed that among the glycerides used, the highest
release was achieved with Geleol when compared with
Compritol and Precirol. Being the lipid of highest
monoglyceride content, Geleol showed the highest
release efficiency and consequently lower t50%. In case
of Compritol and Precirol, the relatively slow release and
higher t50% can be attributed to the hydrophobic long
chain fatty acids of the triglycerides that retain the
lipophilic drug, resulting in a more sustained release [23,34]. This effect was evident in Tween 80
SLN formulations, whereas in case of Cremophor RH40
SLNs the difference between the three lipids was less
pronounced (Fig. 5 and Table 4).
33.74 ± 2.50 33.10
50.18 ± 1.70 23.28
37.08 ± 4.31 44.17
34.97 ± 0.89 39.39
48.00 ± 0.88 20.65
24.67 ± 1.52 68.74
20.78 ± 1.51 117.54
48.95 ± 3.49 24.03
23.35 ± 3.23 45.501
The results also indicate the effect of lipid concentration
on SLNs’ release profile: increasing the lipid concentration from 5 to 10% (w/w) resulted in a corresponding
decrease in the percentage of MLX released and a
consequent increase in t50% for Tween 80 and Cremophor
RH40 SLNs (Fig. 5 and Table 4). However, in case of
Geleol SLNs (SLN1, SLN6, SLN19, and SLN24), a
slight increase in RE% was observed (Table 4). This
observed decrease in the release profile can be attributed
to the higher lipid content encapsulating the drug, thus
reducing drug partition in the outer phase and consequently its release in the receiver media. The release
profiles of these SLNs resemble the drug-enriched core
model [35]. In such a model, the drug-enriched core is
surrounded by a practically drug-free lipid shell. Because
of the increased diffusional distance and hindering effects
by the surrounding solid lipid shell, the drug has a
sustained release profile.
RE, release efficiency; SLN, solid lipid nanoparticle; t50% (h), time
required to release 50% of the drug.
a
See Tables 1 and 2 for the description of the formulations.
The release pattern of the drug from all SLN formulations followed the Higuchi’s equation. The R2 values
Table 4 Release efficiency and t50% (h) of the selected
meloxicam solid lipid nanoparticles formulations
Surfactants
Tween 80
Formulasa
SLN1
SLN4
SLN6
SLN7
SLN10
SLN12
SLN13
SLN16
SLN18
RE 48 (%)
Cremophor RH40
t50% (h) Formulaa
32.42 ± 1.28 33.72
55.55 ± 1.70 18.73
38.93 ± 4.37 43.46
26.62 ± 0.72 56.19
39.98 ± 1.82 25.12
20.58 ± 1.98 92.44
29.05 ± 1.77
47.14
51.08 ± 0.77 20.14
21.58 ± 2.57 132.75
SLN19
SLN22
SLN24
SLN25
SLN28
SLN30
SLN31
SLN34
SLN36
RE 48 (%)
t50% (h)
72 Egyptian Pharmaceutical Journal
ranged from 0.9151 to 0.9977 in case of Tween 80 and
from 0.9115 to 0.9984 in case of Cremophor RH40. This
result is generally in agreement with many studies that
reported that drug-loaded SLNs provide a controlled release
pattern following Higuchi’s square root model [36,37].
Conclusion
In this study, the MLX-loaded SLNs were successfully
prepared using modified high-shear homogenization and
ultrasound techniques. Physicochemical characterization
revealed that the prepared drug-loaded SLNs were of
spherical shape and homogenously distributed. The DSC
analysis showed the amorphous state of MLX in SLNs.
SLNs achieved high drug incorporation with small-sized
particles (nanosize) and showed shear-thinning rheological behavior. The in-vitro release behavior was greatly
affected and can be controlled by optimizing the
compositional variables. The sustained release behavior
of MLX-loaded SLNs together with the favorable
physicochemical characteristics supports that SLNs are
promising delivery systems for poorly water-soluble drugs
such as MLX and can form a foundation for further
clinical studies for the topical delivery of MLX.
Acknowledgements
Conflicts of interest
There are no conflicts of interest.
12 Venkateswarlu V, Manjunath K. Preparation, characterization and in vitro
release kinetics of clozapine solid lipid nanoparticles. J Controll Rel 2004;
95:627–638.
13 Hou D, Xie C, Huang K, Zhu C. The production and characteristics of solid
lipid nanoparticles (SLNs). Biomaterials 2003; 24:1781–1785.
14 Teeranachaideekul V, Souto EB, Junyaprasert VB, Müller RH. Cetyl palmitate-based NLC for topical delivery of coenzyme Q10 – development, physicochemical characterization and in vitro release studies. Eur J Pharmaceut
Biopharmaceut 2007; 67:141–148.
15 Li Y, Dong L, Jia A, Chang X, Xue H. Preparation and characterization of solid
lipid nanoparticles loaded traditional Chinese medicine. Int J Biol Macromol
2006; 38 (3–5):296–299.
16 Yang SC, Lu LF, Cai Y, Zhu JB, Liang BW, Yang CZ. Body distribution in mice
of intravenously injected camptothecin solid lipid nanoparticles and targeting
effect on brain. J Controll Rel 1999; 59:299–307.
17 Rahman Z, Zidan AS, Khan MA. Non-destructive methods of characterization
of risperidone solid lipid nanoparticles. Eur J Pharmaceut Biopharmaceut
2010; 76:127–137.
18 Lv Q, Yu A, Xi Y, Li H, Song Z, Cui J, et al. Development and evaluation of
penciclovir-loaded solid lipid nanoparticles for topical delivery. Int J Pharm
2009; 372 (1–2):191–198.
19 Jenning V, Gohla SH. Encapsulation of retinoids in solid lipid nanoparticles
(SLN). J Microencapsul 2001; 18:149–158.
20 Shah KA, Date AA, Joshi MD, Patravale VB. Solid lipid nanoparticles (SLN)
of tretinoin: potential in topical delivery. Int J Pharm 2007; 345 (1–2):
163–171.
21 Yang Y-Y, Chung T-S, Bai X-L, Chan W-K. Effect of preparation conditions on
morphology and release profiles of biodegradable polymeric microspheres
containing protein fabricated by double-emulsion method. Chem Eng Sci
2000; 55:2223–2236.
22 Liu J, Gong T, Wang C, Zhong Z, Zhang Z. Solid lipid nanoparticles
loaded with insulin by sodium cholate-phosphatidylcholine-based mixed
micelles: preparation and characterization. Int J Pharm 2007; 340
(1–2):153–162.
23 Kumar VV, Chandrasekar D, Ramakrishna S, Kishan V, Rao YM, Diwan PV.
Development and evaluation of nitrendipine loaded solid lipid nanoparticles:
Influence of wax and glyceride lipids on plasma pharmacokinetics. Int J
Pharm 2007; 335 (1–2):167–175.
24 Freitas C, Müller RH. Effect of light and temperature on zeta potential and
physical stability in solid lipid nanoparticle (SLN) dispersions. Int J Pharm
1998; 168:221–229.
25 Ahlin P, Kristl J, Šmid-Korbar J. Optimization of procedure parameters and
physical stability of solid lipid nanoparticles in dispersions. Acta Pharm
1998; 48:259–267.
References
1 Sarathchandiran I. A review on nanotechnology in solid lipid nanoparticles.
Int J Pharm Develop Technol 2012; 2:45–61.
2 Fang J-Y, Fang C-L, Liu C-H, Su Y-H. Lipid nanoparticles as vehicles for
topical psoralen delivery: solid lipid nanoparticles (SLN) versus nanostructured lipid carriers (NLC). Eur J Pharmaceut Biopharmaceut 2008;
70:633–640.
3 Mei Z, Chen H, Weng T, Yang Y, Yang X. Solid lipid nanoparticle and
microemulsion for topical delivery of triptolide. Eur J Pharmaceut Biopharmaceut 2003; 56:189–196.
4 Jenning V, Gysler A, Schäfer-Korting M, Gohla SH. Vitamin A loaded solid
lipid nanoparticles for topical use: occlusive properties and drug targeting to
the upper skin. Eur J Pharmaceut Biopharmaceut 2000; 49:211–218.
5 Sharma A, Jindal M, Aggarwal G, Jain S. Development of a novel method for
fabrication of solid lipid nanoparticles: using high shear homogenization and
ultrasonication. Res J Pharm Biol Chem Sci 2010; 1:265–274.
6 Chen H, Chang X, Du D, Liu W, Liu J, Weng T, et al. Podophyllotoxin-loaded
solid lipid nanoparticles for epidermal targeting. J Controll Release 2006;
110:296–306.
7 Sivaramakrishnan R, Nakamura C, Mehnert W, Korting HC, Kramer KD,
Schäfer-Korting M. Glucocorticoid entrapment into lipid carriers – characterisation by parelectric spectroscopy and influence on dermal uptake.
J Controll Release 2004; 97:493–502.
8 Engelhardt G. Pharmacology of meloxicam, a new non-steroidal antiinflammatory drug with an improved safety profile through preferential inhibition of COX-2. Br J Rheumatol 1996; 35 (Suppl 1):4–12.
9 Kaplan-Machlis B, Klostermeyer BS. The cyclooxygenase-2 inhibitors: safety
and effectiveness. Ann Pharmacother 1999; 33:979–988.
10 Pairet M, Van Ryn J, Schierok H, Mauz A, Trummlitz G, Engelhardt G.
Differential inhibition of cyclooxygenases-1 and -2 by meloxicam and its
40 -isomer. Inflamm Res 1998; 47:270–276.
11 Mehnert W, Mäder K. Solid lipid nanoparticles: production, characterization
and applications. Adv Drug Deliv Rev 2001; 47 (2–3):165–196.
26 Liu F, Yang J, Huang L, Liu D. New cationic lipid formulations for gene
transfer. Pharm Res 1996; 13:1856–1860.
27 Plasencia I, Norlén L, Bagatolli LA. Direct visualization of lipid domains in
human skin stratum corneum’s lipid membranes: effect of pH and
temperature. Biophys J 2007; 93:3142–3155.
28 Araújo J, Gonzalez-Mira E, Egea MA, Garcia ML, Souto EB. Optimization
and physicochemical characterization of a triamcinolone acetonide-loaded
NLC for ocular antiangiogenic applications. Int J Pharm 2010; 393
(1–2):167–175.
29 Illing A, Unruh T. Investigation on the flow behavior of dispersions of solid
triglyceride nanoparticles. Int J Pharm 2004; 284 (1–2):123–131.
30 Liu W, Hu M, Liu W, Xue C, Xu H, Yang X. Investigation of the carbopol gel of
solid lipid nanoparticles for the transdermal iontophoretic delivery of triamcinolone acetonide acetate. Int J Pharm 2008; 364:135–141.
31 Barnes HA. Thixotropy – a review. J Non-Newtonian Fluid Mech 1997;
70 (1–2):1–33.
32 Lee CH, Moturi V, Lee Y. Thixotropic property in pharmaceutical formulations.
J Controll Rel 2009; 136:88–98.
33 Zur Mühlen A, Mehnert W. Drug release and release mechanism of
prednisolone loaded solid lipid nanoparticles. Pharmazie 1998; 53:
552–555.
34 Harivardhan Reddy L, Murthy RSR. Etoposide-loaded nanoparticles made
from glyceride lipids: formulation, characterization, in vitro drug release, and
stability evaluation. AAPS PharmSciTech 2005; 6:E158–E166.
35 Wissing SA, Kayser O, Müller RH. Solid lipid nanoparticles for parenteral
drug delivery. Adv Drug Deliv Rev 2004; 56:1257–1272.
36 Tiyaboonchai W, Tungpradit W, Plianbangchang P. Formulation and characterization of curcuminoids loaded solid lipid nanoparticles. Int J Pharm
2007; 337 (1–2):299–306.
37 Vivek K, Reddy H, Murthy RSR. Investigations of the effect of the lipid
matrix on drug entrapment, in vitro release, and physical stability of
olanzapine-loaded solid lipid nanoparticles. AAPS PharmSciTech 2007;
8:E1–E9.
Original article 73
Effect of pollution on the chemical content and secondary
metabolites of Zygophyllum coccineum and Tamarix nilotica
Hanan E. Osmana and Reham K. Badawyb
a
Department of Plant and Microbiology, Faculty
of Science (Girls Branch), Al-Azhar University and
b
Environmental Pollution Unit, Department of Plant
Ecology and Range Management, Desert Research
Center, Cairo, Egypt
Correspondence to Hanan E. Osman, PhD,
Department of Plant and Microbiology, Faculty
of Science (Girls Branch), Al-Azhar University,
Nasr City 11651, Cairo, Egypt
Tel: + 20 22633998; fax: + 20 22633101;
e-mail: [email protected]
Received 4 November 2012
Accepted 14 February 2013
Egyptian Pharmaceutical Journal
2013,12:73–82
Objectives
This study investigated the uptake and translocation pattern of trace metals from two
medicinal plant species namely: Zygophyllum coccineum and Tamarix nilotica from two
contaminated sites and a noncontaminated (NC) site. The effects of heavy metals on
the amino acids and secondary metabolites of the tested plant species were assessed.
Materials and methods
Medicinal plant samples and soil samples were collected from three different sites: two
contaminated and one NC site. The concentration levels (mg/kg) of the selected trace
metals (Al, B, Cr, Cu, Fe, Mn, Mo, Pb, V, and Zn) were estimated in the tested plant
species and associated soil.
Results
Heavy metal contents in the investigated plant species reflected the metal
concentration in the soil samples. The highest content of the determined heavy metals
were detected in both tested plants from contaminated sites in comparison with those
from the NC site.
The concentrations of free amino acids in T. nilotica and Z. coccineum plants from the
contaminated sites were higher compared with those in plants from the NC site. Moreover,
the concentration of free amino acids in plants from the wastewater-contaminated sites
was higher compared with that in plants from the Suez industrial emission site.
The content of secondary metabolites (tannins, saponins, and alkaloids) was
decreased in plants from polluted sites compared with those from the NC site. The
concentration of tannins ranged from 0.07 to 0.33 g, saponins from 9.99 to 8.22%,
and alkaloids from 7.95 to 1.00%. Moreover, the maximum tannins and alkaloid content
was detected in Z. coccineum from the noncontaminated site.
Conclusion
The plants collected from the investigated sites pose a serious danger. However, a
periodical assessment of plants used for traditional medicine should be encouraged as
this will assist in ensuring their quality and safety in herbal use, especially for people
living in urban areas where the level of pollution may be very high.
Keywords:
free amino acid, heavy metals, medicinal plant, secondary metabolites, Tamarix nilotica,
Zygophyllum coccineum
Egypt Pharm J 12:73–82
& 2013 Division of Pharmaceutical and Drug Industries Research,
National Research Centre
1687-4315
Introduction
Medicinal plants are widely used as home remedies and
raw materials for pharmaceutical industries. The past
decade has seen a significant increase in the use of herbal
medicine. The environmental conditions in developing
countries; pollution in irrigation water, atmosphere, and
soil; sterilization methods; and storage conditions all play
an important role in the contamination of medicinal
plants by pesticides and heavy metals. The sources of
environmental pollution with toxic metals are quite
varied, ranging from industrial and traffic emissions to
the use of purification mud and agricultural expedients,
such as cadmium-containing dung, organic mercury
fungicides, and the insecticide lead arsenate [1].
Heavy metal contamination in agricultural environments
can result from an atmospheric fallout, pesticide for-
mulations, contamination by chemical fertilizers, and
irrigation with water of poor quality [2]. Heavy metals
rank high among the chief contaminants of leafy
vegetables and medicinal plants [3].
Uptake of trace elements by plants varies and depends
largely on several factors such as soil pH and organic matter
content. Plant uptake is one of the major routes of exposure
of the food chain to trace elements in the soil [4].
Trace elements play an important role in the chemical,
biological, metabolic, and enzymatic reactions in the
living cells of plants, animals, and human beings [5].
However, the release of trace metals through human
activities into the environment has increased over the
years, and the excess of these metals in the environment
has been reported to be extremely dangerous to human
health [6]. The accumulation of trace metals by plants is
1687-4315 & 2013 Division of Pharmaceutical and Drug Industries Research,
National Research Centre
DOI: 10.7123/01.EPJ.0000428268.89779.59
74 Egyptian Pharmaceutical Journal
one of the most serious environmental concerns. This is
as a result of the harmful effects of toxic metals on animal
and human health [7].
Evidence of severe poisoning caused by some metal
compounds and the proven carcinogenicity of some metal
ions has fostered intensive research into the different
uptake and translocation patterns in food crops [8]. The
broad use of traditional medicines by rural communities
because of the accessibility and affordability of herbal
medicine has also necessitated a further research into the
uptake and translocation pattern of trace metals by some
medicinal plants from urban areas [3].
Zygophyllum coccineum belongs to the Zygophyllaceae
family. The leaves, stems, and fruits of this plant are
used in folk medicine as a drug active against rheumatism, gout, asthma, and hypertension. It is also used as a
diuretic, local anesthetic, antihistaminic, and antidiabetic
agent [9].
Several species of plants belonging to the genus Tamarix
(Family: Tamaricaceae) have been used in traditional
medicine. Antioxidant and antimicrobial activities of T.
hispida [10] and T. aphyla [11] have also been described.
Tamaricaceous plants produce a unique class of hydrolysable tannins with diverse structures [12].
The environmental conditions, atmosphere, pollution,
soil, and harvesting and handling are some of the factors
that may play important roles in the contamination of
medicinal plants by metals and microbial growth [3]. It is
therefore of major interest to evaluate the composition
of some metallic elements in herbal plants, because at
elevated levels, these metals can be dangerous and
toxic [13,14].
Although some trace metals may have both curative and
preventive roles in combating diseases, it has been
established that an overdose or prolonged ingestion of
medicinal plants may lead to chronic accumulation of
different elements that may cause various health
problems [15].
The overall objectives of this research were to determine
the concentrations of the 10 tested heavy metals in
Tamarix nilotica and Z. coccineum plant biomass from
contaminated and noncontaminated (NC) sites and to
determine the effect of heavy metal contamination from
industrial emissions or by wastewater irrigation on the
content of secondary metabolites and amino acids of both
tested plant species.
Materials and methods
Site description
This study was carried out at three sites: two contaminated and one NC. The NC site was located at Sokhna
Road, 35 km from Cairo governorate.
The first contaminated site is a wastewater-contaminated
(WWC) site near the domestic wastewater channel. This
site is located at El-Saff, Cairo governorate, which is
south of the industrial complex of Helwan (including the
Iron and Steel Factory and Weaving, Coke, and fertilizer
industries). These industrial activities produce large
amount of wastes that are usually dumped into an
artificial canal extending over a large area behind the
factories. The source of irrigation in this site is the
sewage effluent, which comes from the sewage treatment
station at Helwan since the past 23 years (according to
the report of the committee preparing the Egyptian code
for reuse of wastewater, 2004). The second contaminated
site, the Suez industrial emission (SIE) contaminated
site (SEC), is located near the fertilizer and ceramic
factories in Ain Sokhna, Suez governorate. The fertilizer
plant of the Egyptian Fertilizers Company (EFC)
manufactures granulated urea.
Soil and plant sampling
During June 2009, Z. coccineum and T. nilotica plant
samples, based on their coverage at the site, together
with the associated soil samples were collected. The
tested medicinal plants were collected from their natural
habitats. The plants were not exposed to any agricultural
treatments. Five random samples were collected from
each site to obtain a comprehensive profile of the site for
statistical analysis.
The soil samples were collected from a depth of 0–60 cm.
The collection of plant samples was based on plant
coverage at the site and plant health.
Soil and plant analysis
Soil samples were air dried at room temperature and then
sieved using a 2-mm stainless steel sieve. The soil : water
extracts (1 : 2.5) were prepared and used in the
determination of pH, electrical conductivity, and cationic
and anionic compositions according to the methods
described by Richards [16] and by Jackson [17]. The
total carbonates were determined according to the
methods described by Piper [18]. The organic matter
was determined according to the method described by
Nelson and Sommers [19]. The available nitrogen in
the soil was extracted using a solution of 2 mol/l KCl
according to the method described by Keeney and
Nelson [20]. The available phosphorus was extracted
using a solution of 0.5 mol/l NaHCO3, pH 8.5, according
to the method described by Watanabe and Olsen [21].
The soil samples were analyzed for the total content of
the studied elements in the filtered soil extracts obtained
from samples digested by HNO3, H2SO4, and 60%
HClO4, as outlined by Hesse [22]. Total tested heavy
metals were determined by inductively coupled plasma
optical emission spectrometry (ICP).
The plant samples were washed with distilled water to
remove any adhering soil. After washing, the plant
samples were oven dried at 651C and then ground to a
powder. The plant samples were digested with H2O2 and
H2SO4 [23] and then subjected to analysis of nitrogen
and phosphorus. The nitrogen content was determined
using a modified Micro-Kjeldahl method, as described by
Peach and Tracey [24]. The phosphorous content was
Pollution effect on two wild plants Osman and Badawy 75
determined according to the method described by
Rowell [25]; this method depends on the formation of a
blue complex between phosphate and ammonium molybdate in the presence of ascorbic acid (reducing agent). The
samples were measured with a spectrophotometer at an
absorbance of 880 nm. The plant samples were analyzed
for the total content of the studied elements using the
digested extracts, which were obtained with 0.5 g of
concentrated HNO3 and H2O2 [26]. The heavy metal
content in all the samples was determined by aspirating
directly to ICP. The alkaloid content was determined
according to the method described by Jenkins et al. [27].
The saponin content was determined according to the
method described by Wall et al. [28]. The tannin content
was determined according to the method described by
Claus [29]. The free amino acid content was determined
according to the method described by Block et al. [30].
Metal translocation factor
The root-to-shoot translocation factor (TF) was described as the ratio of heavy metals in the plant shoot
to that in the plant root [31]. The TF is determined
according to the equation: BF = C [HM in shoot]/C [HM
in root].
Statistical analysis
The experiment was laid out in a randomized complete
block design with three replications. There were two
factors in the study: three sites (NC, WWC, and SEC)
and two types of plant species (Z. coccineum and T. nilotica).
Data were subjected to analyses using M-STATC., as
described by Russell [32]. The mean values were
compared using the Duncan New Multiple range test
as described by Waller and Duncan [33]. Mean values
indicated by the same alphabetical letters in the same
column are not significantly different at P = 0.05.
The data on the TF, alkaloid content, tannin content,
and saponin content of the samples were presented as
mean ± SD of the three replicates and were analyzed
using Excel 2007 for Windows.
Results and discussion
Soil properties and heavy metal concentrations
Chemical properties of the soil from the three tested sites
are presented in Table 1. The data shows that salinity of
the saturated extract from the soil, as evidenced by the EC
values, was very high in soil from the WWC site
(11.28 mMho). The values of soil pH ranged from 8.83 in
the soil from the WWC site to 8.71 in that from the
industrial emission site, indicating that the soils are alkaline
in these locations. The soil from the NC site was slightly
alkaline with a pH of 7.97. Schipper et al. [34] reported that
after long-term wastewater irrigation, the soil pH increased
and that this may be due to the high content of cations
such as Na, Ca, and Mg in the wastewater.
The organic matter content was high in the soil from the
contaminated sites; it was 1.24% at the WWC site and
0.69% at the SIE site compared with 0.43% at the NC
site. The cationic composition of the total salts is mostly
dominated by Na + , followed by Ca2 + and Mg2 + , and
then by K + . The most dominant anion was SO24 – ,
followed by Cl – , and then by HCO3. The highest OM,
Ca2 + , Mg2 + , Na2 + , K + , Cl – , and SO4– concentrations
were detected in the WWC sample, whereas the highest
HCO3 content was detected in the SIE sample.
Accumulation of K in the soil with wastewater application
was attributed to the original content of this nutrient in
the wastewater applied [35]. Irrigation with wastewater
increased the total cation concentration of Ca and
Mg [36].
As shown in Table 2, the available N and P content in the
soil samples from the contaminated sites is significantly
higher compared with those from the NC site as a result
of contamination with wastewater at the WWC site and
Table 1 Electrical conductivity (EC), pH, concentration organic matter content (OM) and some anions and cations (mEq/l) in the
studied soil samples from the noncontaminated (NC) site, El-Saff wastewater-contaminated (WWC) site, and Suez industrial
emission (SIE) site
Cations (mEq/l)
Sites
NC
WWC
SIE
Anions (mEq/l)
EC (mMho)
pH
OM (%)
Ca2 +
Mg2 +
Na +
K+
CO23 –
HCO3–
Cl –
SO24 –
1.206
11.28
0.468
7.97
8.83
8.71
0.43
1.24
0.69
5.5
32
3
3
22
2.5
3.65
140.50
9.90
1.01
2.05
0.75
0
0
0
0.8
0.8
1.2
3.125
86.25
1.25
9.235
109.5
13.7
Table 2 Interaction effects of the site and plant species on nitrogen, phosphorus, and heavy metal contents (mg/kg) of the studied
soil samples from the noncontaminated (NC) site, El-Saff wastewater-contaminated (WWC) site, and Suez industrial emission (SIE)
site
Site
NC
Plant
T. nilotica
Z. coccineum
WWC T. nilotica
Z. coccineum
SIE
T. nilotica
Z. coccineum
N
P
Al
B
Cr
Cu
Fe
Mn
24.4 d
21.0 d
65.1 c
194.5 b
228.9 a
34.9 d
4.6 b
2.7 b
5.5 b
4.9 b
18.8 a
5.0 b
449.4 d
368.0 d
3221.0 b
3493.1 a
2306.6 c
2303.3 c
51.1 d
42.8 d
111.7 b
128.7 a
94.4 c
103.3 bc
23.0 d
17.3 d
54.8 a
49.2 ab
36.8 c
41.6 bc
32.4 d
35.8 d
117.9 a
112.3 a
96.2 b
83.9 c
163.4 c
119.5 c
1533.5 a
1215.1 b
1264.3 ab
1510.7 a
22.8 c
30.4 c
131.0 ab
127.6 ab
116.6 b
141.3 a
Mean values for each column having common letters are not significantly different at the 0.05 level.
Mo
1.4
1.6
4.1
5.0
6.1
5.3
d
d
c
b
a
b
Pb
V
Zn
21.7 b
21.7 b
123.3 a
121.9 a
113.9 a
115.6 a
3.3 b
3.9 b
16.3 a
15.1 a
12.6 a
14.3 a
25.5 b
25.3 b
122.5 a
124.6 a
135.2 a
125.8 a
76 Egyptian Pharmaceutical Journal
with fertilizer factory effluent at the SIE site. These
elements are essential nutrients for plant growth.
Figure 1
Heavy metal contents of the three sites are represented
in Table 2. The total heavy metal contents were increased
significantly many folds in the samples from the
contaminated sites compared with those from the NC
site. Heavy metal concentrations of the contaminated
sites were increased by 8.21, 2.56, 2.58, 3.38, 9.72, 4.86,
3.14, 5.65, 4.36, and 4.86 times at the WWC site, whereas
they were increased by 5.64, 2.11, 1.95, 2.64, 9.81, 4.84,
3.94, 5.29, 3.74, and 5.14 times at the SIE site for Al, B,
Cr, Cu, Fe, Mn, Mo, Pb, V, and Zn, respectively compared
with the NC site.
The results show a great variability in the heavy metal
content according to site of plant collection. The
maximum concentrations of Al, B, Cr, Cu, Mn, Pb, and
V were found at the WWC site: significantly for Al, B, Cr,
and Cu and nonsignificantly for Mn, Pb, and V. Meanwhile, the maximum but not significant concentrations of
Fe, Mo, and Zn were detected in plants from the SIE site.
Soils, especially those found in or near the metalliferous sites
and metal smelters, are highly contaminated with heavy
metals, including Cd, Cr, Cu, Pb, Ni, and Zn. For example,
soils sampled from a former Zn/Cd smelter site contained up
to 99 500 mg/kg Zn in addition to 1005–7220 mg/kg Pb,
2500–4500 mg/kg Cu, and 28–578 mg/kg Cd [37].
Effect of plant species on root heavy metal content (mg/kg) of Tamarix
nilotica and Zygophyllum coccineum. Values followed by different
letters within columns are significantly different at the 0.05 probability
level.
Figure 2
Heavy metal concentrations in plants
Metal concentrations in plants vary with plant species [38]. Plant uptake of heavy metals from soil occurs
either passively with the mass flow of water into the roots
or through active transport across the plasma membrane of
root epidermal cells. Under normal growing conditions,
plants can potentially accumulate certain metal ions an order
of magnitude greater than the surrounding medium [39].
The plant species has a considering effect on the heavy
metal content in both roots and shoots of T. nilotica and Z.
coccineum plants. The contents of Al, B, and Fe in T. nilotica
roots and those of Al, B, Cr, Cu, Fe, Pb, and Zn in T.
nilotica shoots were significantly higher compared with
those in Z. coccineum roots and shoots, respectively.
Meanwhile, the contents of Cu, Mn, and Zn in Z.
coccineum roots were higher compared with those in
T. nilotica roots (Figs 1 and 2). The contents of B, Cr,
Mo, and V and Mn, Mo, and Zn in roots and shoots,
respectively for both plants were the same.
The effect of the site on the heavy metal concentrations
in both T. nilotica and Z. coccineum plants are depicted
in Figs 3 and 4. The results showed that, in most cases,
the concentrations of the tested heavy metals in plants
from the WWC site were significantly higher compared
with those in plants from the SIE site. The increase in Al,
B, Cr, Cu, Fe, Mn, Mo, Pb, V, and Zn concentrations was
7.74, 3.10, 4.36, 3.81, 4.17, 7.42, 4.22, 9.30, 6.10, and 5.30fold, respectively in plant shoots from the WWC site and
was 6.57, 1.96, 3.39, 2.73, 3.91, 5.35, 6.31, 7.35, 5.55, and
4.39-fold, respectively in plants from the SIE site
compared with that in plants from the NC site.
Effect of plant species on shoot heavy metal content (mg/kg) of Tamarix
nilotica and Zygophyllum coccineum. Values followed by different
letters within columns are significantly different at the 0.05 probability
level.
Figure 3
Effect of the site on shoot heavy metal content (mg/kg) of Tamarix
nilotica and Zygophyllum coccineum. Values followed by different
letters within columns are significantly different at the 0.05 probability
level.
Pollution effect on two wild plants Osman and Badawy 77
On comparing the two contaminated sites, mostly there
was a significant increase in the determined heavy metal
content in plants from the WWC site compared with
plants from the SIE site (Figs 3 and 4).
The data in Table 3 shows the interaction effect of the
plant species and site on the tested heavy metal contents
for T. nilotica and Z. coccineum. The high heavy metal
contents for both roots and shoots, mostly, were detected
in plants from the WWC site.
The content of heavy metals in industrialized regions were
determined by Januz et al. [40], who reported that the plants
growing in an industrialized region have higher contents of
heavy metals compared with plants growing in a second less
industrialized region. Some metals such as Cu, Mn, and
Zn are the natural essential components of enzymes and
coenzymes and are important for growth, photosynthesis,
and respiration, although other metals such as Pb and Cd
have no biochemical or physiological importance, therefore
they are considered as very toxic pollutants.
Figure 4
Effect of the site on root heavy metal contents (mg/kg) of Tamarix
nilotica and Zygophyllum coccineum. Values followed by different
letters within columns are significantly different at the 0.05 probability
level.
Although the concentrations of the tested heavy metals
in soils at contaminated sites were above the critical
concentrations in soil for these elements [41], no visual
phytotoxicity symptoms on both tested plants were
observed.
The Al, Cr, Cu, Fe, Mn, Mo, and Pb concentrations were
all above the normal range for roots and shoots of both
tested plants from the contaminated sites, whereas the
concentrations of B and Zn were within the permissible
level (Table 3).
The variation in the elemental content from plant to
plant is mainly attributed to the differences in the
botanical structure and mineral composition of the soil in
which the plants are cultivated. Other factors responsible
for a variation in the elemental content are preferential
absorbability of the plant, use of fertilizers, irrigation
water, and climatic conditions [38].
Translocation factor of heavy metals
A plant’s ability to translocate metals from the roots to
shoots is measured using the TF, which is defined as the
ratio of metal concentration in the shoots to that in the
roots. The TF index showed that the both tested plant
species most efficiently translocated the tested heavy
metals to the shoot system. The mean TF (average TF
values for each metal in different sites for both tested
plants) values revealed that T. nilotica showed great
efficiency for translocating metals from the roots to
shoots. The TF values for T. nilotica for all tested metals
under study were higher than 1, except for B and V
(Figs 5 and 6). The trends of the TF values for heavy
metals in T. nilotica were in the order of Cr4Cu4Mo4
Fe4Pb4Zn4Mn4Al4V4B. Meanwhile, Z. coccineum
had a TF higher than 1 for Cr, Cu, Pb, and V. The results
in Figs 5 and 6 show that TF of Z. coccineum for these
considered
metals
were
in
the
order
of
Cr4Cu4Pb4V4Zn4Fe = Mo4Al = B4Mn. A TF
higher than 1 indicated a very efficient ability to transport
metals from the roots to shoots, most likely due to
efficient metal transport systems [43].
Table 3 Interaction effect of the site and different plant species on heavy metal contents (mg/kg) in roots and shoots of T. nilotica
and Z. coccineum plants from the noncontaminated (NC) site, El-Saff wastewater-contaminated (WWC) site, and Suez industrial
emission (SIE) site
Site
Root
NC
WWC
SIE
Shoot
NC
WWC
SIE
PL
Plant
Al
B
Cr
Cu
T. nilotica
Z. coccineum
T. nilotica
Z. coccineum
T. nilotica
Z. coccineum
84.83 d
104.00 d
644.20 a
545.10 b
438.80 c
402.20 c
24.37 b
24.12 b
67.26 a
44.28 ab
43.75 ab
33.78 b
1.29
1.66
6.02
5.19
5.65
5.01
T. nilotica
Z. coccineum
T. nilotica
Z. coccineum
T. nilotica
Z. coccineum
77.80 e
58.92 e
570.00 a
488.20 c
530.5 b
368.30 d
135a
15.49 d
19.11 d
66.18 a
41.04 b
31.79 c
35.97 bc
14–78a
1.47 c
1.77 c
8.70 a
5.42 b
5.73 b
5.24 b
5b
c
c
a
b
ab
b
Fe
Mn
19.68 c
23.84 c
58.88 b
74.65 a
55.55 b
68.31 b
113.6
161.1
564.8
494.4
556.2
502.9
d
c
a
b
a
b
20.38 e
16.00 e
79.72 a
58.79 c
63.88 b
35.52 d
1.1–33.1a
131.6 d
139.30 d
655.8 a
474.50 c
559.5 b
498.90 c
450b
Mo
Pb
V
Zn
8.32 d
11.7 d
82.97 a
85.28 a
41.08 c
52.32 b
0.33 d
0.37 d
1.84 b
1.39 c
1.7 b
2.45 a
6.92 c
6.88 c
82.24 a
86.38 a
81.25 a
60.54 b
0.75 d
1.47 d
8.49 a
7.59 b
6.62 c
6.55 c
21.64
32.25
58.23
62.21
56.55
69.80
d
c
b
ab
b
a
10.71 d
8.17 d
69.28 a
67.54 a
43.27 c
55.30 b
44.25b
0.45 e
0.26 f
1.37 d
1.67 c
2.39 a
2.15 b
Up to 1a
8.55 d
9.04 d
88.89 a
74.84 b
71.84 b
57.43 c
0.3–18.8a
0.74 e
1.62 d
7.60 a
6.71 bc
6.92 ab
6.55 c
–
11.39 d
11.79 d
67.84 a
55.05 b
53.88 b
47.98 c
6–126a
PL, permissible limits according to Kabata Pendias & Pendias [41]a and FAO/WHO [42]b standards for metal concentrations in consumable
vegetables and edible parts.
Mean values for each column having common letters are not significantly different at the 0.05 level.
78 Egyptian Pharmaceutical Journal
Figure 5
Translocation factors with SDs of Al, B, Cr, Cu, Fe, Mn, Mo, Pb, V, and
Zn in Tamarix nilotica from the noncontaminated (NC) site, El-Saff
wastewater-contaminated (WWC) site, and Suez industrial emission
(SIE) site. Error bars represent ± SE of the mean values for three
separate plant extractions.
The mean TF for the tested heavy metals ranged from 0.62
to 1.21 and 0.83 to 1.21 for T. nilotica and Z. coccineum,
respectively. According to Baker [44], there are three basic
types of tolerance strategies to heavy metals (accumulation,
exclusion, and indication), which describe the relationship
between the total soil and plant metal concentration and
that excluder and accumulator plants could grow together
in the same environment. The relationships between the
soil and plant metal concentrations should be thoroughly
tested for each plant species separately to understand the
physiological mechanisms.
Accumulation and exclusion are two basic strategies by
which plants respond to elevated concentrations of heavy
metals [45]. In metal accumulator species, TFs greater
than 1 were common, whereas in metal excluder species
the TFs were typically lower than 1 [44].
Nitrogen and phosphorus content in plants
Figure 6
Nitrogen (N) is the essential mineral element required
in the greatest amount by plants, comprising 1.5–2% of
plant dry matter [46]. Phosphorus (P) is the second
nutritional element after nitrogen that limits plant growth,
having a concentration of about 0.2% of the total plant dry
weight [47]. P is a macronutrient that is a key component
in many molecules (i.e. nucleic acids, phospholipids, and
ATP) that participates in basic plant processes [48].
The concentration of nitrogen and phosphorus were
significantly higher in tested plants from the contaminated sites compared with those from the NC site. The
highest content of N was detected in plants from the
WWC site, whereas the highest P content was detected
in plants from the SIE site (Fig. 7).
Translocation factors with SDs of Al, B, Cr, Cu, Fe, Mn, Mo, Pb, V, and
Zn in Zygophyllum coccineum plants from the noncontaminated (NC)
site, El-Saff wastewater-contaminated (WWC) site, and Suez industrial
emission (SIE) site. Error bars represent ± SE of the mean values for
three separate plant extractions.
Amino acid contents
Under heavy metals stress, plants exhibit a number of
physiological changes in their cells [49,50]. Several
mechanisms allow plants to tolerate the presence of
Figure 7
Interaction effect of the site [noncontaminated (NC) site, El-Saff wastewater-contaminated (WWC) site, and Suez industrial emission (SIE) site] and
plant species (Tamarix nilotica and Zygophyllum coccineum) on nitrogen and phosphorus contents (ppm) in plants. Values followed by different
letters within columns are significantly different at the 0.05 probability level.
Pollution effect on two wild plants Osman and Badawy 79
Table 4 Mean free amino acid (FAA) contents of Tamarix nilotica and Zygophyllum coccineum from the noncontaminated (NC) site,
El-Saff wastewater-contaminated (WWC) site, and Suez industrial emission (SIE) site
NC
FAA (%)
Aspartic
Glutamic
Histidine
Arginine
Lysine
Threonine
Serine
Proline
Glycine
Alanine
Valine
Methionine
Isoleucine
Leucine
Tyrosine
Phenylalanine
Acidic
Alkali
Neutral
WWC
SIE
T. nilotica
Z. coccineum
T. nilotica
Z. coccineum
T. nilotica
Z. coccineum
0.3109
0.3549
0.1642
0.1828
0.1658
0.1224
0.1446
0.4923
0.1314
0.1796
0.1193
0.0005
0.0947
0.1860
0.0543
0.1192
0.3637
0.4244
0.2343
0.2866
0.2139
0.1481
0.1756
0.4405
0.1756
0.1914
0.1455
0.0026
0.1145
0.2191
0.0601
0.1400
0.4752
0.6011
0.2725
0.3877
0.2912
0.2433
0.2816
0.6181
0.2669
0.2899
0.2327
0.0005
0.1336
0.3622
0.1538
0.2240
0.6929
0.9076
0.3422
0.3321
0.4333
0.3333
0.3608
0.8159
0.2898
0.4014
0.3260
0.0210
0.2393
0.4993
0.1623
0.3264
0.3893
0.4433
0.2005
0.4278
0.2768
0.1931
0.1926
0.5669
0.2017
0.2510
0.2070
0.0147
0.1453
0.3056
0.0868
0.1488
0.6369
0.6742
0.2515
0.2527
0.2422
0.1568
0.2127
0.6279
0.2086
0.2735
0.1451
0.0014
0.1125
0.2357
0.0794
0.1596
Table 5 Correlation coefficients between the contents of free amino acids and heavy metals in shoots of Tamarix nilotica
Amino acid
Al
B
Cr
Cu
Fe
Mn
Mo
Pb
V
Zn
Aspartic
Threonine
Serine
Glutamic
Proline
Glycine
Alanine
Valine
Methionine
Isoleucine
Leucine
Tyrosine
Histidine
Lysine
Arginine
0.888
0.938
0.813
0.818
– 0.958
0.909
0.960
0.989*
0.436
0.957
0.969
0.797
0.767
0.802
0.955
0.984
0.955
0.999*
0.999*
– 0.586
0.975
0.932
0.873
– 0.202
0.582
0.918
1.00**
0.999*
1.00**
0.577
0.992*
1.00**
0.963
0.965
– 0.803
0.997*
0.998*
0.980
0.103
0.800
0.995*
0.955
0.940
0.958
0.797
0.958
0.986
0.907
0.911
– 0.887
0.971
0.996*
0.999*
0.260
0.885
0.998*
0.896
0.873
0.900
0.883
0.930
0.968
0.667
0.872
– 0.924
0.947
0.984
0.999*
0.343
0.923
0.989*
0.854
0.827
0.959
0.920
0.996*
0.999*
0.972
0.975
– 0.779
0.999*
0.995*
0.971
0.064
0.776
0.990*
0.966
0.952
0.968
0.773
0.449
0.556
0.315
0.325
– 0.944
0.491
0.613
0.716
0.881
0.945
0.641
0.290
0.243
0.299
0.947
0.940
0.975
0.881
0.886
– 0.913
0.956
0.988*
1.00**
0.315
0.911
0.993*
0.869
0.844
0.873
0.908
0.896
0.944
0.823
0.828
– 0.953
0.916
0.965
0.992
0.420
0.951
0.974
0.807
0.778
0.813
0.950
0.952
0.983
0.898
0.903
– 0.897
0.966
0.993*
1.00**
0.280
0.895
0.997*
0.886
0.863
0.891
0.892
*Correlation is significant at the level 0.05.
**Correlation is significant at the level 0.01.
Table 6 Correlation coefficients between the contents of free amino acids and heavy metals in shoots of Zygophyllum coccineum
Amino acid
Al
B
Cr
Cu
Fe
Mn
Mo
Pb
V
Zn
Aspartic
Threonine
Serine
Glutamic
Proline
Glycine
Alanine
Valine
Methionine
Isoleucine
Leucine
Tyrosine
Histidine
Lysine
Arginine
0.993
0.744
0.835
0.974
0.969
0.883
0.931
0.714
0.677
0.706
0.752
0.828
0.779
0.811
0.993
0.998*
0.709
0.806
0.961
0.955
0.858
0.911
0.677
0.638
0.669
0.717
0.799
0.746
0.780
0.985
0.993
0.572
0.687
0.896
0.887
0.753
0.822
0.536
0.491
0.526
0.581
0.679
0.616
0.657
0.940
0.915
0.908
0.960
0.998*
0.999*
0.982
0.997*
0.889
0.864
0.884
0.913
0.957
0.930
0.948
0.984
0.976
0.483
0.608
0.845
0.834
0.680
0.759
0.445
0.397
0.434
0.493
0.598
0.530
0.574
0.899
0.999*
0.693
0.792
0.954
0.948
0.846
0.901
0.661
0.620
0.652
0.701
0.785
0.731
0.766
0.981
0.919
0.313
0.450
0.731
0.717
0.532
0.625
0.271
0.220
0.260
0.324
0.440
0.364
0.413
0.802
0.995
0.732
0.825
0.969
0.964
0.874
0.924
0.701
0.663
0.693
0.739
0.818
0.768
0.801
0.991
0.998*
0.615
0.725
0.918
0.910
0.786
0.851
0.580
0.536
0.570
0.623
0.717
0.656
0.696
0.956
1.00**
0.658
0.762
0.939
0.932
0.820
0.880
0.625
0.583
0.615
0.666
0.755
0.698
0.735
0.971
*Correlation is significant at the level 0.05.
**Correlation is significant at the level 0.01.
heavy metals inside the cells, and synthesis of phytochelatins has been particularly concerned, as phytochelatins may chelate heavy metals, leading to detoxification
of these metals in cells [51]. The interaction of heavy
metals with sulfhydryl-containing amino acids and pep-
tides/proteins plays a major role in their environmental
and biochemical behavior [52].
Sixteen types of amino acids were detected in the shoots
of the tested plant species from the three sites (NC,
80
Egyptian Pharmaceutical Journal
WWC, and SIE) (Table 4). Amino acids are divided into
three types (i.e. acidic, alkali, and neutral) on the basis of
their characters [53].
The concentrations of amino acids in T. nilotica and Z.
coccineum plants from the contaminated sites were higher
compared with those in plants from the NC site. The
most abundant amino acid in all the plant tissues was
glutamic acid. Moreover, the concentration of amino acids
in plants from the domestic wastewater site was higher
compared with that in plants from the SIE site for both
tested plants. These results are in agreement with those
of Wu et al. [54] and of Kováčik et al. [55].
On computing correlation coefficients it was revealed that
levels of aspartic acid and threonine in shoots of T. nilotica
were significantly positively correlated with their respective
Cr and Mn concentrations (Table 5). As regards the levels
of serine, glutamic acid, tyrosine, histidine, and lysine,
only boron (B) showed a positive relationship. In case of
levels of proline, methionine, isoleucine, and arginine, no
correlations were detected. Levels of valine, alanine, and
leucine were positively and significantly correlated with
more than one metal. Concentrations of Al, Cu, Fe, Pb, and
Zn; Cr, Cu, Fe, Mn, Pb, and Zn; and Cr, Cu, Mn, Pb, and
Zn, respectively were correlated with levels of valine,
leucine, and alanine, respectively.
In Z. coccineum, a significant positive correlation was
detected between levels of aspartic acid and concentration of B, Mn, V, and Zn in the shoot, whereas levels of
glutamic, proline, and alanine correlated with shoot
concentrations of Cu (Table 6).
In most agricultural soils, nitrate (NO3– ) is the most
important source of N for plants [56]. For nitrogen
metabolism, the nitrate must be taken up across the
plasma membrane. Once inside the symplast of a plant,
Figure 8
Content of secondary metabolites (alkaloids, saponins, and tannins) and fat (%) of Tamarix nilotica and Zygophyllum coccineum plants from the
noncontaminated (NC) site, El-Saff wastewater-contaminated (WWC) site, and Suez industrial emission (SIE) site. Mean values for each column
having common letters are not significantly different at the 0.05 level.
Pollution effect on two wild plants Osman and Badawy 81
NO3– is reduced to NO2– by nitrate reductase (NR), and
NO2– is converted to NH4-N by nitrite reductase. The
resulting NH4-N is then assimilated into amino acids,
nucleic acids, proteins, chlorophylls, and other metabolites [57]. Factors influencing the enzymatic regulation
responsible for N assimilation include: contents of
Mo [58] and Cu [59].
The content of amino acids in shoots of T. nilotica and Z.
coccineum plants from the three tested sites were in the
order of WWC4SIE4NC, in line with the nitrogen and
phosphorus concentrations in plants. The amino acid
content (acidic, alkali, and neutral amino acids) showed
an increase in plants from the WWC site compared with
those from the other sites, which may be due to an
elevation of nitrogen, phosphorus, Mo, and Cu concentrations in shoots of the plants (Table 3).
Cruz et al. [60] reported that activities of nitrogen
metabolism-related enzymes such as nitrate reductase are
considerably lower in a low nitrate supply compared with
a high supply of nitrates.
Mo, one of the essential microelements for plant growth
and the metal component of the Mo cofactor, is
responsible for the catalytic activity of NR, aldehyde
oxidase, xanthine dehydrogenase, and sulfite oxidase. Mo
promotes N accumulation and utilization in wheat plants,
which is directly related to nitrate reductase. A higher Mo
status also results in higher accumulation and utilization
of plant N [58]. Cu exposure results in increase in the
concentration of free amino acids [59]. It can be observed
that there is superiority of Z. coccineum plants in terms of
amino acid content compared with T. nilotica; this may be
due to the higher content of shoot Mo in Z. coccineum
compared with T. nilotica and a genetic variation between
the two plants.
Effect of heavy metals on secondary metabolites
Phytochemicals are divided into two main groups
according to their function in the plant body: primary
and secondary constituents. The primary constituents are
sugars, amino acids, proteins, and chlorophyll and the
secondary constituents consist of alkaloids, terpenoids,
saponins, flavonoids, tannins, and phenolic compounds [61].
The content of secondary metabolites (tannins, saponins,
and alkaloids) and fat were lower in plants from the
polluted sites compared with those from the NC site.
The tannin content ranged from 0.07 to 0.33 g, saponin
from 9.99 to 8.22%, and alkaloids from 7.95 to 1.00%.
Moreover, the maximum tannin and alkaloid contents
were detected in Z. coccineum from the NC site (Fig. 8).
Heavy metal-induced changes in the phenolic compounds may further affect their functions in plant cells.
Phenolic compounds, including tannins, are often involved in responses to different kinds of abiotic and biotic
stresses [62].
Cobbett and Goldsbrough [63] hypothesized that secondary metabolism may be an integral part of the plant’s
capacity to modify metabolic processes to survive and
grow in adverse conditions, including in the presence of
phytotoxic metals.
Individual plant species differ in their capacity to modify
their metabolism to tolerate or accumulate heavy metals.
The modifications may involve sequestration of the
metals in vacuoles, biosynthesis of organic compounds
that detoxify these metals, or synthesis of modified
tissues to exclude the contaminant [64]. These processes
often alter the uptake and distribution of other metal
ions, as was seen in the present study with altered heavy
metal concentrations in both tested plant tissues. A
consequence of this modified metabolism may include
the loss of specific enzymes or nonessential biomolecular
synthetic processes such as secondary metabolite biosynthesis.
Conclusion
These results prove that industrial pollutants and their
metal contamination can change the chemical composition of the soil and its properties, which reflects on some
medicinal plants, thereby, seriously impacting the quality,
safety, and efficacy of natural plant products produced by
medicinal plant species. The plants from polluted areas
cannot be used as herbal medicine. It is also important to
implement good quality control practices for screening
of herbal medicines to protect consumers from toxicity.
The data presented in this study provide the evidence
of the detrimental effects of naturally occurring or
industrially generated metal contamination in T. nilotica
and Z. coccineum.
The plants collected from the investigated sites pose a
serious danger; however, a periodical assessment of plants
used for traditional medicine should be encouraged as
this will assist in ensuring their quality and safety in
herbal use, especially for people living in urban areas
where the level of pollution may be very high.
Amino acids are well-known biostimulants that have
positive effects on plant growth and yield. The higher
content of amino acids in the studied plant species from
the contaminated sites led us suggest extraction of amino
acid and their usage as foliar sprays for different plant
species (agricultural uses), especially plants of Z. coccineum
that have a short life cycle. Further studies are warranted
to extract these amino acids and to ensure the safety and
heavy metal-free status of these amino acids for their use.
Acknowledgements
Conflicts of interest
There are no conflicts of interest.
References
1 Schilcher H, Peters H, Wank H. Pestiszide and Schmermretalle in Arzneipmanzen and Arzneiplanzen Zubereitun-gen. Pharm Ind 1987; 49:202–211.
2 Marcovecchio JE, Botté SE, Freije RH. Heavy metals, major metals, trace
elements. In: Nollet LML, editor. Handbook of water analysis. 2nd ed. Boca
Raton: CRC Press; 2007. pp. 275–311.
82 Egyptian Pharmaceutical Journal
3 Ajasa AMO, Bello MO, Ibrahim AO, Ogunwande IA, Olawore NO. Heavy
trace metals and macronutrients status in herbal plants of Nigeria. Food
Chem 2004; 85:67–71.
36 Zhang LP, Mehta SK, Liu ZP, Yang ZM. Copper-induced proline synthesis is
associated with nitric oxide generation in Chlamydomonas reinhardtii. Plant
Cell Physiol 2008; 49:411–419.
4 Logan TJ, Goins LE, Lindsay BJ. Field assessment of trace element uptake by
six vegetables from N-Viro Soil. Water Environ Res 1997; 69:28–33.
37 Reeves RD, Schwartz C, Morel JL, Edmondson J. Distribution and metalaccumulating behavior of Thlaspi caerulescens and associated metallophytes in France. Int J Phytoremediation 2001; 3:145–172.
5 Hashmi DS, Ismail S, Shaikh GH. Assessment of the level of trace uptake by
six vegetables from N-Viro soil. Water Environ Res 2007; 69:28–33.
6 Olowoyo JO, van Heerden E, Fischer JL, Baker C. Trace metals in soil and
leaves of Jacaranda mimosifolia in Tshwane area, South Africa. Atmos
Environ 2010; 44:1826–1830.
7 Olowoyo JO, Okedeyi OO, Mkolo NM, Lion GN, Mdakane STR. Uptake and
translocation of heavy metals by medicinal plants growing around a waste
dump site in Pretoria, South Africa. S Afr J Bot 2012; 78:116–121.
8 Vélez D, Devesa V, Súñer MA, Montoro R. Metal contamination in food. In:
Nollet LML, editor. Handbook of food analysis. 2nd ed. New York: Marcel
Dekker; 2004. pp. 1485–1511.
9 Rimbau V, Cerdan C, Vila R, Iglesias J. Antiinflammatory activity of some
extracts from plants used in the traditional medicine of North-African
countries (II). Phytother Res 1999; 13:128–132.
10 Saı̈dana D, Mahjoub MA, Boussaada O, Chriaa J, Chéraif I, Daami M, et al.
Chemical composition and antimicrobial activity of volatile compounds of
Tamarix boveana (Tamaricaceae). Microbiol Res 2008; 163:445–455.
11 Abdel-Mogib M, Basaif SA, Al-Garni SM. Antimicrobial activity and chemical
constituents of leaf extracts of Tamarix aphylla. Alex J Pharm Sci 2001;
15:121–123.
38 Alloway BJ. Toxic metals in soil-plant systems. Chichester, UK: John Wiley
and Sons; 1994.
39 Kim IS, Kang KH, Johnson-Green P, Lee EJ. Investigation of heavy metal
accumulation in Polygonum thunbergii for phytoextraction. Environ Pollut
2003; 126:235–243.
40 Januz IM, Danutra W, Jerzy K, Robart R, Krysztof L, Jerzy C. The occurrence
of Pb, Cd, Cu, Mn, Ni, Co and Cr in selected species of medicinal plants in
Poland. Bromatol Toksykol 1994; 28:363–368.
41 Kabata-Pendias A, Pendias H. Trace elements in soils and plants. Boca
Raton, FL: CRC Press Inc.; 1992.
42 FAO/WHO. Joint Expert Committee on Food Additives, Summary and
Conclusions, 3rd Meeting, Rome, 1–10 June 1999.
43 Zhao F-J, Hamon RE, Lombi E, McLaughlin MJ, McGrath SP. Characteristics
of cadmium uptake in two contrasting ecotypes of the hyperaccumulator
Thlaspi caerulescens. J Exp Bot 2002; 53:535–543.
44 Baker AJM. Accumulators and excluder-strategies in the response of plants
to heavy metals. J Plant Nutr 1981; 3:643–646.
12 Quideau S. Chemistry and biology of ellagitannins: an underestimated class
of bioactive plant polyphenols. Singapore: World Scientific Publishing; 2009.
45 Vogel-Mikuš K, Drobne D, Regvar M. Zn, Cd and Pb accumulation and
arbuscular mycorrhizal colonisation of pennycress Thlaspi praecox Wulf.
(Brassicaceae) from the vicinity of a lead mine and smelter in Slovenia.
Environ Pollut 2005; 133:233–242.
13 Somers E. The toxic potential of trace metals in foods. A review. J Food Sci
1983; 39:215–217.
46 Frink CR, Waggoner PE, Ausubel JH. Nitrogen fertilizer: retrospect and
prospect. Proc Natl Acad Sci USA 1999; 96:1175–1180.
14 Schuhmacher M, Bosque MA, Domingo JL, Corbella J. Dietary intake of lead
and cadmium from foods in Tarragona Province, Spain. Bull Environ Contam
Toxicol 1991; 46:320–328.
47 Raghothama KG. Phosphate acquisition. Annu Rev Plant Biol 1999;
50:665–693.
15 Jabeen S, Shah MT, xKhan S, Hayat MQ. Determination of major and trace
elements in ten important folk therapeutic plants of Haripur basin, Pakistan.
J Med Plants Res 2010; 4:559–566.
16 Richards LA. Diagnosis and improvement of saline and alkali soils. U.S.
Department of Agriculture Handbook. No. 60. Washington DC; 1954.
17 Jackson ML. Soil chemical analysis. UK: Constable and Comp. Ltd; 1963.
p. 963.
18 Piper CS. Soil and plant analysis. New York: Inter Science Inc; 1950.
19 Nelson DW, Sommers LE. Total carbon, organic carbon, and organic matter.
In: Page AL, editor. Methods of soil analysis, Part II, 2nd ed., agronomy. 9
Madison, WI: Am Soc Agron; 1982. pp. 539–580.
20 Keeney DR, Nelson DW. Methods of soil analysis. Parts 2. In: Page AL, et al,
editor. Nitrogen inorganic forms. Madison, WI: Am Soc Agron; 1982.
pp. 643–698.
21 Watanabe FS, Olsen SR. Test of an ascorbic acid method for determination
in water and NaHCO3 extracts from soil. Soil Sci Soc Am J 1956; 33:
226–230.
22 Hesse PR. A textbook in soil chemical analysis. London: William Glowe; 1971.
23 Nicholson G. Methods of soil, plant and water analysis. N Z Forest Service.
FRI Bulletin No. 70: 1984.
24 Peach K, Tracey MV. Modern method of plant analysis. Vol. 1. Berlin:
Springer-Verlag; 1956. p. 4.
25 Rowell DL. Soil science: methods and applications. New York: Department
of Soil Science, University of Reading. Co-published in the US with John
Wiley and Sons Inc.; 1993.
26 Norvell WA. Comparison of chelating agents as extractants for metals in
diverse soil materials. Soil Sci Am J 1984; 48:1285–1292.
27 Jenkins GL, Christina JE, Hager GP. Quantitative pharmaceutical chemistry.
5th ed. New York, London: McGraw-Hill Book Co. Inc.; 1957.
28 Wall ME, Krider MM, Krewson CF, Eddy CR, Willaman JJ, Corell DS, Gentry
HS. Steroidal sapogenins. VII. Survey of plants for steroidal sapogenins and
other constituents. J Am Pharm Assoc 1954; 43:1–7.
29 Claus ER. Pharmacognosy. 5th ed. London: Herny Kimpton Co. Inc.; 1967.
30 Block RJ, Dorrum EL, Zweeg B. Annual paper chromatography and paper
electrophoresis. 2nd ed. New York: Academic Press Inc. Publishers; 1998.
31 Yanqun Z, Yuan L, Jianjun C, Haiyan C, Li Q, Schvartz C. Hyper accumulation
of Pb, Zn and Cd in herbaceous grown on lead-zinc mining area in
Yunnan, China. Environ Int 2005; 31:755–762.
32 Russell DF. MSTATC. USA: Directory Crop Soil Science Department,
Michigan University; 1991.
48 Schachtman DP, Reid RJ, Ayling SM. Phosphorus uptake by plants: from soil
to cell. Plant Physiol 1998; 116:447–453.
49 Malik D, Sheoran IS, Singh R. Carbon metabolism in leaves of cadmiumtreated wheat seedlings. Plant Physiol Biochem 1992; 30:223–229.
50 Moya JL, Ros R, Picazo I. Influence of cadmium and nickel on growth, net
photosynthesis and carbohydrate distribution in rice plants. Photosynth Res
1993; 36:75–80.
51 Rauser WE. Phytochelatins and related peptides. Structure, biosynthesis,
and function. Plant Physiol 1995; 109:1141–1149.
52 Dı́az-Cruz MS, Mendieta J, Monjonell A, Tauler R, Esteban M. Study of the
zinc-binding properties of glutathione by differential pulse polarography and
multivariate curve resolution. J Inorg Biochem 1998; 70:91–98.
53 Chang E-H, Zhang S-F, Wang Z-Q, Wang X-M, Yang J-C. Effect of
nitrogen and phosphorus on the amino acids in root exudates and
grains of rice during grain filling. Acta Agronomica Sinica 2008; 34:
612–618.
54 Wu F-B, Chen F, Wei K, Zhang G-P. Effect of cadmium on free amino acid,
glutathione and ascorbic acid concentrations in two barley genotypes
(Hordeum vulgare L.) differing in cadmium tolerance. Chemosphere 2004;
57:447–454.
55 Kováčik J, Klejdus B, Hedbavny J. Effect of aluminium uptake on physiology,
phenols and amino acids in Matricaria chamomilla plants. J Hazard Mater
2010; 178 (1–3):949–955.
56 Hirsch RE, Sussman MR. Improving nutrient capture from soil by the genetic
manipulation of crop plants. Trends Biotechnol 1999; 17:356–361.
57 Stitt M, Müller C, Matt P, Gibon Y, Carillo P, Morcuende R, et al. Steps
towards an integrated view of nitrogen metabolism. J Exp Bot 2002;
53:959–970.
58 Yu M, Hu C-x, Sun X-c, Wang Y-h. Influences of Mo on nitrate reductase,
glutamine synthetase and nitrogen accumulation and utilization in Moefficient and Mo-inefficient winter wheat cultivars. Agric Sci China 2010;
9:355–361.
59 Mazen AMA. Accumulation of four metals in tissues of Corchorus olitorius
and possible mechanisms of their tolerance. Biologia Plantarum 2004;
48:267–272.
60 Cruz JL, Mosquim PR, Pelacani CR, Araújo WL, DaMatta FM. Effects of
nitrate nutrition on nitrogen metabolism in cassava. Biologia Plantarum
2004; 48:67–72.
61 Krishnaiah DR, Sarbatly U, Bono A. Photochemical and antioxidants for
health and medicine. A move toward nature. Mol Boil Rev 2007; 1:97.
33 Waller RA, Duncan DB. A Bayes rule for the segmmetric multiple comparison problem. J Am Stat Assoc 1969; 64:1485–1502.
62 Rivero RM, Ruiz JM, Garcı́a PC, López-Lefebre LR, Sánchez E,
Romero L. Resistance to cold and heat stress: accumulation of phenolic
compounds in tomato and watermelon plants. Plant Sci 2001; 160:
315–321.
34 Schipper LA, Williamson JC, Kettles HA, Speir TW. Impact of land-applied
tertiary-treated effluent on soil biochemical properties. J Environ Qual 1996;
25:1073–1077.
63 Cobbett C, Goldsbrough P. Phytochelatins and metallothioneins: roles in
heavy metal detoxification and homeostasis. Annu Rev Plant Biol 2002;
53:159–182.
35 Monnett GT, Reneau RB Jr, Hagedorn C. Evaluation of spray irrigation for onsite wastewater treatment and disposal on marginal soils. Water Environ Res
1996; 68:11–18.
64 Boyd RS, Davis MA. Metal tolerance and accumulation ability of the Ni
hyperaccumulator Streptanthus polygaloides Gray (Brassicaceae). Int J
Phytoremediation 2001; 3:353–367.
Original article 83
Optimization of growth conditions and continuous production
of inulinase using immobilized Aspergillus niger cells
Nagwa A. Atwaa and Enas N. Daniala,b
a
Department of Chemistry of Natural and Microbial
Products, Division of Pharmaceutical Industries,
National Research Centre, Cairo, Egypt and
b
Department of Biochemistry, Faculty of Girls Science,
King Abdulaziz University Jeddah, Saudi Arabia
Correspondence to Nagwa A. Atwa, PhD, Department
of Chemistry of Natural and Microbial Products,
Division of Pharmaceutical Industries, National
Research Centre, El-Behoos St. 33, Dokki, Cairo
12622, Egypt
Tel: + 20 100 522 7200; fax: + 20 234 25490;
e-mail: [email protected]
Received 7 January 2013
Accepted 17 March 2013
Egyptian Pharmaceutical Journal
2013,12:83–89
Aim
The aim of the study was the optimization of growth conditions for the production
of inulinase as well as the continuous production of the enzyme in an airlift bioreactor
using Aspergillus niger cells.
Methods
First, inulinase production by A. niger cells, using different carbon and nitrogen
sources, was studied on a shake flask level. Second, the cells were adsorbed onto
different carriers, and their production over several successive batches was tested.
Finally, the economically-favorable continuous production of inulinase by A. niger cells
immobilized onto linen fibers was carried out in an airlift bioreactor using crude inulin
juice as the fermentation medium.
Results
Although all tested substances resulted in the biosynthesis of certain amounts of
inulinase enzyme, the highest titer of 163.5 U/ml was obtained when the producing
cells were incubated for 96 h at 271C and 180 rpm in a fermentation medium
containing both inulin and peptone as sole carbon and nitrogen sources, respectively.
Moreover, when the cells of the tested microorganism were adsorbed onto different
carriers, especially linen fibers, their productivity was also successfully maintained, to
different extents, for five successive batches. However, as commercially pure inulin is
very expensive and available in only small quantities, the fermentation medium was later
substituted by a crude inulin extract obtained by mechanical crushing and filtration of
Jerusalem artichoke tubers. The crude inulin juice was able to sustain inulinase
production during the second batch cultivation of A. niger cells that were immobilized
by their adsorption onto linen fibers to a satisfactory level of about 122 U/ml.
Furthermore, the use of the previously mentioned crude inulin preparation was also
compared with the use of either complete or minimal media, composed solely of 1%
pure inulin, for the continuous production of inulinase enzyme by A. niger cells that
were immobilized in their maximum production phase and packed inside an external
loop airlift bioreactor. The results of this experiment were very encouraging as, using
this technique, an inulinase production of about 838 U/ml over an incubation period of
48 h was obtained compared with a production of about 996 and 1013 U/ml, which
resulted from the use of either minimized or complete media, respectively, for the same
incubation period.
Conclusion
The method adopted in this study for inulinase production is simple, economic, time
saving, and nontoxic to the microorganism. Moreover, the loaded linen fiber pads are
reusable.
Keywords:
airlift bioreactor, Aspergillus niger, inulin, inulinase
Egypt Pharm J 12:83–89
& 2013 Division of Pharmaceutical and Drug Industries Research,
National Research Centre
1687-4315
Introduction
Inulin is a widespread polyfructan, naturally occurring in
more than 30 000 edible plant species [1]. It consists of
linear chains of b(2,1)-linked fructose residues attached
to a terminal sucrose molecule [2]. Apart from its role as a
food component, inulin has also received great importance as a raw material for the production of fructose
syrup [3,4] and inulooligosaccharides [5]. Fructose is a
safe alternative to sucrose, which is known to be the
cause of many health problems including corpulence,
carcinogenicity, atherosclerosis, and diabetes [2]. In
addition, fructose also increases the absorption of iron,
as it forms an iron–fructose complex whose absorption
was found to be much better than that of inorganic
1687-4315 & 2013 Division of Pharmaceutical and Drug Industries Research,
National Research Centre
DOI: 10.7123/01.EPJ.0000428964.32893.44
84
Egyptian Pharmaceutical Journal
iron [6]. Fructose can be produced from inulin either
enzymatically or chemically through acid hydrolysis.
The latter method is not recommended because of
the undesirable coloring of inulin hydrolysate and the
formation of difructose anhydride, which has practically
no sweetening properties [2]. Moreover, the enzymatic
production of organic products, especially those used in
food and pharmaceutical industries, has many advantages
over chemical processes: The productivity is generally
higher, because of the high specificity of the enzymes for
their substrates; the production cost is relatively lower;
and most importantly it creates less pollution. Therefore,
many efforts have been made to replace chemical
processes with enzymatic ones [7]. Unfortunately, the
conventional enzymatic production of fructose from
inulin involves many steps and results in only a 45%
fructose yield. In contrast, the almost complete hydrolysis
of inulin (90–95%) into fructose can be performed in a
single step using inulinase enzyme [b(2,1)-fructan
fructanohydrolase] [6,8]. Inulinase is produced by many
microorganisms, including filamentous fungi, yeasts, and
bacteria. The fermentation of inulinase by these microorganisms can be greatly improved by modifying some
parameters, including the physiochemical and nutritional
conditions of growth required by the producing cells. In
this study, Jerusalem artichoke (Helianthus tuberosus) was
used as a cheap source of inulin, as about 80% w/w (dry
weight basis) of the tuber acts as a store for carbohydrates [7]. Moreover, in comparison with conventional
fermentations, immobilization of living cells provides
several important advantages such as a faster production
rate, easier purification of products, and a higher
productivity over a certain period of time [9]. One of
the most reliable, safe, and easy methods of immobilization is the adsorption of the producing cells onto an inert,
suitable support [10–12]. Therefore, the present study
was carried out to examine the inulinase productivity of
Aspergillus niger cells under different cultivation conditions
and to study the effect of adsorption immobilization of
these cells onto different carriers on the productivity.
Finally, the continuous production of inulinase by the
producing cells that were immobilized onto linen fibers
and packed inside an external loop airlift bioreactor was
also investigated over a number of successive batches,
using either complete or minimal media as well as crude
inulin juice.
Materials and methods
Microorganism
The production of inulinase was carried out using a locally
isolated strain of A. niger. The microorganism was
maintained on Czapek’s Dox (CD) agar medium [13]
at 301C for 7 days and then stored in the refrigerator
until use.
Authentic enzyme and chemicals
Inulinase (EC.3.2.1.7) was supplied by NOVO Industry
(A/S, Seoul, Korea). Pure inulin and the remaining
chemicals used were obtained from Sigma (St. Louis,
Missouri, USA). All solvents (analytical grade) were
obtained from Merck (Darmstadt, Germany).
Supports tested for cell immobilization
The immobilization of Aspergillus cells, and eventually
their inulinase productivity, was tested by the adsorption
method using three different carriers: glass wool (Pyrex
fiber glass, Sliver 8 mm; Corning Glass Work, Corning,
New York, USA), linen, and synthetic fibers (the latter
two were locally provided).
Preparation of crude inulin solution
Twenty grams of Jerusalem artichoke tubers (H. tuberosus),
collected locally, were washed, sliced, and grinded using a
blender along with 100 ml of distilled water, then filtered
through a fine gauze. The pH of the solution was
adjusted to 6.2 by addition of concentrated sodium
hydroxide. The resulting juice was sterilized at 1211C and
1.5 atmospheric pressure for 15 min [14]. The raw inulin
extract was analyzed, and its inulin content concentration
was estimated, according to the method described by
Ashwell [15], to be B1.5% (w/v).
Recovery and activity assay of inulinase
Inulinase activity was assayed by measuring the amount
of reducing sugars released from inulin [16]. The
fermentation broth was centrifuged at 3000g and 41C
for 5 min. The obtained supernatant was used as the
crude enzyme. A reaction mixture of 0.1 ml of the enzyme
sample and 0.9 ml of acetate buffer (0.1 mol/l, pH 5.0)
containing 2% inulin was incubated at 501C in a water
bath for 15 min. The mixture was then kept at 1001C for
10 min to inactivate the enzyme. The same mixture to
which the same amount of inactivated crude enzyme
(heated at 1001C for 10 min) was added before the
reaction was used as a control. The reaction mixture was
assayed for reducing sugars according to the method of
Nelson–Somogyi cited by Spiro [17]. The calibrating
curve was drawn with fructose (10–100 mg). One unit of
inulinase was defined as the amount of enzyme that
released one micromole of fructose from inulin per min
under assay conditions.
Media and cultivation conditions
Shake flask fermentation of free cells
Unless otherwise mentioned, inulinase production was
carried out in 250 ml Erlenmeyer flasks, each containing
50 ml of basal CD medium [13] comprising (g/l): inulin,
10; NaNO3, 3; K2HPO4, 1; MgSO40.7H2O, 0.5; KCl, 0.5;
and Fe2SO40.7H2O, 0.01 (pH 6.5). The flasks were then
sterilized, inoculated with about 2 109 spores/ml of the
producing microorganism, and incubated for 96 h at
120 rpm and 301C. The effect of various carbon sources,
such as fructose, glucose, maltose, starch, and lactose, was
investigated. Each carbon source was added to the basal
medium (without inulin) at a concentration of 10 g/l
either individually or in combination with inulin, which
was then supplemented at a concentration of either 1 or
5 g/l. Similarly, various organic and inorganic nitrogen
sources were individually added to the basal medium as a
substitute for NaNO3 in order to study their effect on
Optimization of inulinase production Atwa and Danial 85
inulinase production. The tested organic nitrogen sources
(peptone, urea, and yeast, beef, and meat extracts) were
added at a concentration of 50 g/l. The inorganic nitrogen
sources under study (NH4SO4 and NH4Cl) were added
according to their nitrogen content such that it was
equivalent to that of NaNO3, which was omitted from the
medium.
Shake flask fermentation of immobilized cells
The immobilization of A. niger was studied using the
adsorption method [11,12]. A total of 1.4 g of each tested
support (glass wool and synthetic and linen fibers) was
added to a 250 ml flask containing 50 ml of the optimized
medium composed of (g/l): inulin, 10; peptone, 50; K2HPO4,
1; MgSO40.7H2O, 0.5; KCl, 0.5; and Fe2SO40.7H2O, 0.01
(pH 6.5). The flasks were then sterilized, inoculated with
about 2 109 spores/ml of the producing microorganism,
and incubated for 96 h at 120 rpm and 301C. To assess the
productivity of the immobilized cells for another batch, the
loaded pads were washed thoroughly with normal saline,
carefully squeezed under sterile conditions, and used to
inoculate 50 ml of a fresh sterile medium, which was then
reincubated under the former conditions but for a shorter
incubation period of 72 h.
An experiment was carried out as an attempt to reduce
the quantity of the constituents of the fermentation
medium used during repeated batch cultivation of the
cells previously adsorbed onto linen fibers in their
maximum inulinase production phase. This was achieved
to decrease the growth of the escaped cells and to
produce inulinase using the cheapest possible medium.
Therefore, as described previously, A. niger cells were
inoculated in 50 ml of sterile medium along with 1.4 g of
linen fibers in each flask. After 96 h of incubation at 301C
and 120 rpm, the linen fiber pads saturated with the cells
in their maximum production phase were washed
thoroughly with normal saline solution and carefully
squeezed using sterilized forceps. These pads were then
transferred to new flasks containing different ratios of the
constituents of the main medium as shown in Table 1.
Crude inulin, obtained by mechanical crushing and
filtration of Jerusalem artichoke tubers, as previously
explained, was also tested. These flasks were then
reincubated at 301C and 120 rpm for another 72 h. At
the end of this incubation period, inulinase production
and the cell dry weight of unadsorbed cells in each flask
were estimated.
Airlift bioreactor fermentation of immobilized cells
The production of inulinase enzyme by A. niger cells
immobilized onto linen fibers was investigated in an
external loop airlift bioreactor [12], using either complete
or minimized fermentation media as well as raw inulin
juice. A schematic diagram of the designed apparatus is
illustrated in Fig. 1. The bioreactor consists mainly of a
riser column with a height of 40 cm and a downcomer
tube with a diameter of 1 cm. The riser column is
composed of an inner perforated column with an internal
diameter of 3 cm jacketed by an outer column that has an
internal diameter of 4 cm. The inner column was
Table 1 Optimization of the fermentation medium used in the
second batch production of inulinase by Aspergillus niger cells
immobilized onto linen fibers
Inulin (g/l)
Control medium number
1
10
2
10
Second batch medium number
1
10
2
10
3
10
4
10
5
10
6
10
7
10
8
10
9
8
10
5
11
2
12
7.5
13
5
14
2.5
15
10
16
10
Peptone (g/l)
Salt content (%)
50
50
100
100
50
25
5
1
0
50
50
50
50
50
50
37.5
25
12.5
0
0
100
100
100
100
100
50
25
0
100
100
100
75
50
25
0
0
Controls 1 and 2, inulinase production by free cells and by the first
batch of cultivated immobilized cells, respectively.
Figure 1
Schematic diagram of the airlift bioreactor and its accessories used for
the production of inulinase by immobilized Aspergillus niger cells. 1,
riser column; 2, inner perforated column; 3, downcomer tube; 4, oneway valve; 5, air sparger; 6, air inlet; 7, air outlet; 8, medium inlet; 9,
medium outlet; C1–C5, clamps; F1, F2, F3, air filters; P1, P2, peristaltic
pump; P3, air pump; R1, medium feeding reservoir; R2, product
collection reservoir.
designed to hold up the linen fiber pads, on which the
producing cells were previously immobilized, and to
prevent their fluidization. The perforation of the column
allowed the fermentation broth to come in contact with
the immobilized cells in many parts and also helped
achieve a good oxygen transfer to the packed fibers.
86
Egyptian Pharmaceutical Journal
The whole system was mounted inside an incubator
adjusted at 301C.
The inoculum was in the form of eight firmly squeezed
linen pads supporting Aspergillus cells that were previously
immobilized by their cultivation in the optimized
medium for 96 h at 120 rpm and 301C. The linen pads
loaded with the immobilized cells were packed, under
aseptic conditions, inside the inner column of the
bioreactor. A working volume of 360 ml of each tested
medium was fed one at a time. The aeration rate of the
bioreactor was adjusted at 0.5 v/v/m. The fermentation
medium once introduced by a peristaltic pump P1 into
the riser column was left without circulation for 30 h in
contact with the immobilized cells. This phase was
performed to reinitiate the inulinase production of the
immobilized cells. Thereafter, peristaltic pump P2 was
adjusted such that the medium could circulate at a rate of
30 ml/h. During the experiment, 20 ml aliquots of the
culture were systematically withdrawn with a syringe
through an inline air filter. These samples were assayed to
monitor inulinase production and cell escapement.
Results and discussion
Optimization of growth and inulinase production
parameters of free cells on shake flask level
Effect of different incubation periods
The production of inulinase by A. niger cells on inulin
basal CD fermentation medium was monitored over a
period of 120 h under the previously mentioned shaking
cultivation conditions of 120 rpm and 301C. The results
illustrated graphically in Fig. 2 show that the activity of
inulinase was 0.88 U/ml in the fermentation medium after
6 h of incubation. This recorded enzyme activity was
found to increase linearly with time by a production rate
(Qp) of 1.6 U/ml/h and reached a maximum volumetric
production of 139.349 U/ml after about 96 h of incubation. After this incubation period, a gradual decrease in
inulinase activity was observed. The reported production
Figure 2
Effect of different incubation periods on the growth of and inulinase
production by free Aspergillus niger cells cultivated in basal Czapek’s
Dox medium.
Figure 3
Effect of different carbon sources, added either individually or in
addition to 0.1 or 0.5% inulin, on inulinase production by free
Aspergillus niger cells.
decrease rate (– Qp) was about 0.69 U/ml/h. Cell growth
was also studied during the course of fermentation and
was found to increase gradually with time by a specific
growth rate (m) of about 0.35 g/l/h. A maximum cell dry
weight (Xmax) of about 9.98 g% was recorded after 96 h of
incubation. Thereafter, a slight cell lysis was observed
with a specific degradation rate (– m) of about 0.02 g/l/h,
resulting in a cell dry weight of 9.51 g/l after 120 h of
incubation. This result showed that inulinase production
was growth-dependent and that the maximum inulinase
productivity of the producing organism was just before
the onset of its stationary phase of growth. Moreover, a
maximum yield coefficient (units of inulinase per gram of
cell mass formed) of 1396.3 U/g cells was recorded after
96 h of incubation.
Effect of different carbon sources
Different carbon sources were tested for their ability to
sustain substantial amounts of inulinase enzyme production (Fig. 3). Among them, inulin resulted in a maximum
enzyme production of about 140 U/ml, followed by
sucrose, which resulted in B114 U/ml of the enzyme.
Lower enzyme titers ranging between 96 and 80 U/ml
were recorded upon using other carbon sources including
(in descending order of enzyme activity recorded):
fructose, glucose, maltose, starch, and finally lactose.
However, because the use of inulin as a sole carbon source
in the fermentation medium was inconvenient owing to
its high cost, it was therefore added to the medium
containing each individual carbon source, in small
percentages of 0.1 and 0.5%, as an attempt to initiate
higher inulinase production. This goal was achieved as
the addition of inulin in these percentages resulted in
significant increases in enzyme production (results
ranging from 5.6 to 12%). However, none of these
enzyme titers could exceed the level obtained when
inulin was added as a sole carbon source in the
fermentation medium.
Optimization of inulinase production Atwa and Danial 87
Figure 4
Figure 5
Effect of different organic and inorganic nitrogen sources on inulinase
production by free Aspergillus niger cells.
Inulinase production during repeated batch cultivation of Aspergillus
niger cells immobilized onto different support materials. Control,
inulinase production by free cells.
Effect of different nitrogen sources
Different nitrogen sources, either organic or inorganic,
were also tested for inulinase productivity. The results
in Fig. 4 show that a maximum production of about
163.5 U/ml could be achieved when peptone was used as
a sole nitrogen source in the fermentation medium. Much
lower yields ranging between 124 and 93 U/ml were
recorded upon using other organic nitrogen sources
including (in descending order of enzyme productivity):
yeast and beef extracts, urea, and then finally meat
extract. In contrast, the use of inorganic nitrogen sources
such as NaNO3, NH4SO4, and NH4Cl resulted in enzyme titers of approximately 131, 125, and 99.5 U/ml,
respectively.
Optimization of growth and inulinase production
parameters of immobilized cells on shake flask level
Effect of immobilizing Aspergillus niger cells on different
carriers
A. niger cells were tested for their ability to produce
inulinase while immobilized by adsorption onto different
carriers including glass wool and synthetic and linen
fibers. The enzyme production results were compared
with those obtained when free cells of the fungus were
cultivated on the same optimized culture medium under
similar cultivation conditions. The results in Fig. 5
indicate that the cells immobilized onto linen fibers
were only slightly affected by the immobilization process,
as they were able to produce a satisfactory enzyme
concentration of about 131 U/ml compared with 164 U/ml
produced by cultivation of free cells for the same
incubation period of 96 h. Successive batch cultivation
of the immobilized cells was performed to test their
inulinase productivity. This result was very promising
because this previously mentioned inulinase titer was,
more or less, attained after a much shorter incubation
time of 72 h, as the cells were inoculated in their
maximum production phase. The obtained yield of
inulinase could also be more or less sustained within
appropriate ranges for five consecutive batches, resulting
in a total enzyme yield of 630 U/ml within a combined
serial incubation period of 384 h. In contrast, the first
Figure 6
Optimization of the fermentation medium used in the second batch
production of inulinase by Aspergillus niger cells immobilized onto linen
fibers. Controls 1 and 2, inulinase production by free cells and by the
first batch of cultivated immobilized cells, respectively.
batch cultivation of the A. niger cells adsorbed onto either
synthetic fibers or glass wool resulted in lower inulinase
yields of 112 and 103 U/ml, respectively. It was observed
that these titers were maintained, with only a slight
decrease, during the experiment.
Optimization of the fermentation medium used for the second
batch production of inulinase by Aspergillus niger cells
immobilized onto linen fibers
The results illustrated in Fig. 6 show that a maximum
inulinase production of about 162 U/ml was obtained
when free cells were cultivated for 96 h (control 1).
Moreover, the first batch cultivation of immobilized cells
(control 2) resulted in a satisfactory inulinase production
of 134.5 U/ml for the same incubation period. However,
the results showed no significant differences between the
inulinase titers estimated in the fermentation broths of
media no. 1 to 8 (used in the second batch cultivation of
72 h), which ranged between 130.28 and 120.44 U/ml.
88
Egyptian Pharmaceutical Journal
This means that the inulinase productivity of the cells
was more or less maintained in the second batch even
when the peptone or salt content of the medium was
reduced or even eliminated. However, it was found that
the inulin content of the medium was critical for both the
growth of the producing organism and its productivity, as
its reduction, keeping the percentage of the other
constituents constant, affected the inulinase titer and
cell growth greatly (media no. 9 to 11). The critical effect
of inulin on inulinase production was also revealed when
different percentages ranging between 75 and 25% were
used in the media (media no. 12 to 14), as the production
of inulinase decreased to 87.16 and 47.21 U/ml, respectively. However, medium 15, composed of only pure
inulin (10 g/l), and medium 16, composed of crude inulin
solution (15 g/l), resulted in satisfactory inulinase levels
of 126 and 122 U/ml, respectively. Relying on these
results, the complete medium could be substituted by
either medium 15 (minimal medium) or medium 16 (raw
inulin extract) for the production of inulinase during the
repeated batch cultivation of A. niger cells immobilized
onto linen fibers.
Optimization of fermentation medium used for the
continuous production of inulinase by immobilized cells
in an airlift bioreactor
Inulinase production using complete medium
The results illustrated in Fig. 7 show that inulinase
production increased gradually at the rate of 1.56 U/ml/hr
and reached a volumetric production of 102.6 U/ml after
only 48 h of incubation. This maximum inulinase
production level was maintained until 78 h of incubation.
Inulinase production using minimal medium
The experiment was repeated using minimal medium as
previously described. Although this medium was only
composed of 10 g/l pure inulin, lacking any other media
component, the inulinase productivity of the cells, of
about 100 U/ml, was satisfactorily restored after only 24 h
Figure 7
of incubation wherein the recorded productivity rate was
2.54 U/ml/h. This titer was more or less maintained until
the end of the fermentation time (Fig. 7). The obtained
results could be attributed to the fact that immobilized
cells need nutrients that will only maintain their
inulinase productivity on the expense of their growth.
It was also observed that the use of a minimal medium
resulted in a reduction in unwanted growth of escaping
cells, which favors recovery of the produced enzyme.
Inulinase production using crude inulin solution
The experiment was finally performed using the crude
inulin solution, prepared as previously mentioned in the
Material and methods section. The latter resulted in a
slightly reduced inulinase yield compared with that
obtained using either complete or minimal media
(Fig. 7). The recorded productivity rate under these
conditions was 2.03 g/l/h for the first 36 h of the
incubation period. However, a satisfactory production
level ranging between 85.9 and 79.9 U/ml was then
reached and approximately sustained for another 42 h.
These results were very encouraging as, using this
technique, a combined production of about 838 U/ml of
inulinase was obtained from a very economic crude
extract of inulin in only 48 h, which is comparable with
yields of 996 and 1013 U/ml that were obtained when
immobilized cells were cultivated using pure inulin in
either minimized or complete media, respectively, for the
same incubation period.
Conclusion
From these experiments, we can conclude that the
production of inulinase by A. niger cells immobilized by
their adsorption onto the surface of linen fibers, using
crude inulin extraction, is a very promising method that
could be performed on large scales for economic,
industrial production of the enzyme. The main advantage
of this method is the higher productivity of the
immobilized cells compared with that of the free cells,
considering the possibility of their repeated batch
cultivation. It was also observed that the production time
during the repeated batch cultivations reduced by more
than half. Moreover, with the use of crude inulin juice, a
low percentage of cell growth, and eventually cell
escapement, was attained. The latter made the recovery
and purification of the enzyme much easier. As a final
conclusion, this method is simple, economic, time saving,
and nontoxic to the microorganism. In addition, the
loaded linen pads are reusable.
Acknowledgements
The authors express their deepest gratitude to Prof. Dr. A.I. El-Diwany
and Prof. Dr. M.A. Farid for their generous participation in designing and
funding the airlift bioreactor and for their continuous invaluable support.
Continuous production of inulinase by Aspergillus niger cells immobilized onto linen fibers in an airlift bioreactor, using either complete or
minimized media as well as crude inulin juice.
Conflicts of interest
There are no conflicts of interest.
Optimization of inulinase production Atwa and Danial 89
References
1 Singh RS, Sooch BS, Puri M. Optimization of medium and process parameters for the production of inulinase from a newly isolated Kluyveromyces
marxianus YS-1. Bioresour Technol 2007; 98:2518–2525.
2 Vandamme EJ, Derycke DG. Microbial inulinases: fermentation
process, properties, and applications. Adv Appl Microbiol 1983; 29
(C):139–176.
3 Zhang L, Wang Y. Gene engineering of producing fructose by
inulase hydrolyzing Helianthus tuberosus. Chinese Patent 02132446;
2002.
4 Gill PK, Manhas RK, Singh P. Hydrolysis of inulin by immobilized thermostable extracellular exoinulinase from Aspergillus fumigates. J Food Eng
2006; 76:369–375.
5 Zhengyu J, Jing W, Bo J, Xueming X. Production of inulooligosaccharides
by endoinulinases from Aspergillus ficuum. Food Res Int 2005; 38:
301–308.
6 Gupta AK, Singh DP, Kaur N, Singh R. Production, purification and
immobilisation of inulinase from Kluyveromyces fragilis. J Chem Technol
Biotechnol 1994; 59:377–385.
7 Kim D-M, Kim H-S. Continuous production of gluconic acid and sorbitol from
Jerusalem artichoke and glucose using an oxidoreductase of Zymomonas
mobilis and inulinase. Biotechnol Bioeng 1992; 39:336–342.
8 Vranesic D, Kurtanjek Z, Santos AMP, Maugeri F. Optimisation of inulinase
production by Kluyveromyces bulgaricus. Food Technol Biotechnol 2002;
40:67–73.
9 Kennedy JF, Cabral MS. Immobilized living cells and their applications. Applied biochemistry and bioengineering. In: Chibata I, Wingard LB, editors.
Immobilized microbial cells. Vol. 4 NY: Academic Press; 1983. pp. 189–280.
10 Cabral MS, Kennedy JF. Covalent and coordination immobilization of
proteins. In: Taylor RF, editor. Protein immobilization. NY: Marcel Dekker;
1991. pp. 73–138.
11 Farid MA, El-Batal AI, El-Diwany AI, El-Anshasy HA. Optimization of glutamic
acid production with immobilized cells of Corynebacterium glutamicum.
Adv Food Sci 1996; 18:34–38.
12 Atwa NA. Microbial and biochemical studies on the production of antibiotics for veterinary uses [PhD thesis]. Microbiology Department, Faculty of
Sciences, Cairo University; 2003..
13 Sharma AD, Kainth S, Gill PK. Inulinase production using garlic (Allium
sativum) powder as a potential substrate in Streptomyces sp. J Food Eng
2006; 77:486–491.
14 Aboo Baker DHA, El-Genaihi SE, Aboul Enein AM, Danial EN. Comparative
study of inulin in two Asteraceae plants Chicory and Jerusalem artichoke. Ain
Shams Univ Res bull 2009; 22:1–11.
15 Ashwell G. Colorimetric analysis of sugars. Methods Enzymol 1957;
3 (C):73–105.
16 Sheng J, Chi Z, Li J, Gao L, Gong F. Inulinase production by the marine yeast
Cryptococcus aureus G7a and inulin hydrolysis by the crude inulinase.
Process Biochem 2007; 42:805–811.
17 Spiro RG. Analysis of sugars found in glycoproteins. Methods Enzymol
1966; 8 (C):3–26.
90
Short communication
Chemical constituents from the aerial parts of Salsola inermis
Fatma S. Elsharabasya,c and Ahlam M. Hosneyb
Departments of aChemistry of Natural and Microbial
Products, bTheraputic Chemistry, National Research
Center, Dokki, Egypt and cCollage of Science and
Humanities, Salman bin Abdul Aziz University, Alkharj
City, Kingdom of Saudi Arabia
Correspondence to Fatma S. Elsharabasy, PhD,
Department of Chemistry of Natural and Microbial
Products, National Research Center, El-Behooth St,
Dokki 12311, Egypt
Tel: + 20 101 468 5611; fax: + 20 233 370 931;
e-mail: [email protected]
Received 22 July 2012
Accepted 31 October 2012
Egyptian Pharmaceutical Journal
2013,12:90–94
Background and objective
The hydroalcoholic extract from the aerial parts of Salsola inermis exhibited antioxidant,
anti-inflammatory, and antinociceptive effects. The present study deals with the
isolation and identification of the chemical constituents of this hydroalcoholic extract.
Materials and methods
The aerial parts of S. inermis (Forsskal) were collected from wild plants growing near
the El-Alamein area in October 2005. Air-dried and powdered aerial parts of S. inermis
were extracted with 70% alcohol in H2O. The extract was partitioned successively with
CHCl3, EtOAc, and n-BuOH. The structures of the isolated compounds were
determined by chemical and spectroscopic analyses.
Results and conclusion
Phytochemical investigation of the alcoholic extract from the aerial parts of
S. inermis revealed 12 compounds, identified as long chain hydroxyl fatty acid
9,12,13-trihydroxydecosan–10,15,19-trienoic acid; trans-N-feruloyl tyramine-4000 -O-bD-glucopyranoside; umbelliferone; scopoletin; 3-methyl kaempferol; olean-12-en-3,28diol; olean-12-en-28-oic acid; stigmasterol-3-b-O-D-glucopyranoside; 3-O-[b-Dglucopyranosyl]oleanolic acid; kaempferol 3-O-b-glucopyranoside; and isorhamnetin
3-O-b-glucopyranoside, in addition to b-sitosterol, stigmasterol, and stigmastanol.
Some of these compounds have hydroxyl groups, which help in scavenging free
radicals and inhibit COX and various mediators involved in the pathogenesis of
pain relief.
Keywords:
aerial parts, coumarins, flavonoids, NMR, Salsola inermis, terpenes
Egypt Pharm J 12:90–94
& 2013 Division of Pharmaceutical and Drug Industries Research,
National Research Centre
1687-4315
Introduction
The genus Salsola, family Chenopodiaceae (Goosefoot
family), includes over 100 species found in the dry
regions of Asia, Europe, and Africa [1]. The Salsola
species represents 16 species in Egypt, most of which
grow in the Egyptian deserts [2]. Previous phytochemical
investigation of the genus resulted in the isolation of
alkaloids, saponins, sterols and their glucosides, comarinolignan, isoflavonoids, and flavonoids [3–10]. Some Salsola
plants are widely used as folk medicine for the treatment
of hepatitis [11] or infections caused by tapeworm and
parasites [12]; they also have pronounced vasoconstrictive, hypertensive, and cardiac stimulant action [13]
and can act as an allergenic substance [14,15]. Reactive
oxygen species (ROS) are always present in cells as
metabolic products of normal cellular respiration. However, oxidative stress, an imbalance caused by excessive
ROS originating from endogenous and exogenous sources,
might cause inflammation and therefore play a pivotal
role in many diseases [16]. Cytopreventive antioxidants
prevent the formation of free radicals and scavenge them
or promote their decomposition [17]. In chemical terms,
polyhydroxy flavonoids efficiently modulate the redox
status and thus may play a critical role in regulating the
inducible gene expression of inflammatory mediators in
the lipopolysaccharide-stimulated mouse leukemic monocyte macrophage cell line (RAW 2647macrophages) [18].
As a continuation of our previous studies that showed that
the ethanol extract of Salsola inermis has antioxidant and
anti-inflammatory properties [19], the present study
deals with the isolation and identification of chemical
constituents of the hydroalcoholic extract from the aerial
parts of S. inermis.
Materials and methods
Electron impact mass spectra (EIMS) were obtained using
Varian MAT 711 (Germany), Finnigan SSQ 7000 (San Jose,
California, USA), and OMM 7070 E spectrometers
(Maryland, USA). 1H-NMR and 13C-NMR spectra were
recorded at 500 MHz on a JEOL 500 A spectrometer
(JEOL Inc., USA). The 1H-NMR and 13C-NMR chemical
shifts are expressed in ppm relative to tetramethylsilane.
Infrared (IR) spectra were measured on a Perkin Elmer FTIR1700 spectrometer (Perkin Elmer, USA) at the National
Research Centre, Cairo, Egypt. Ultraviolet (UV) spectra
were recorded on a Shimadzu UV-Vis spectrophotometer
(Shimadzu, USA). Thin layer chromatography (TLC)
plates (aluminum sheets) precoated with silica gel G 60
(F 254; Merck) were used for chromatography. Special
reagents used were iodine–potassium iodide for detection
of coumarins and chlorosulfonic acid spray reagent for the
detection of sterols and triterpens. The two-dimensional
paper chromatographic technique using the solvent system
1687-4315 & 2013 Division of Pharmaceutical and Drug Industries Research,
National Research Centre
DOI: 10.7123/01.EPJ.0000428060.24957.95
Chemical constituents of Salsola inermis Elsharabasy and Hosney 91
BuOH : HOAc : H2O (4 : 1 : 5) and HOAc (15%) was also
used [20].
and 0–10) afforded compounds IX, X, XI, and XII.
The physical and spectral data of the isolated compounds
are as follows.
Plant material
The aerial parts of S. inermis (Forsskal) were collected
from wild plants growing near the El-Alamein area in
October 2005. The plant specimen was authenticated by
Dr N. El-Hadidi, Faculty of Science, Cairo University, and
was compared with reference herbarium specimens.
General procedure for extraction and isolation
Air-dried and powdered aerial parts of S. inermis were
extracted with 70% alcohol in H2O after evaporation of
the solvent under reduced pressure. It was essential that
the extract (200 g) be partitioned successively with
CHCl3, EtOAc, and n-BuOH.
The CHCl3 fraction (8 g) was applied onto a silica gel
column and eluted with a gradient of n-hexane, CHCl3, and
MeOH (100–0, 90–10, 80–20, 70–30, 60–40, 50–50, 40–60,
20–80, 0–100) to give five fractions A1–A5. Further
purification of A1 (0.8 g) by preparative TLC with
n-hexane/CHCl3 as an eluent afforded compounds I
(0.05 g) and II (0.02 g). Moreover, column chromatography
of A2 with CHCl3 afforded compounds III (0.28 g) and IV
(0.10 g). Column chromatography of A4 with gradient
elution using EtOAc/MeOH yielded compound V
(0.18 g). The EtOAc fraction (7 g) was chromatographed
over silica gel with successive petroleum ether/EtOAc
(80–20, 20–80, 0–100) and EtOAc/MeOH (90–10, 0–100)
elution to give eight fractions, B1–B8. Column chromatography of B6 (1.24 g) with CHCl3/MeOH elution (9–1 and
9–2) afforded compounds VI (5 mg) and VII (3 mg).
Further, column chromatography of B8 with CHCl3/MeOH
elution (9–1 and 8–2) afforded compound VIII (6 mg).
BuOH (12 g) applied onto a flash column chromatography
column with H2O/MeOH gradient elution afforded three
fractions, C1–C3. Purification of C2 and C3 carried out on a
Sephadex LH-20 column with CHCl3/MeOH elution (1–9
1
Table 1 H-NMR (300 MHz) and
compound II (CHCl3-d6)
Position
1
2
3
10
20
30
40
50
60
100
200
1000
2000 , 6000
3000 , 5000
4000
Glc-1
2
3
4
5
6
OCH3
13
6.47
7.43
7.17 br d (1.6)
6.74 d (8.5)
6.95 dd (8.2, 1.7)
3.34 t (7.3)
2.76 t (7.3)
7.17 d (8.5)
7.1 2 d (8.5)
4.2 d (7.4)
3.33 (overlap)
3.39 (overlap)
3.25 (overlap)
3.25 (overlap)
3.99 dd (12.0, 1.9)
3.73 dd (12.0, 5.5)
3.81s
Gummy white solid, EIMS, m/z 386: [M] + calculated for
C22H42O5; IR (KBr) nmax cm – 1 3437, 2925, 2854, 1740,
929; 1H-NMR (500 MHz, CDCl3) dH 0.9 (3H, t, J = 7.3,
H-22), 1.35 (11 H, bs, H-4, H-5, H-6, H-7, H-8a), 1.45
(1H, m, H-8b), 1.61 (2H, m, H-3), 2.05 (2H, t, J = 6,
8 Hz, H-21), 2.17 (1H, m, H-14a), 2.33 (1H, m, H-14b),
3.46 (1H, m, H-13), 3.98 (1H, t, J = 5.3 Hz, H-12), 4.05
(1H, m, H-9), 5.42, O, (1H, J = 11.2, 5.2 Hz, H-16), 5.47,
O, (1H, J = 11.2, 5.2 Hz, H-15), 5.68 (1H, dd, J = 15.7,
5.2 Hz, H-11), 5.73 (1H, dd, J = 15.7, 5.2 Hz, H-10).
Compound II
Amorphous powder, IR (KBr) nmax cm – 1 3416, 2925,
1725, 1646, 1515, 1269, 1074. EIMS, m/z: 476 [M] + calcd
for C24H30NO9. UV lmax (MeOH) nm (loge): 225 (3.12),
278 (2.99), 311 (3.1). 1H-NMR and 13C-NMR spectral
data are presented in Table 1.
Compound III
White crystals, m.p. 225–2281C, Rf 0.42 (TLC, S1); UV
lmax nm (MeOH) 217, 245, 260sh, 279sh, and 322 nm;
EIMS m/z 162 [M] + , C9H6O3. 1H-NMR (500 MHz,
CDCl3) dH 6.15 (1H, d, J = 9.6 Hz, H-3), 6.58 (1H, d,
J = 2.6 Hz, H-8), 6.85 (1H, dd, J = 8.6 Hz, H-6), 7.35
(1H, d, J = 8.6 Hz, H-5), 7.81 (1H, d, J = 9.3 Hz, H-4).
Compound IV
Colorless needle crystals (CHCl3), m.p. 221–2231C, Rf
0.5 (TLC, S1); UV lmax nm (MeOH) 229, 250sh, 260sh,
295sh and 342 nm. 1H-NMR (500 MHz, CDCl3) dH 6.26
and 7.58 (2H, d, J = 9.6 Hz, H-3 and H-4), 6.85 and 6.92
(2H, s, H-8 and H-5) and 3.92 (3H, s, Me-6).
Compound V
Colorless needles, m.p. 131–1321C, showed [M + ] peak at
m/z 412 (25.0%), 414 (17%), and 416 (1.40%) and
characteristic fragmentation peaks at m/z 275, 255, 231, 213.
C-NMR (300 MHz) for
dH
Compound I
dC
175
121.7
140.98
129.18
114.0
148
148.6
115.64
121.4
40.55
40.39
132.0
130
115.0
156
101.3
74.01
77.44
70.62
67.93
63.61
56.42
Compound VI
White needles (0.22 g), m.p. 2541C. M + peak at m/z 441
(9.30%), corresponding to C30H50O, and an intensive
peak at m/z 411 (18.92%), corresponding to M + –
CH2OH. IR showed characteristic absorption bands at
3395 (OH), 2925 cyclic (CH2), 1730, and 1446 (C = C).
D12 double bond proved to be readily recognizable by
mass spectra and 1H-NMR shows seven tertiary methyl
proton singlets at 0.81, 0.82, 0.84, 0.86, 1.18, 1.22, and
1.84, an olefin proton at d 5.4 (br.s.), and a hydroxyl
methylene proton at 5.14.
Compound VII
Isolated as white crystals (0.01 g), m.p. 259–601C, IR
spectrum showed strong bands near 3415 cm – 1 (OH),
1735 cm – 1 (CO), two bands 1390–1375 and 1369–1354
cm – 1 in the ‘A-region’, and three bands at 1328–1318,
1303–1296, and 1267–1248 cm – 1 in the ‘b-region’; its
92 Egyptian Pharmaceutical Journal
mass gave an M + + 1 peak at m/z 457 (2.02%), corresponding to C30H48O3, fragmentation characteristic with
respect to oleanane triterpenoids having D12 : 13 unsaturation.
The ion at m/z 189 stands for rings A and B in the
dehydrated form [21].
Compound VIII
Yellow powder, m.p. 275–2781C, Rf 0.47 (TLC, S2); UV
lmax nm (MeOH) 268 and 363, (MeOH/NaOMe) 279
and 423, (MeOH/AlCl3) 269 and 423; 1H-NMR
(500 MHz, DMSO) dH 6.16 and 6.42 (2H, d,
J = 2.2 Hz, H-6 and H-8), 6.90 and 8.0 (each 2H, d,
J = 8.7 Hz, H-30 , -50 and H-20 ,-60 ).
Compound IX
White powder, m.p. 265–2681C, Rf 0.47 (TLC, S2);
EIMS, M + peak at m/z 576, 9.45%, corresponding to the
molecular formula C35H60O6, m/z 163 (13.54) of one
hexose sugar; IR spectrum (KBr) Vmax cm – 1, 3421 (OH),
1730–1446 (C = C), 1129, 1076, 1055, and 1015 (ether
linkage of glycoside); 1H-NMR (500 MHz, DMSO-d6) dH
0.64 and 1.02 (each 3H, s, H-18 and H-19), 0.78–0.85
(9H, m, H-26, 27 and H-29), 0.89 (3H, d, J = 6.6 Hz,
H-21), 0.94 (3H, m, H-29), 4.39 (1H, m, H-3), 5.38 (1H,
broad s, H-6), and 4.30 (1H, d, J = 7.7 Hz, H-10 ).
Compound X
White powder, m.p. 260–2631C, Rf 0.57 (TLC, S2); EIMS,
M + at m/z 618 compatible with C36H58O8, m/z 456 ascribe
to the mass of triterpene (aglycone), corresponding to
C30H47O3, m/z 438 (aglycone-H2O), 426 (aglycone-2Me),
410 (aglycone-COOH + Me), 248 and 189, 133, the ion at
m/z 161 stands for a hexose sugar.
Compound XI
Yellow powder, UV lmax nm: (MeOH) 256, 267.1, 292sh,
357; MeOH + NaOMe, 272, 291, 325sh, 415; MeOH +
NaOAc, 273, 315, 390; MeOH + AlCl3, 274, 292, 340sh,
425 MeOH + AlCl3 + HCl; 272, 303sh, 360sh, 403.
1
H-NMR (500 MHz, DMSO) dH 7.82 (2H, d,
J = 8.2 Hz, H-20 , 60 ), 6.84 (2H, d, J = 8.2 Hz, H-30 ,50 ),
6.28 (1H, d, J = 1.9 Hz, H-6), 5.30 (1H, d, J = 7.6 Hz,
H-100 of glucose), 3.27–3.57 (m, rest of glucose protons).
Acid hydrolysis gave kaempferol and glucose.
Compound XII
Yellow powder, m.p. 224–226oC; brown fluorescence in
UV, Rf 0.34, UV lmax nm: (MeOH) 254, 265sh, 353;
(NaOMe) 270, 331sh, 415; (NaOAc) 271, 311sh, 394;
(AlCl3) 264, 296, 366sh, 400; (AlCl3 + HCl) 262, 300, 366,
400. Acid hydrolysis gave isorhamnetin and glucose.
Chemical constituents of Salsola inermis Elsharabasy and Hosney 93
The two coumarins III and IV were isolated from the
CHCl3 extract. Umbelliferone (III) showed shine blue
fluorescence under UV light (366) and when sprayed
with I2/KI reagent turned into a colorless spot. From
the results of 1H-NMR analysis and by cochromatography with the reference substance, compound III was
identified.
Results and discussions
The aqueous ethanolic extract was successively partitioned in H2O/CHCl3, H2O/EtOAc, and H2O/n-BuOH.
The three fractions were then subjected to a sequence of
column chromatography procedures to yield compounds
I–V, VI–VIII, and IX–XII, respectively.
9,12,13-Trihydroxydocosan–10,15,19-trienoic acid (I) was
isolated as a white solid with the molecular formula
C22H42O5, calculated from the [M + ] peak at m/z 386. Its
IR spectrum showed OH and CO absorptions at 3437 and
1740 cm – 1, respectively. 13C-NMR was characteristic of
an unsaturated long chain fatty acid with a methyl group
at dC 14.3, several methylene carbons from 23.2 to 39.91,
two sp2carbons at 139.3 and 157.4, and a substituted
carboxyl carbon at dC 166.98, in addition to three lowfield oxygenated carbons at dC 72.99 and 77.0, bearing
methane protons at dH 4.2, 3.89, and 3.58, respectively,
which confirmed the presence of three hydroxyl groups;
an olefinic proton signal appeared at d 5.27. Analysis of
the spectra provided evidence for the fragment and
established the structure of compound I [22].
000
Trans-N-feruloyl tyramine-4 -O-b-D-glucopyranoside (II)
showed EIMS, M + at m/z 476 calculated for the
molecular formula C24H30NO9. Its IR spectrum exhibited
characteristic absorption bands for a hydroxyl group
(3416 cm – 1), conjugated carbonyl group (1646 cm – 1),
and conjugated double bond (1515 cm – 1). Acid hydrolysis
of II afforded D-glucose as determined by comparing the
Rf of the hydrolysis product with that of an authentic
sample using the paper chromatographic technique. The
1
H-NMR spectrum (Table 1) indicated the presence of
one 1,4-disubstituted aromatic ring at dH 7.19 (2H, d,
J = 8.4 Hz, H-2000 , 6000 ) and dH 7.19 (2H, d, J = 8.5 Hz, H3000 , 5000 ); one 1,3,4-trisubstituted aromatic ring at dH 6.95
(1H, dd, J = 8.2, 1.7 Hz, H-60 ) and dH 6.75 (1H, d,
J = 8.2 Hz, H-50 ); one trans olefin at dH 6.68 (1H, d,
J = 15.2 Hz, H-3) and dH 4.22 (1H, d, J = 12.1 Hz, H-2);
and one methoxy proton at dH 3.99 (3H). From the
coupling constant of the anomeric proton at dH 4.24
(1H, d, J = 7.4 Hz, Glc-1), C-1 of the D-glucopyranose
was determined to be in the b-configuration. Analysis of
the 13C-NMR (Table 1; dC-1" 175, dC-1 40.5) and the
molecular formula of II revealed that C-100 and C-1were
linked by a nitrogen atom. The current analysis and
comparison with the data in the literature suggested the
structure of compound II [23].
Scopoletin (IV) showed strong blue fluorescence under
UV light (366) and when sprayed with I2/KI reagent
turned into brown spot. The UV spectrum of IV in
MeOH showed absorption bands at 229, 250sh, 260sh,
295sh, and 342 nm, which suggested a 6,7-dioxgenated
coumarin skeleton. From the results of 1H-NMR analysis
and by cochromatography with the reference substance,
compound IV was identified [24].
Three known sterols (V) isolated from the CHCl3 extract
gave positive results for the Liebermann test for sterols
and showed an [M + ] peak at m/z 412 (25.0%), 414
(17%), and 416 (1.40%) corresponding to C29H48O,
C29H50O, and C29H52O, respectively. Because of its
occurrence with the identified sterols [25], the sterol
with M + at m/z 414 (17.0%) was identified as bsitosterol, the sterol with M + at m/z 412 (25.0%) was
identified as stigmasterol, and the sterol with M + at m/z
416 was identified as sitostanol.
Three compounds VI, VII, and VIII were isolated from
the EtOAc extract.
Olean-12-en-3,28 diol (VI) gave a positive Liebermann
test for triterpenes. The compound with M + at m/z 441
(8.02%) was identified as C30H50O2, with a peak at 411
(45.0%). Spectral analysis suggested the structure of the
compound [21].
Olean-12-en-28-oic acid (VII): the IR spectrum showed
strong bands near 3415 (OH) and 1735 cm – 1 (CO): two
bands, 1390–1375 and 1369–1354 cm – 1, in the so called
‘A-region’ and three bands at 1328–1318, 1303–1296, and
1267–1248 cm – 1 in the ‘b-region’; its mass gave an
M + + 1 peak at m/z 457 (2.02%), corresponding to
C30H48O3, fragmentation characteristic with respect to
oleanane triterpenoids having D12 : 13 unsaturation. The
ion at m/z 189 represents rings A and B in the dehydrated
form. Previous spectral data and chemical analysis
elucidate the structure of this compound [21].
3-Methyl kaempferol (VIII) was identified from the
analysis of its UV spectra in MeOH before and after the
addition of different shift reagents and from the analysis
of its 1H-NMR spectral data [20]; this was further
confirmed by cochromatography with a reference substance.
Stigmasterol-3-b-O-D-glucopyranoside (IX) showed an
EIMS M + peak at m/z 576 (9.45%), corresponding to
the molecular formula C35H60O6, m/z 163 (13.54) of one
hexose sugar. IR spectroscopy revealed bands Vmax cm – 1,
3421 OH, 1730–1446 (C = C), 1129, 1076, 1055, and
1015 (ether linkage of glycoside). 1H-NMR revealed one
anomeric proton at 4.43 (d, J = 6.78), indicating the sugar
to be in the b-configuration. Thus, from the large JH1,H2
94
Egyptian Pharmaceutical Journal
coupling constant, the structure of this compound was
elucidated as stigmasterol-3-b-O-D-glucopyranoside [25].
3 Karawya MS, Wassel GM, Baghdadi HH, Ahmed ZF. Isolation of methyl
carbamate from four Egyptian Salsola species. Phytochemistry 1972;
11:441–442.
3-O-[b-D-glucopyranosyl]oleanolic acid (X) showed an
M + peak at m/z 618, corresponding to the molecular
formula C36H58O8, and a fragment ion at m/z 456,
corresponding to H30H48O3. This is ascribed to the mass
of triterpene acid having a D12 aglycone. IR revealed
bands Vmax cm – 1, 3421 OH, 1730–1446 (C = C), 1129,
1076, 1055, and 1015 (ether linkage of glycoside).
4 Wassell GM, Baghdadi HH, El Difrawy SM. Phyto-ecdysone from some wild
Egyptian Salsola species. Fitoterapia 1979; 50:51–52.
Kaempferol 3-O-b-glucopyranoside (XI) and isorhamnetin 3-O-b-glucopyranoside (XII) gave typical brown
fluorescence under UV for the C-3-substituted flavonoid
glycosides. Acid hydrolysis yielded glucose and kaempferol or isorhamnetin, respectively. The structures of
compounds XI and XII were confirmed by 1H-NMR and
cochromatography with authentic reference samples [26].
Isolation of these compounds from S. inermis has not been
reported previously.
Conclusion
Twelve compounds were isolated and identified for the
first time from the 70% ethanolic extract of S. inermis.
Some of these compounds contain different hydroxyl
groups and the others were terpenoids, which help
scavenge free radicals and inhibit COX and various
mediators involved in the pathogenesis of pain relief.
The chloroform fraction showed more potent inhibitory
activity than the ethanol extract, whereas the 70%
ethanolic extract was more potent than the chloroform
fraction in antinociceptive activity.
Acknowledgements
Conflicts of interest
There are no conflicts of interest.
References
1 El Hadidi MN, Fayed AA. Materials for excursion flora of Egypt. Taeckholmia
1994–95; 15:57.
2 Boulos L. Flora of Egypt, checklist. 1 Cairo, Egypt: Al Hadara Publisher;
1995. p. 118.
5 Woldu Y, Abegaz B. Isoflavonoids from Salsola somalensis. Phytochemistry
1990; 29:2013–2015.
6 Tundis R, Menichini F, Conforti F, Loizzo MR, Bonesi M, Statti G, Menichini F.
A potential role of alkaloid extracts from Salsola species (Chenopodiaceae)
in the treatment of Alzheimer’s disease. J Enzyme Inhib Med Chem 2009;
24:818–824.
7 Beyaoui A. New antioxidant bibenzyl derivative and isoflavonoid from the
Tunisian Salsola tetrandra Folsk. Nat Prod Res 2012; 26:235–242.
8 Tundis R. Inhibitory effects on the digestive enzyme alpha-amylase of three
Salsola species (Chenopodiaceae) in vitro. Pharmazie 2007; 62:473–475.
9 Oueslati MH, Jannet HB, Mighri Z, Chriaa J, Abreu PM. Phytochemical
constituents from Salsola tetrandra. J Nat Prod 2006; 69:1366–1369.
10 Swart P. Biological activities of the shrub Salsola tuberculatiformis Botsch.:
contraceptive or stress alleviator? Bioessays 2003; 25:612–619.
11 Abegaz BM, Woldu Y. Isoflavonoids from the roots of Salsola somalensis.
Phytochemistry 1991; 30:1281–1284.
12 Saratikov AS, Vengerovskiľ AI. New hepatoprotective agents of natural origin.
Eksp Klin Farmakol 1995; 58:8–11.
13 Beyaoui A, Chaari A, Ghouila H, Ali Hamza M, Ben Jannet H. New antioxidant
bibenzyl derivative and isoflavonoid from the Tunisian Salsola tetrandra Folsk.
Nat Prod Res 2012; 26:235–242.
14 Nofal SM. Evaluation of some biological activities of the alcoholic extracts of
Salsola volkensii and Salsola villosa in rats. Egypt J Pharm Sci 2004;
45:41–52.
15 Assarehzadegan MA. Allergy to Salsola Kali in a Salsola incanescens-rich
area: role of extensive cross allergenicity. Allergol Int 2009; 58:261–266.
16 Flora SJS. Role of free radicals and antioxidants in health and disease.
Cell Mol Biol 2007; 53:1–2.
17 Si H, Liu D. Isoflavone genistein protects human vascular endothelial cells
against tumor necrosis factor-a-induced apoptosis through the p38b mitogen-activated protein kinase. Apoptosis 2009; 14:66–76.
18 Blonska M, Czuba ZP, Krol W. Effect of flavone derivatives on interleukin-1b
(IL-1b) mRNA expression and IL-1b protein synthesis in stimulated RAW
264.7 macrophages. Scand J Immunol 2003; 57:162–166.
19 Ahlam HM, Fatma SE. Antioxidant, anti-inflammatory and analgesic effects of
Salsola inermis. J Arab Soc Med Res 2007; 2:45–52.
20 Mabry TJ, Markham KR, Thomas MB. The systematic identification of flavonoids. New York: Springer Verlag; 1970.
21 Djerassi C, Budzikiewicz H, Wilson JM. Mass spectrometry in structural and
stereochemical problems unsaturated pentacyclic triterpenoids. Tetrahedron
Lett 1962; 3:263–270.
22 Kato T, Yamaguchi Y, Abe N, Uyehara T, Namai T, Kodama M, Shiobara Y.
Structure and synthesis of unsaturated trihydroxy c 18 fatty. Acids in rice
plants suffering from rice blast disease. Tetrahedron Lett 1985; 26:
2357–2360.
23 Yahagi T, Yamashita Y, Daikonnya A, Wu J-B, Kitanaka S. New feruloyl tyramine glycosides from Stephania hispidula Yamamoto. Chem Pharm Bull
(Tokyo) 2010; 58:415–417.
24 Murray ROH, Mendez J, Brown SA. The natural coumarins. Chichester,
New York, Brisbane, Toranto and Singapore: John Wiley & Sons Ltd; 1982.
25 Elgamal MHA, Abdel Hady FK, Soliman HSM. Constituents of local plants.
Phytochemical screening of some selected local saponin bearing plants. Bull
Natl Res Centre (Cairo) 1990; 15:215–220.
26 Kojima H, Sato N, Hatano A, Ogura H. Sterol glucosides from Prunella
vulgaris. Phytochemistry 1990; 29:2351–2355.