Kwame Nkrumah University of Science and
Technology, Kumasi
COLLEGE OF SCIENCE
DEPARTMENT OF CHEMISTRY
The Antimicrobial Activities of the Stem Extract of
Strophanthus gratus, Apocynaceae
DANIEL HENNEH
JUNE, 2013
THE ANTIMICROBIAL ACTIVITIES OF THE STEM EXTRACT OF
STROPHANTHUS GRATUS, APOCYNACEAE
By
Daniel Henneh
BPharm(Hons), KNUST, Kumasi
A Thesis submitted to the Department of Chemistry, Kwame Nkrumah University of
Science and Technology in partial fulfillment of the requirements for the award of the
degree of
MASTER OF SCIENCE (ORGANIC CHEMISTRY)
College of Science
OCTOBER, 2013
CERTIFICATION
I hereby declare that this submission is my own work towards the Master of Science degree and
that to the best of my knowledge, it neither contains material previously published by another
person nor material which has been accepted for the award of any degree of the university,
except where due acknowledgement has been made in the text.
Daniel Henneh (PG2851708)
……………………
……………………..
Candidate
Signature
Date
Dr. Sylvester K. Twumasi
……………………
……………………..
Supervisor
Signature
Date
Certified by:
Certified by:
Mr. R. B. Voegborlo
……………………
……………………..
Head of Department
Signature
Date
DEDICATION
This thesis is dedicated to my wife, Janet Henneh, and my elder brother, Hon. Kwasi AmeyawCheremeh, MP (Sunyani East Constituency) whose unflinching encouragement helped me to
enrol in the MSc program and also to be able to complete the program. Thank you for your
invaluable assistance.
ACKNOWLEDGEMENT
First, I thank God for helping, strengthening and guiding me throughout the program. Whenever
the challenges abounded, you were there to take me through. You sent men to encourage and
strengthen me. I am grateful to you.
Second, to my dynamic supervisor, Dr. S. K. Twumasi, I say a big thank you for your
commitment and support. You were always available to help, guide and advise. Without your
unreserved assistance, I would never have completed my MSc work.
I specially thank Dr. S. Osafo Acquaah of the Chemistry Department, College of Science,
KNUST. Your encouragement always echoed in my mind and produced desire and energy for
the completion of this work.
I thank all the lecturers of the Department of Chemistry, College of Science, KNUST, for their
variety of assistance to me. I also, thank Mr Adu and all lecturers and technicians of the
Microbiology Department, Faculty of Pharmacy, KNUST for their assistance in my laboratory
work.
Lastly, I thank my course mates, especially David and Maxwell for their co-operation.
ABSTRACT
The ethanolic and aqueous extracts of the stem of Strophanthus gratus from the Botanical
Gardens, KNUST, were tested for in vitro antimicrobial activities. Traditional herbal
practitioners use the decoctions of the stem of this plant to treat gonorrhoea and syphilis.
Phytochemical tests on the extracts showed that they contained saponins, flavonoids, steroids,
alkaloids, anthraquinone glycosides, cyanogenetic glycosides, cardiac glycosides and tannins.
Test organisms used included E. faecalis, Pr. vulgaris, Staph aureus, B. subtilis, B. thuringiensis,
S. typhi, Ps aeruginosa and Neisseria gonorrhoeae. The results showed that both the ethanolic
and aqueous extracts were active against the test organisms. However, the ethanolic extract
recorded lower Minimum Inhibitory Concentrations against the organisms when compared with
the aqueous extract. When the activities of the extracts were compared with those of
ciprofloxacin under the same experimental conditions, it was realized that the extracts were more
active than ciprofloxacin against the test organisms with the exceptions of Neisseria
gonorrhoeae, S. typhi and E. coli. Auxiliary tests on the ethanolic extract of the plant showed
that the plant has some antioxidant activity in addition to its antimicrobial activity.
TABLE OF CONTENTS
TITLE
PAGE
CERTIFICATION……………………………………………………………………….ii
DEDICATION……………………..……………………………………………………iii
ACKNOWLEDGEMENT………….…………………………………………………..iv
ABSTRACT……...……………………………………………………………………….v
TABLE OF CONTENTS……………………………….………………………………vi
LIST OF TABLES……………………………………………….……………………....x
LIST OF FIGURES……………………………………………………………………...x
1 INTRODUCTION……………………………………………………………………....1
1.1 BACKGROUND……………….…………………………………………….…….1
1.2 STATEMENT OF THE PROBLEM…………….………………………………....2
1.3 OBJECTIVES…………………………………………………………………...….3
1.4 JUSTIFICATION OF THE PROJECT………………………………………..……4
2 LITERATURE REVIEW …………………………………………………………...….7
2.1 PHYTOCHEMICAL PRINCIPLES IN PLANTS……………………………...….7
2.1.1 Alkaloids …………………………………………………………...……….7
2.1.2 Flavonoids ……………………………………………………………...…...9
2.1.3 Tannins ……………………………………………………………...……..10
2.1.4 Terpenoids ……………………………………………………………....…11
2.1.5 Steroids from plants……………………………………………………..…11
2.1.6 Anthracene and anthraquinone derivatives…………………………..….…12
2.1.7 Cyanogenetic glycosides ……………………………………………….….12
2.2 STROPHANTHUS …………………………………………………………….…12
2.2.1 Uses of strophanthus gratus ……………………………...……………….13
2.2.2 Phytochemicals from Strophanthus gratus ………………………………..13
2.3 THE PRINCIPLE OF SOXHLET EXTRACTION ……..…………………….….15
2.4 DISEASE CAUSING BACTERIA …………………………………..………..…16
2.4.1 Staphylococci……………………………………...……………………….16
2.4.2 Neisseria ……………………………………………………………….......16
2.4.3 Bacillus.………………………………………………...……………….…17
2.4.4 Pseudomonas …………………………………………………..………….17
2.4.5 Escherichia………………………………………………………….…...…17
2.4.6 Salmonella …………………………….……………………………..……18
2.4.7 Proteus ……………………………………………………..……….….….18
2.4.8 Klebsiella …………………………………………………...………….….18
2.5 ANTIBIOTICS ………………………………………………………………......18
2.5.1 Mechanisms of action of antimicrobial agents ……………………..……..21
2.5.2 Assay of antibiotics……………………………………………….…….…21
2.6 ANTIOXIDANT ACTIVITY OF PLANTS ………………………………….….22
3 MATERIALS AND METHODS ……………………………………………………..25
3.1 Materials ……………………………………………………………………….....25
3.1.1 Collection of plant material …………………………………………….…25
3.1.2 Chemicals…………………………………………………………………..25
3.1.3 General cleaning and sterilization of glassware ……………………….…..26
3.2 METHODS ……………………………………………………………………….....26
3.2.1 Preparation of extracts…………………………………………………..…26
3.2.2 Phytochemical screening……………………………………………….….27
3.2.2.1 Test for saponins ………………………………………………...27
3.2.2.2 Test for general glycosides …………………………………...…27
3.2.2.3 Test for flavonoids ………………………………………………28
3.2.2.4 Test for terpenoids and steroids …………………………………28
3.2.2.5 Test for carotenoids …………………………………………..….28
3.2.2.6 Test for coumarins …………………………………………...….29
3.2.2.7 Test for alkaloids ………………………………………………...29
3.2.2.8 Test for anthraquinones………………………………….…….....29
3.2.2.9 Test for anthraquinone glycosides ……………………………....30
3.2.2.10 Test for cyanogenetic glycoside …………………..……………30
3.2.2.11 Test for cardiac glycosides………………………….…………..30
3.2.2.12 Test for tannins ……………………………………..……….…31
3.2.3
THIN LAYER CHROMATOGRAPHY ….…………………….….…...31
3.2.4
IR METHODOLOGY ……………………………..……………….…...31
3.2.5
PREPARATION OF SOLUTIONS OF EXTRACT ………………..…..32
3.2.6
PREPARATION OF MEDIA …………………………….…………..…32
3.2.6.1 Sterile Distilled Water …………………….……………………..32
3.2.6.2 Nutrient Agar …………………………………………….……...32
3.2.6.3 Nutrient Broth ……………….………………………………..…32
3.2.7
ANTIMICROBIAL ACTIVITY TESTS …………………….……….…33
3.2.7.1 Preparation of broth culture …………………………….…….…33
3.2.7.2 Preparation of nutrient agar culture ……………………………..33
3.2.8 ANTIOXIDANT ACTIVITY TESTS ……………………………….…....34
3.2.8.1 Preparation of solutions………………………………………….34
3.2.8.1 Total Phenol Assay …………………………………………...…35
3.2.8.2 Total Antioxidant Capacity Assay ………………………………35
3.2.8.3 Reducing Power …………………………………………………36
4.
4.1
RESULTS AND DISCUSSION ……………………………………………...…38
RESULTS …………..…………………………………………………………...38
4.1.1 Results for phytochemical screening………………………………………38
4.1.2 Results for extraction………………………………………………………39
4.1.3 Results for thin layer chromatography……………………………………..40
4.1.4 Results for antibiotic activity tests………………………………………....40
4.1.5 Results for antioxidant activity tests…………………………………….…47
4.1.6 Results for the IR spectrophotometry…………………………………...…51
4.2
GENERAL DISCUSSIONS ………………………..…………………………...54
4.3
ANTIMICROBIAL ACTIVITY ……………………..…………………………56
4.4
ANTIOXIDANT ACTIVITY ……………………..……………………………60
5.
5.1
CONCLUSIONS AND RECOMMENDATIONS………………………………62
CONCLUSIONS………………………………………………………………...62
5.2 RECOMMENDATIONS………………………………………………...…………62
References ……………………………………………………………………………....63
Appendix 1: Anova: Two-Factor Without Replication……………………………….…68
Appendix 2: MIC Exploratory plots……………………………………………………..69
Appendix 3: t-Tests……………………………………………………………………....70
Appendix 4: Correlation of MICs between test drugs……………….....………………..71
LIST OF TABLES
Table 4.1.1: Results for phytochemical screening……………………………………….39
Table 4.1.2 Extraction yields………………………………………………………….…39
Table 4.1.3: Results for thin layer chromatography…………………………………….40
Table 4.1.4a: Antibiotic activity test results for ethanol extract…………………………41
Table 4.1.4b: Antibiotic activity test results for water extract………………………...…41
Table 4.1.4c: Antibiotic activity test results for ciprofloxacin…………………………..42
Table 4.1.4e: Minimum Inhibitory Concentrations (MIC) and Ratios…………………..46
Table 4.1.4f: Minimum Inhibitory Concentrations and Ratios…………………………..46
Table 4.1.5a: Total Phenolic Content test results………………………………………..47
Table 4.1.5b: Total antioxidant capacity test results………………………………….…48
Table 4.1.5c: Reducing Power test results for S. gratus and n-propylgallate…………....50
LIST OF FIGURES
Figure 2.1: Chemical structures of some useful alkaloids ………………………………..8
Figure 2.2: Basic flavonoid rings …………………………………………………………9
Figure 2.3: Chemical structures of some tannins ……………………………………..…10
Figure 2.4: Chemical structures of some terpenoids ………………………………….…11
Figure 2.5: chemical structures of anthracene and anthraquinone ………………………12
Figure 2.6: Chemical structure of Ouabain……………………………………………....14
Figure 2.7: Chemical structures of lignans from S. gratus………………………………15
Figure 4.1.4a: Plots of Zone of Growth Inhibition against Log Concentration of Ethanol
Extract……………………………………………………………………………………43
Figure 4.1.4b: Plots of Zone of Growth Inhibition against Log Concentration of Ethanol
Extract…………………………………………………………………………………....43
Figure 4.1.4c: Plots of Zone of Growth Inhibition against Log Concentration of Water Extract
…………………………………………………………………………………...44
Figure 4.1.4d: Plots of Zone of Growth Inhibition against Log Concentration of Water
Extract……………………………………………………………………………………44
Figure 4.1.4e: Plots of Zone of Growth Inhibition against Log Concentration of
Ciprofloxacin…………………………………………………………………………….45
Figure 4.1.4f: Plots of Zone of Growth Inhibition against Log Concentration of
Ciprofloxacin…………………………………………………………………………….45
Figure 4.1.5a(A): A plot of Absorbance of tannic acid against concentration of tannic
acid.....................................................................................................................................47
Figure 4.1.5a(B): A plot of the total phenolic content present in S. gratus expressed as tannic
acid equivalent (TAE) against concentration of S. gratus......................................48
Figure 4.1.5b(A): A plot of the Absorbance of PMoV4MoVI8O407−(formed in ascorbic acid
solutions) against concentration of ascorbic acid..............................................................49
Figure 4.1.5b(B): A plot of the total antioxidant capacity (TAC expressed as Ascrobic acid
equivalent - AAE) of S. gratus against the respective concentrations of S. gratus...49
Figure 4.1.5c: A plot of total phenolic content expressed as tannic acid equivalents (TAE) against
total
antioxidant
capacity
(TAC)
expressed
as
ascorbic
acid
equivalents
gratus………………………………………………………………………………….….50
Figure 4.1.5d: Reducing power of S. gratus compared to n-propyl gallate ……………..51
of
S.
CHAPTER ONE
1 INTRODUCTION
This chapter covers the background of the work, the statement of the problem, objectives
of the work and justification of the work.
1.1 BACKGROUND
Plants do exhibit various therapeutic activities (Taleb-Contini et al; Shargel et al, 2007).
There is a worldwide renewed interest in natural products for therapy and health promotion
(Cseke et al, 2006) because from 1981 to 2002, no combinatorial compounds became approved
drugs, although several are currently in late-stage clinical trials (Cseke et al, 2006). Meanwhile
plant chemical constituents have served as lead compounds for discovery of drugs including
aspirin, atropine, belladonna, capsaicin, cascara, colchicines, digoxin (lanoxin), ephedrine,
ergotamine, ipecac, opium, physostigmine, pilocarpine, podophyllum, psyllium, quinidine,
reserpine, scopolamine, senna, taxol, tubocrarine, viblastine and vincristine (Shargel et al, 2007).
Various plants in Ghana and Africa are traditionally used for their therapeutic benefits (Ghana
Herbal Pharmacopoeia, 2007; Gulla et al., 2001). The challenge however, remains that not
enough research has been done into these plants and their preparations used in Ghana. The Food
and Drugs Board focuses research on the safety of the use of herbal preparations and not on their
therapeutic abilities. An investigation into the therapeutic ability of plants can lead to the
discovery of new drug leads (Shargel et al, 2007). A recent survey revealed that 61% of the 877
drugs introduced worldwide can be traced to or were inspired by natural products (Cseke et al,
2006).
Some plants in Ghana are used because of their antimicrobial actions. This is evidenced
by the labeled uses of the various herbal preparations certified by the Food and Drugs Board.
Many uncertified preparations are also used for their antimicrobial activity. Usually, several
phytochemical principles with different pharmacological targets are involved in the medicinal
actions of herbal preparations. This characteristic may be advantageous or disadvantageous when
compared with single isolated compounds. It is advantageous when the constituents work
together to bring about therapy but disadvantageous when they work antagonistically or when
some of the components are responsible only for toxic effects (Katzung, 2007).
This work focuses on the antimicrobial activity of the aqueous and ethanolic stem
extracts of the indigenous shrub Strophanthus gratus. However, investigations would also be
made to ascertain additional benefits of the use of Strophanthus gratus in the area of antioxidant
effects.
1.2
STATEMENT OF THE PROBLEM
The stem of Strophanthus gratus is used by traditional herbal practitioners to treat
gonorrhea and syphilis (Houghton et al, 2006). These claims are yet to be scientifically verified.
The Ghana Herbal Pharmacopoeia and other books contain literature on indications,
pharmacological actions and secondary metabolites of certain plants (Ghana Herbal
Pharmacopoeia, 2007). But Strophanthus gratus is not listed in the Ghana Herbal
Pharmacopoeia. Literature has documented works mostly on the seeds of Strophanthus gratus
with a few on the stem and leaves. Literature states that the seeds contain 4 -8% of ouabain (Gstrophanthin), a useful cardiac glycoside (Evans, 1989; Burkill, 1985; Cowan et al, 2001).
According to literature, the stem also contains minimal quantities of ouabain. Other isolated
secondary metabolites from the stems are lignans (Cowan et al, 2001).
Therefore, there are two problems to be solved regarding the use of the stem of
Strophanthus gratus as herbal preparation to treat infections. These are verification of the
antimicrobial actions and identification of the phytochemical principles responsible for the
antimicrobial actions of the plant; both in vitro and in vivo works are necessary. However, this
research work focuses on the in vitro antimicrobial and antioxidant activities of the plant.
1.3
OBJECTIVES
The objectives are:
1. To obtain aqueous and ethanolic extracts of the stem of Strophanthus gratus: Traditional
herbal practitioners use the aqueous decoction in the treatment of the infections.
However, since the solubilities of various metabolites in water differ from the solubilities
of the same metabolites in ethanol, this study would explore the antimicrobial properties
of both extracts.
2. To screen the extracts for phytochemical principles: The extracts would be screened for
the presence or absence of alkaloids, various glycosides, tannins and all major groups of
phytochemical principles.
3. To determine the in vitro antimicrobial activities of the extracts: The antimicrobial
activities of the extracts would be determined using water and methanol as the solvents
since these solvents do not have any antimicrobial actions of themselves.
Dimethylsulfoxide (DMSO) would be used in case of solubility problems
4. To establish Thin Layer Chromatograph of the extracts.
5. To determine the in vitro antimicrobial activities of prototype antibiotic under similar
conditions as (3) above and compare them to the antimicrobial activities of the crude.
Crude herbal antibiotics should not be used unless they have some form of advantage
over orthodox medication
6. To obtain the IR spectra of the extracts. The IR spectrum would be a profile against
which future extracts’ spectroscopic profile can be checked. Depending on the season and
or age of the plant certain specific metabolites may be absent or present and that may
affect the antimicrobial activities of the plant. For instance, a specific alkaloid may be
present or absent depending on the age and or season of the year but the test for alkaloids
in general may always be positive. IR spectrum would assist in discovering any
differences between the extracts used in this study and also between the extracts used in
this study and the extracts of future studies.
7. To determine some antioxidant activities of the extracts if proven to have antimicrobial
activities.
1.4
JUSTIFICATION OF THE PROJECT
Infectious diseases are by far the most important agents of diseases in Africa today.
Millions of children suffer and die from malaria, respiratory infections and diarrheal disease each
year. Sexually Transmitted Infections are also common in Africa (Parry et al, 2004).
Approximately 80% of the people in developing countries depend on traditional medicine (Trape
et al, 2005). In Ghana, it is estimated conservatively that between 60-90% of the general
population rely on medicinal plants either totally or partially for their health care needs (Parry et
al, 2004). The major problems normally encountered in the use of orthodox medicines are drug
resistance and high cost (Parry et al, 2004). Herbal medicines are usually cheap with lesser
incidence of drug resistance (Evans, 1989).
Strophanthus gratus stem is believed to treat a number of infections including gonorrhea
and syphilis. However, no experimental data is available on the medicinal uses and
phytochemical principles of the stem. In Nigeria, scientific study has been conducted on the
leaves of Strophanthus hispidus (a closely related species to Strophanthus gratus) because it was
traditionally claimed to cure diabetes. The research established that the leave extracts have in
vivo hypoglycaemic effects and therefore the leaves are potential antidiabetic drug. The research
also established the following phytochemical principles: alkaloids, flavonoids, saponins, and
cardiac and cyanogenic glycosides (Ojiako et al, 2009)
Ciprofloxacin, the first line orthodox drug for the treatment of gonorrhoea in Ghana is
currently facing serious resistance challenges from Neisseria gonorrhoeae. A more effective
alternative and cheaper drug from a plant would therefore make a great impact on the health of
Ghanaians. Once, Strophanthus gratus allegedly treats gonorrhea, it is important that the extract
is assayed with this causative organism. However, antimicrobial agents could have broad
spectrum actions and therefore it also becomes important to screen the extract against other
organisms (Shargel et al, 2007).
This study is aimed at establishing the groups of phytochemical principles and
antimicrobial activities of the stem extracts of Strophanthus gratus. Antioxidant activity work
would be done on the extracts if proven to have antimicrobial activity. This is because
antioxidant activity may augment antimicrobial activity. This work would create the platform for
further research into the stem of Strophanthus gratus to possibly standardize a herbal preparation
from it.
CHAPTER TWO
2
LITERATURE REVIEW
The literature review includes phytochemical principles such as alkaloids, tannins,
glycosides etc which are responsible for plant action, uses of Strophanthus gratus and
phytochemical principles from S. gratus, disease causing bacteria, soxhlet extraction principle,
antibiotics: types, mechanisms of action and assay methods, and antioxidant activity of plants
and its assay methods.
2.1
PHYTOCHEMICAL PRINCIPLES IN PLANTS
The actions of any herbal medicine is due to certain phytochemicals or secondary
metabolites within the herb. Not all the chemical compounds elaborated by plants are of equal
interest to medicinal chemists, herbalists and pharmacognosists. The active phytochemicals are
usually alkaloids, tannins, saponins, glycosides, etc and such active principles deserve special
attention. Other groups of compounds such as carbohydrates, fats and proteins are usually of
dietetic importance, and many such as starch and gums are used in pharmacy although lacking
any marked pharmacological action (Evans, 1996).
2.1.1 Alkaloids
Alkaloids are chemically heterogenous group of basic nitrogen containing substances
found predominantly in higher plants. However, such basic substances also occur in lower plants,
animals, microorganisms, and marine organisms (Bhat et al, 2005)
Alkaloids usually contain one or two nitrogen atoms although some like ergotamine may contain
up to five nitrogen atoms. True alkaloids meet the following criteria:

Nitrogen is part of a heterocyclic ring

The occurrence of the compound is restricted to plant kingdom

The compound has complex molecular structure

The compound manifests significant physiological activity
Many alkaloids possess curative properties and are therefore important in the
pharmaceutical industry. Alkaloids display a variety of pharmacological activities: analgesic
potentiator (cocaine), antiamebic (emetine), anticholinergic (atropine) antimalarial (quinine),
antihypertensive (reserpine), cardiac depressant (quinidine), central stimulant (caffeine), diuretic
(theophylline), gout suppressant (colchicine), and many more (Bhat et al, 2005). Some of these
alkaloids are illustrated below:
Figure 2.1: Chemical structures of some useful alkaloids
2.1.2 Flavonoids
Flavonoids are the largest group of naturally occurring phenolic compounds, which occur
in different plant parts both in free state and as glycosides. They are also known as plant
pigments or co-pigments. The presence of these pigments is responsible for the various colours
exhibited by plant parts. They exhibit various biological activities including: antimicrobial,
antiulcer, antiarthritic, estrogenic, anticancer. The basic rings upon which the flavonoids are built
include flavones, flavonol, flavonone and dihydroflavonol (Bhat et al, 2005; Ojiako et al, 2009).
Figure 2.2: Basic flavonoid rings
2.1.3 Tannins
Tannins are group of polymeric phenolic compounds which have tanning properties.
They are classified into hydrolysable, condensed and pseudotannins. Hydrolysable tannins can
be hydrolysed either by acids or enzymes known as tannase. Hydrolysable tannins are formed by
combination of several molecules of gallic acid and ellagic acid through ester-linkages to central
glucose molecule. Some hydrolysable tannins have antitumour, anti-viral, anti-HIV,
antimicrobial or other biological activities (Bhat et al, 2005; Ojiako et al, 2009).
Condensed tannins are polymeric proanthocyanidins and occur commonly in woody and
some herbaceous plants. Condensed tannins reportedly have these actions: antioxidant (radical
scavenging) and protection against cardiovascular disorders. Pseudotannins are simpler phenolic
compounds of low molecular weight co-occuring with tannins e.g. gallic acid (Bhat et al, 2005).
Figure 2.3: Chemical structures of some tannins
2.1.4 Terpenoids
Terpenoids are polyisoprene compounds synthesized by some plants, marine organisms
and fungi. They exhibit various biological activities including antimalarial, antihypertensive,
anti-tumour and antiglaucoma. Some skeletal types of terpenoids include farnesol, farnesene and
labdane (Bhat et al, 2005; Evans, 1989).
Figure 2.4: Chemical structures of some terpenoids
2.1.5 Steroids from Plants
Steroids from plants exist as conjugates with sugars. Such conjugates are known as
glycosides. The two main steroidal glycosides are cardiac glycosides and saponins. Cardiac
glycosides exhibit characteristic stimulatory effect on mammalian hearts. Saponins lower the
surface tension of water causing their aqueous solutions to froth readily (Bhat et al, 2005; Evans,
1989).
2.1.6 Anthracene and Anthraquinone Derivatives
Anthracene consists of three fused benzene nuclei while the anthraquinone is 9,10-oxoanthracene. Anthraquinones may occur in the free state and as glycosides. Anthracene and
anthraquinones are purgatives (Bhat et al, 2005; Evans, 1989).
Figure 2.5: chemical structures of anthracene and anthraquinone
2.1.7 Cyanogenetic glycosides
Cyanogenetic glycosides produce hydrogen cyanide upon hydrolysis. E.g amygdalin.
2.2
STROPHANTHUS
Strophanthus is a genus of 35-40 species of flowering plants in the family Apocynaceae.
The genus includes vines, shrubs and small trees. They are native mainly to tropical Africa
extending to South Africa with some few species in Asia (Philippines, Southern China and
Southern India) (Endress et al, 2000). Strophanthus gratus is a scandent shrub which can grow to
25 feet or more. It is glabrous with leaves oblong and can be up to 6 inches long. The leaves are
short-acuminate with veins spreading at right angles to midrib. The sepals are broad and the
seeds glabrous (Endress et al, 2000).
2.2.1 Uses of Strophanthus gratus
The leaves are used in Sierra Leone for gonorrhea. The leaf-sap is put onto ulcerated
sores in Ivory Coast. The leaves are mashed and applied to guinea-worm sores in Ghana. They
are used as a dressing for sores in Nigeria. The leaf and stem decoctions are taken for
constipation in Ghana and Nigeria. A decoction of the crushed stem is taken in Ghana for severe
sickness with weakness poison. The decoction is also used to treat gonorrhea and syphilis in
Ghana and Nigeria. The seeds are used as arrow poisons across various parts of Africa (Burkill,
1985).
2.2.2 Phytochemicals from Strophanthus gratus
Strophanthus gratus contains ouabain, a cardiac glycoside (Cowan et al, 2001). The
cardiac glycosides are present mainly in the seeds (4-8% weight by weight) but in minimal
quantities in other parts of the plant (Burkill, 1985).
Figure 2.6: Chemical structure of Ouabain
Until Cowan et al’s work, the only known isolated and characterized phytochemical in S. gratus
was ouabain, a cardiac glycoside. In 2000, Cowan et al isolated lignans from the stem of
Strophanthus gratus (Cowan et al, 2001). Three lignans; pinoresinol, 8-hydroxypinoresinol and
olivil have been isolated from the stem. The seeds contain glycosides, saponins, steroids; tannins,
astringents (Burkill, 1985).
Figure 2.7: Chemical structures of lignans from S. gratus
pinoresinol, R = H; 8-hydroxypinoresinol, R = OH
olivil
A related species, Strophanthus hispidus has been worked on in Nigeria. All parts of
Strophanthus hispidus contain alkaloids, flavonoids, saponins, and cardiac and cyanogenic
glycosides (Ojiako et al, 2009). A time trend hypoglycemic study of both ethanolic and
chloroformic extracts from various parts of the plant showed that it has in vivo hypoglycemic
activity. Probably, this is why local folks in Nigeria use it to treat diabetes (Ojiako et al, 2009).
2.3
THE PRINCIPLE OF SOXHLET EXTRACTION
The soxhlet exractor is for the extraction of solids such as dried leaves or seeds. The solid
is put in a clean thimble. The solvent vapor rises in the side tube and condensate drops onto the
solid in the thimble, leaches out soluble material, and after initiating an automatic siphon, carries
it to the flask where nonvolatile extracted material accumulates. Substances of low solubility can
be extracted by prolonged operation (Williamson et al, 2007)
2.4
DISEASE CAUSING BACTERIA
Several bacteria are disease causing organisms. The bacteria discussed below are
common bacteria in the Ghanaian environment (GNDP, 2004)
2.4.1 Staphylococci
They are Gram-positive cocci. Staphylococci are non-motile and non-sporing and can
grow aerobically or anaerobically. Staphylococcus aureus produces a golden yellow pigment. It
causes skin lesions such as boils and can affect bone tissues in the case of staphylococcal
osteomyelitis. It produces a toxin which, if ingested with food in which the organism has been
growing can give rise to food poisoning. A common manifestation of its infection is the
production of pus i.e. it is pyogenic. Other common conditions associated with staphylococcal
infections are impetigo and conjunctivitis (Hugo et al, 1992; Parry et al, 2004)).
2.4.2 Neisseria
The Gram-negative pathogenic cocci belongs to the genus Neisseria. The cells are
slightly curved rather than true spheres and have been likened to a kidney bean in shape. They
often occur in pairs and are embedded in pus cells. Neisseria gonorrhoeae is the causal organism
of the venereal disease gonorrhea. The organism can also affect the eyes, causing a purulent
ophthalmia.
Neisseria meningitides
is a cause of cerebrospinal fever or meningococcal
meniningitis (Shagel et al, 2007; Hugo et al, 1992).
2.4.3 Bacillus
They are Gram-positive rods. Members of this genus are widespread in air, soil and water
and in animal products such as hair, wool and carcasses. It occurs characteristically as a large rod
with square ends. It is aerobic and spore-forming. Bacillus cerus has been implicated as a cause
of food poisoning. Bacillus polymyxa is the source of the antibiotic polymyxin B. Bacillus
subtilis and Bacillus licheniformis are the source of bacitracin (Shagel et al, 2007; Hugo et al,
1992).
2.4.4 Pseudomonas
Pseudomonas aeruginosa is a Gram-negative rod and has in recent years assumed the
role of a dangerous pathogen. It has long been a troublesome secondary infection of wounds,
especially burns, but was not necessarily pathogenic. With the advent of immunosuppressive
therapy following organ transplant, systemic infections including pneumonia have resulted from
infection by this organism. It has also been implicated in eye infections resulting in the loss of
sight. It is resistant to many antibacterial agents and is biochemically very versatile, being able to
use many disinfectants as food sources (Shagel et al, 2007; Hugo et al, 1992).
2.4.5 Escherichia
Escherichia are Gram-negative organisms. Escherichia coli and the organisms
(Salmonella, Shigella, Proteus, Serratia marcescens, Klebsiella) are known as enterobacteria, so
called because they inhabit the intestines of humans and animals. They are of great significance
in public health. Escherichia coli is the cause of enteritis in young infants and the young of farm
animals where it can cause diarrhoea and fatal dehydration. It is a common infectant of the
urinary tract and bladder in humans, and is a cause of pyelitis, pyelonephritis and cystitis (Shagel
et al, 2007; Hugo et al, 1992).
2.4.6 Salmonella
Salmonella are Gram-negative rods. Salmonella typhi causal organism of typhoid fever,
Salmonella paratyphi causes paratyphoid fever whilst Salmonella typhimurium, Salmonella
enteritidis and very many other closely related organisms are a cause of bacterial food poisoning
(Shagel et al, 2007; Hugo et al, 1992).
2.4.7 Proteus
These are also gram-negative rods. Proteus vulgaris and morganii can infect the urinary
tract of humans. They are avid decomposers of urea producing ammonia and carbon dioxide.
These organisms occasionally cause wound infection (Shagel et al, 2007; Hugo et al, 1992).
2.4.8 Klebsiella
Klebsiella pneumonia subspecies aerogenes is found in the gut and respiratory tract of
man and animals, and in soil and water. It can give rise to acute bronchopneumonia in humans
but is not a common pathogen (Shagel et al, 2007; Hugo et al, 1992).
2.5
ANTIBIOTICS
An antibiotic is a compound or substance that kills or slows down the growth of bacteria
(Nussbaum, 2006). However, with increased knowledge of the causative agents of various
infectious diseases, antibiotic(s) has come to denote a broader range of antimicrobial
compounds, including anti-fungal and other compounds (Nussbaum, 2006). The term
"antibiotic" was coined by Selman Waksman in 1942 to describe any substance produced by a
microorganism that is antagonistic to the growth of other microorganisms in high dilution
(Waksman, 1947). This definition excluded substances that kill bacteria but are not produced by
microorganisms (such as gastric juices and hydrogen peroxide). It also excluded synthetic
antibacterial compounds such as the sulfonamides. Many antibacterial compounds are relatively
small molecules with a molecular weight of less than 2000 atomic mass units.
With advances in medicinal chemistry, most of today's antibiotics chemically are
semisynthetic modifications of various natural compounds (Nussbaum, 2006). These include, for
example, the beta-lactam antibacterials, which include the penicillins (produced by fungi in the
genus 'Penicillium'), the cephalosporins(e.g. cefaclor), and the carbapenems. Compounds that are
still isolated from living organisms are the aminoglycosides (e.g. amikacin), whereas other
antibacterials—for example, the sulfonamides (e.g. sulphamethoxazole), the quinolones (e.g.
ciprofloxacin), and the oxazolidinones—are produced solely by chemical synthesis. Accordingly,
many antibacterial compounds are classified on the basis of chemical/biosynthetic origin into
natural, semisynthetic, and synthetic. Another classification system is based on biological
activity. In this classification antibacterials are divided into two broad groups according to their
biological effect on microorganisms: bactericidal agents kill bacteria, and bacteriostatic agents
slow down or stall bacterial growth.
The chemical structures of some of the named antibiotics are illustrated below.
2.5.1 Mechanisms of action of antimicrobial agents
The penicillins inhibit bacterial growth by interfering with the transpeptidation reaction
of bacterial cell wall synthesis. Beta-lactamase production by microbes is the commonest form of
resistance to penicillins. Cephalosporins are similar to penicillins, but more stable to many
bacterial Beta-lactamases and therefore have a broader spectrum of activity. Tetracyclines are
broad-spectrum bacteriostatic antibiotics that inhibit protein synthesis. Macrolide antibiotics such
as erythromycin can act as bacteriostatic or bactericidal depending on the concentration. They act
by binding to the 50S ribosomal RNA and inhibit protein synthesis. The aminoglycosides are
irreversible inhibitors of protein synthesis (Katzung, 2007)
2.5.2 Assay of antibiotics
In microbiological assays, the response of a growing population of microorganisms to the
antimicrobial agent is measured. An example is the agar diffusion assay. In that type of assay,
the drug diffuses into agar seeded with a susceptible microbial population and produces a zone of
growth inhibition. For two and three-dimensional assays, samples to be assayed are applied in
some form of reservoir (cup or well) to a thin layer of agar seeded with indicator organism. The
drug diffuses into the medium and after incubation a zone of inhibition forms as a circle around
the reservoir. All other factors being constant, the diameter of the zone of inhibition is within,
limits, related to the concentration of the antibiotic in the reservoir (Hugo et al, 1992).
During incubation, the antibiotic diffuses from the reservoir and that part of the microbial
population away from the influence of the antibiotic increases by cell division. The edge of a
zone is formed when the minimum concentration of antibiotic which will inhibit the growth of
the organism on the plate (critical concentration) reaches for the first time a population density
too great for it to inhibit. The position of the edge is thus determined by the initial population
density, growth rate of the organism and the rate of diffusion of the antibiotic (Hugo et al, 1992).
2.6
ANTIOXIDANT ACTIVITY OF PLANTS
Polyphenols (electron-rich compounds) have the ability to go into electron-donation
reactions with oxidizing agents to form stable species (Kang et al., 2005) and thus inhibit or
delay the oxidation of different biomolecules (Amarowicz, 2005; Seidel et al., 2000). Hence
various plant phenols such as vitamin E (α-tocopherol), exhibit antioxidant properties (Kang et
al., 2005; Ozgova et al., 2003; Seidel et al., 2000). Phenolic antioxidants are potent free radical
terminators and this is thought to be due to the ability to donate hydrogen to free radicals and
their presence is a good marker of potential antioxidant activity. The high potential of phenolic
compounds to scavenge free radicals may be explained by their phenolic hydroxyl groups.
Detection of phenols in an extract is a preliminary evidence of its possible antioxidant activity.
The total phenol assay is based on the reduction of phosphomolybdate-phosphotungstate
salts to form a blue complex that is detected quantitively at 760 nm. The reagent used is FolinCicoalteu’s phenol reagent (hexavalent phosphomolydic/phosphotunstatic acid complexes)
illustrated below:
3H2O•P2O5•13WO3•5MoO3•10H2O
3H2OxP2O5•14WO3•4MoO3•10H2O
Folin-Ciocoalteu’s phenol reagent does not contain phenol. Rather, the reagent will react with
phenols and non-phenolic reducing substances to form chromogens that can be detected
spectrophotometrically.
The phosphomolybdenum method of assay of the total antioxidant capacity is based on
the reduction of Mo (VI) to Mo (V) by the antioxidant compound and the formation of a green
phosphate/ Mo (V) complex with a maximal absorption at 695nm. Prieto et al., 1990
successfully used this method to quantify vitamin E in plant extracts. The study revealed that the
antioxidant activity of the extract increased with increasing concentration. This suggests the
presence of vitamin E in the plant extract. Other compounds that might contribute to the total
antioxidant capacity includes carotenoids, flavonoids and cinnamic acid derivatives (Taga et al.,
1984). In this test, ammonium phosphomolybdate, (NH4)3PMo12O40, is formed. This compound
is able to accept more electrons from ascorbic acid or other donors to form a mixed valence
complex that can be detected spectrophotometrically.
PMoVI12O403− + 4e- ⇌ PMoV4MoVI8O407−
A number of in vitro models have been used for the assessment of antioxidant properties
of pharmacologically active agents. Antioxidants may be classified according to their chemical
nature and mode of function. Based on their mode of action, three types have been found to be
consistent; enzyme antioxidants; the preventive antioxidants (Cui et al., 2004), and the
scavenging or chain-breaking antioxidants (Scheibmeir et al., 2005 Reiter, 1997; Shen et al.,
2002). The reducing power measurement, as described by Oyaizu, investigates the ability of an
agent to transform Fe3+ to Fe2+. Other authors have observed a direct correlation between
antioxidant activity and reducing power of certain plant extracts (Duh, 1998; Tanaka et al.,
1988). These reducing properties are generally associated with the presence of reductones (Duh,
1998) which have been shown to exert antioxidant action by breaking the free radical chain
reaction by donating a hydrogen atom (Gordon, 1990). Reductones are also reported to react with
certain precursors of peroxides, thus preventing peroxide formation. The activity of antioxidants
has been attributed to various mechanisms, among which are prevention of chain initiation,
binding of transition metal ion catalysts, decomposition of peroxides, prevention of continued
hydrogen abstraction, reductive capacity, and radical scavenging (Diplock, 1997).
CHAPTER THREE
3
MATERIALS AND METHODS
This chapter addresses two sections; materials and methods.
3.1
MATERIALS
This section encompasses the plant material collection, chemicals and the preparation of
glassware for the work.
3.1.1 Collection of plant material
The stem of the plant, Strophantus gratus was collected from the Botanical Gardens,
Kwame Nkrumah University of Science and Technology, Kumasi, Ghana in October, 2009. It
was then authenticated by Prof. T.C. Fleischer at the Department of Pharmacognosy, Kwame
Nkrumah University of Science and Technology (KNUST), Kumasi.
3.1.2 Chemicals
98% ethanol was from Joseph Mills Ltd (UK). Ascorbic acid, ammonium molybdate,
ferric chloride, n-propyl gallate, potassium ferricyanide, tannic acid, thiobarbituric acid (TBA),
trichloroacetic acid (TCA) and Folin-Ciocalteau reagent were from Sigma-Aldrich Inc. (St.
Louis, MO, USA). Ciprofloxacin was from Dabur Pharma (New Delhi, India). Nutrient agar and
nutrient broth were from Microtrade Ltd (UK). Phytochemical reagents were mostly from
ReAgent Manufacturing Ltd (UK).
3.1.3 General cleaning and sterilization of glassware
All glassware were washed thoroughly with soap solution, rinsed with distilled water and
dried before used. Glassware such as pipettes and spatula were disinfected with Dettol antiseptic
solution, packed into a canister and autoclaved at 121oC for 15 minutes. Petri dishes were
washed, rinsed and packed into suitable canisters to dry. They were then sterilized in an oven at
170oC for 1 hour.
3.2
METHODS
This section covers methods for the preparation of the extracts from Strophanthus gratus,
phytochemical screening of raw plant material and extracts, thin layer chromatography, infra red
spectroscopy, antibiotic activity and antioxidant activity of the extract.
3.2.1 Preparation of extracts
The stems of the Strophanthus gratus were air-dried for 60 days and powdered. For cold
maceration, 50g of the air-dried coarsely powdered material was weighed into a flat bottom
flask. The sample was macerated with 1250mL of the solvent for 24hours at room temperature.
The flask was gently shaken at 2hours intervals for the first 6hours and allowed to stand for 12h
without shaking.
For Soxhlet extraction, accurately 50g of the air dried coarsely powdered material was
accurately weighed into a clean thimble and placed in the column of the soxhlet. 1250ml of
water was added in 250ml batches. After each addition the column was covered with the
condenser and the heating mantle turned on. The total extraction time was 18 hours. The extracts
were concentrated in a rotary evaporator apparatus at approximately 60 °C. The concentrated
extracts were kept in a desiccator until analyses . The procedure was repeated for three more 50g
batches of the powder. The mass of the dried powdered samples and the extracts were weighed
and the percentage yields were calculated as follows.
% yield= mass of extract x 100%
Mass of sample
The procedure was repeated using ethanol (98%) as the solvent.
3.2.2 Phytochemical screening
The phytochemical screening was conducted as per Trease and Evans Pharmacognosy
(Evans, 1996).
3.2.2.1 Test for saponins
2g of extract sample was weighed and boiled in 10ml distilled water for 3-5 minutes. It
was then filtered hot and shaken vigorously. A separation of froth after shaking, which persisted
after some time indicated the presence of saponins, otherwise saponins were absent.
3.2.2.2 Test for general glycosides
0.5g of the extract sample was put into two separate beakers and dried at 60 0C. 5ml of
dilute sulphuric acid was added to one beaker and 5ml of distilled water to the other and heated
on a boiling water bath for 3-5 minutes and contents filtered into two separate test tubes.
The two filterates were then cooled and made alkaline with NaOH solution and heated with
Fehlings solution for 3 minutes. The formation of reddish-brown precipitation in the test tube
containing the filtrate from H2SO4 treatment and the absence of precipitate in the other test tube
indicated the presence of glycosides, otherwise glycosides were absent.
3.2.2.3 Test for flavonoids
(a) A small amount of magnesium ribbon was added to an alcoholic solution of the sample
which was followed by the addition of concentrated HCl dropwise. A brick-red
colouration indicates the presence of flavonoids, otherwise flavonoids are absent.
(b) A small amount of the sample was put on filter paper that has been moistened with dilute
NH3 and viewed under U.V. light. A blue colouration indicated that flavonoids were
present, otherwise flavonoids were absent.
3.2.2.4 Test for terpenoids and steroids
(a) A small amount of the extract was evaporated to dryness in a crucible and redissolved in
chloroform. A few drops of acetic anhydride were added followed by two drops of
concentrated H2SO4. Reddish-pink colouration indicated the presence of terpenoids and
steroids.
(b) A small amount of concentrated H2SO4 was added to a solution of the extract in
chloroform. Red colouration in the chloroform layer indicated terpenoids and steroids.
3.2.2.5 Test for carotenoids
a) About 3ml of antimony trichloride was added to 2ml of the extract. Dark-blue colouration is
indicative of carotenoids and vice versa
b) About 1ml of concentrated H2SO4 was added carefully to 2ml of the extract which formed a
layer under the ethereal solution. The presence or absence of an intense dark-blue or blue-voilet
or greenish-blue colour in the acid layer showed the presence or absence of carotenoids.
3.2.2.6 Test for coumarin
About 3mL of the extract was put into a test tube. The test tube was then covered with a piece of
filter paper moistened with dilute NaOH solution and placed in a hot water bath. After about 15
minutes, the paper was removed and exposed to UV light. Yellow- green fluorescence indicates
the presence of coumarins, otherwise coumarins are absent.
3.2.2.7 Test for alkaloids
10ml of 1% HCl was added to about 2mL of the extract and left to stand for about 30mins
stirring occasionally. The resulting solution was filtered and some few mL of saturated picric
acid was added to 2ml portions of the filtrate. The formation or absence of precipitate indicated
the presence or absence of alkaloids respectively.
3.2.2.8 Test for anthraquinones
A small amount of sample was boiled with 25ml of 0.5M KOH and 4ml of H2O2. The mixture
was cooled, filtered and acidified with a few drops of acetic acid. The acidulated mixture was
extracted with about 15ml of benzene. The benzene was shaken with a small amount of NH4OH.
The formation of red colouration indicated anthraquinone, otherwise anthraquinones are absent.
3.2.2.9 Test for anthraquinone glycosides
20ml of dilute H2SO4 was added to 2mL of the extract and boiled boiled. The mixture was
filtered hot and a portion of the cooled filtrate was shaken with equal volume of benzene. The
benzene layer was separated and shaken with about half its volume of dilute NH 3 solution. A
colourless ammoniacal layer indicates the absence of anthraquinone glycoside and vice versa.
3.2.2.10 Test for cyanogenetic glycoside
A sodium picrate paper was prepared by saturating a strip of filter paper in a solution of 0.5g
Na2CO3 and 0.5g of picric acid dissolved in 100ml of water. The paper was then blotted to dry.
2mL of the extract was placed in a test tube. The material was allowed to hydrolyze (with dil.
HCl) in a stoppered test tube. A few drops of chloroform were then added and the piece of moist
sodium picrate paper was inserted into the test-tube, taking care that it does not come into
contact with the material or touch the inner sides of the tube. The test-tube along with its
contents was kept warm at 35oC for about 3 hours. The presence of the red colour of the sodium
picrate paper after the 3 hours is taken as a positive test for cyanogenetic glycoside and vice
versa.
3.2.2.11 Test for cardiac glycosides
To 2mL of extract was added 2mL of glacial acetic acid containing one drop of ferric chloride
solution. This was underlayed with 1mL of concentrated sulphuric acid. A brown ring at the
interface indicated the presence of a deoxysugar characteristic of cardenolides. A violet ring
appeared below the brown ring, while in the acetic acid layer a greenish ring formed just above
the brown ring and gradually spread throughout this layer.
3.2.2.12 Test for tannins
a) About 0.5 g of the extract was boiled in 10 ml of water in a test tube and then filtered. A few
drops of 0.1% ferric chloride was added and observed for brownish green or a blue-black
colouration.
b) To an aliquot of the extract (dissolved in water), 2ml of sodium chloride (2%) was added,
filtered and mixed with 5 ml 1% gelatin solution. The presence or absence of precipitation
indicated the presence or absence of tannins respectively.
3.2.3
Thin layer chromatography
The extracts (both water and ethanol extracts) were spotted on silica gel plates 1cm from
the bottom and 8cm from the edge. The spots were made concentrated by repeatedly touching the
plates but ensuring that they were as small as possible (1mm in diameter). The plates were then
developed with ethanol, water, acetone and combinations of the three solvents. The developed
plates were exposed first to UV light and secondly to Iodine crystals and measurements taken.
3.2.4
IR spectrophometry
1.7mg of the dry extract and 200mg of spectroscopic-grade KBr were weighed and
transferred into a stainless steel capsule containing a ball bearing. The capsule was shaken for 2
min on a Wig-L-Bug. The sample was evenly distributed over the face of a 13-mm die and
subjected to a pressure of 14,000psi for 4mins while under vacuum in a hydraulic press to
produce a transparent disk which was placed in the IR spectrophotometer and the spectrum run.
3.2.5
Preparation of solutions of extract
2.5g of each extract was weighed and dissolved in 10ml of methanol to obtain 25%w/v
solution. By the method of serial dilution, 15%, 10%, 5%w/v extract solutions were also
prepared. For the water extract, additional concentrations of 50%, 30% and 20% were later
prepared for further investigations. 0.7g of ciprofloxacin powder was weighed and dissolved in
10ml of water to obtain 7%w/v solution. Serial dilution was used to obtain 5%, 3% and 1%
solutions.
3.2.6 Preparation of media
Sterile distilled water was used in the preparation of the nutrient agar and nutrient broth
as follows.
3.2.6.1 Sterile distilled water
Sterile distilled water was prepared by autoclaving distilled water in sealed glass bottles at 121
degree Celsius for 30 minutes.
3.2.6.2 Nutrient agar
28g of nutrient agar powder was weighed and dissolved in 1 liter of distilled water. It was then
allowed to soak for 10 minutes; swirled to mix then sterilized by autoclaving for 15 minutes at
121oC.
3.2.6.3 Nutrient broth
32.5g of nutrient broth powder was weighed and dissolved in 1 liter of distilled water. It was
then allowed to soak for 10 minutes; swirled to mix then sterilized by autoclaving for 15 minutes
at 121oC.
3.2.7 Antimicrobial activity tests
The tests were done as described by Manual of Microbiology (Sawer, 2002). They are
described in the following subsections.
3.2.7.1 Preparation of broth culture
10ml of nutrient broth was inoculated with pure culture of test organism. It was rolled in the
palms for even mixing of contents. The process was repeated for the other test organisms. The
seeded broth was incubated at 37oC for 24 hours. These were pure broth cultures of the
respective test organisms. The following test organisms were used: Enterococcus faecalis, Ps
aeruginosa, Proteus vulgaris, Staph aureus, Bacillus subtilis, Enterococcus coli, Bacillus
thuringiensis, Salmonella typhi, Neisserria gonnorrhoeae
3.2.7.2 Preparation of nutrient agar culture
The experiment was performed as described by the Manual of Microbiology (Sawer, 2002).
20ml nutrient agar in test tubes were melted in boiling water and stabilized at 45oC in
thermostatic water bath for 15 minutes. Each of the molten agar was inoculated with 0.1ml of the
24-hour broth culture of test organism. They were rolled in the palms for even mixing of
contents. The seeded agar were poured into separate sterile petri dishes and allowed to set. Using
sterile cork borer six (6), 4 equidistant cups were created in each of the set agar and the cups
labeled with the four prepared concentrations of the extract. Each cup was filled with the
respective concentration of the crude extract or ciprofloxacin (the standard drug) to three-fourth
full. The plates were covered and left on the Laminar Horizontal Flow Table for one hour for the
extract to penetrate the agar. The plates were then incubated at 37oC for 24 hours.
The zones of growth inhibition were measured and the mean zones of growth inhibition
calculated. The experiment was done in triplicate for each test organism.
Control: Two control experiments were performed. In one control, the agar was not
seeded with test organism. In the other control, the agar was seeded with test organism but the
solvent (methanol) was used in place of the extract solutions.
3.2.8 Antioxidant activity tests
The ethanol extract was used for the antioxidant tests because it gave better antimicrobial activity
test results. The antioxidant tests done included the total phenol assay, total antioxidant capacity
and the reducing power assay.
3.2.8.1 Preparation of solutions
Tannic acid solutions: 3.0003g of Tannic acid powder (99.99%w/w) was weighed and dissolved
in 10mL of distilled water to obtain 0.3mg/mL solution. By the method of serial dilution,
densities of 0.10mg/mL, 0.03mg/mL and 0.010mg/mL of tannic acid solutions were also
prepared.
Ascorbic acid solutions: 3.0303g of ascorbic acid crystals (99.0%w/w) was weighed and
dissolved in 10mL of distilled water to obtain 0.3mg/mL solution. By the method of serial
dilution, densities of 0.10mg/mL, 0.03mg/mL and 0.010mg/mL of ascorbic acid solutions were
also prepared.
n-propyl gallate: 1.2245g and 0.9184g of n-propyl gallate crystals (98%w/w) were weighed and
each one dissolved in 10mL of water to obtain 0.12mg/mL and 0.09mg/mL solutions
respectively. By the method of serial dilution, 0.06, 0.03, 0.01, 0.003 and 0.001mg/mL solutions
were also prepared.
3.2.8.2 Total phenol assay
The total soluble phenols present in the extract was quantitatively determined by colorimetric
assay using the Folin-Cicocalteu’s phenol reagent (Singleton, 1977). Tannic acid (0.01, 0.03, 0.1
and 0.3 mg/mL) was used as the standard drug and water was used to prepare the blank. 1ml of
each extract solution (0.1, 0.3, 1 and 3mg/mL), 1mL each of the tannic acid solutions and 1mL
of water (for the blank) were separately added to 1 ml of Folin-Cicocalteu’s phenol reagent
(diluted five fold in distilled water) in test tubes. The content of the test tubes were mixed and
allowed to stand for five minutes at 25
o
C in the incubator. 1ml of 2 % sodium bicarbonate
solution was added to each mixture. The reaction mixtures were then incubated at 25 oC for 2
hours. The mixtures were then centrifuged at 3000 rpm for 10 min to obtain a clear supernatant.
The absorbance of the supernatants were then determined in triplicates at 760 nm using the UVvisible spectrophotometer (LKB Biochrom, Cambridge, England, Model 4050) against the blank
solution. Tannic acid absorbances were plotted against tannic acid concentrations to obtain a
calibrated concentration absorbance curve using MicroSoft Excel 2007 edition. The absorbances
of the extract solutions were used to deduce the tannic acid equivalents (TAE) from the plot.
3.2.8.3 Total antioxidant capacity assay
Ascorbic acid was used as the standard antioxidant drug and water was used to prepare the blank.
3 mL each of the extract solutions (0.1, 0.3, 1 and 3 mg/mL) and 3mL each of the ascorbic acid
solutions (0.010, 0.03, 0.10, 0.3mg/mL) and 3mL of water (for the blank) were placed in separate
test tubes. 0.3 mL of the reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4
mM ammonium molybdate) was then added to each and the resulting mixtures were incubated at
95°C for 90 min. After the mixtures had cooled to room temperature, the absorbance of each
solution was measured in triplicate using the UV-visible spectrophotometer (LKB Biochrom,
Cambridge, England, Model 4050) at 695 nm against the blank solution. The measurements were
done in triplicates. The absorbances of the ascorbic acid solutions were plotted against ascorbic
acid concentrations to obtain a calibrated concentration absorbance curve using MicroSoft Excel
2007 edition. The absorbances of the extract solutions were used to deduce the ascorbic acid
equivalents (AAE) from the plot.
3.2.8.4 Reducing Power
The reducing capacity of the extract was determined using the method of Fe 3+ -reduction to
Fe2+(Oyaizu, 1986). n-propyl gallate (0.001, 0.003, 0.01, 0.03, 0.06, 0.09 and 0.12 mg/mL ) was
used as the standard antioxidant drug and water was used as the blank. 1mL each of the extract
solutions (0.03, 0.1, 0.3, 1, 2, 3 and 4 mg/mL) and n-propyl gallate as well as water (for the
blank) were placed into separate test tubes. Each test tube content was mixed with 2.5 ml of 0.2
M sodium phosphate buffer (pH 6.6) and 2.5 ml of 1 % potassium ferricyanide solution. The
mixture was incubated at 50oC for 20 min. Following this, 1.5 ml of 10 % trichloroacetic acid
solution (TCA) was added to the incubated mixture, and centrifuged at 3000 rpm for 10 min. The
supernatant (2.5 ml) from each mixture was then mixed with 2.5 ml distilled water and 0.5 ml of
the 0.1 % ferric chloride solution (FeCl3 (aq)) in a test tube. The absorbance was then measured at
700 nm using the UV-visible spectrophotometer (LKB Biochrom, Cambridge, England, Model
4050). The absorbance measurements were done in triplicates. Data was presented as
concentration-absorbance curves with MicroSoft Excel, 2007 edition and the EC50 (concentration
that gives 50% of maximal response) determined.
CHAPTER FOUR
4
RESULTS AND DISCUSSION
The results obtained for phytochemical screening, extraction, antimicrobial and antioxidant tests
are provided in the following subsections. The results for IR spectroscopic measurements and
thin layer chromatography are also provided. The results are further discussed.
4.1 RESULTS
The results are presented in the form of tables and graphs in the following subsections.
4.1.1 Results for phytochemical screening
The results obtained for the raw plant material, cold maceration with water and ethanol and hot
extraction are presented in table 4.1.1 below.
Table 4.1.1: Results for phytochemical screening
TEST
INFERENCE
RELATIVE INTENSITY
raw plant
material
cold
water
extract
cold ethanol
extract
hot water
extract
hot ethanol
extract
Saponins
Present
+
-
+
+
General
glycoside
Present
-
-
+
+
Flavonoids
Present
-
+
+
+++
Steroids and
Terpenoids
Present
+
+
+
+
Carotenoids
Absent
-
-
-
-
Table 4.1.1: Results for phytochemical screening
TEST
INFERENCE
RELATIVE INTENSITY
raw plant
material
cold
water
extract
cold ethanol
extract
hot water
extract
hot ethanol
extract
Coumarins
Absent
-
-
-
-
Alkaloids
Present
+
+
++
++
Anthraquinones
Absent
-
-
-
-
Anthraquinone
glycoside
Present
-
-
+
+
Cyanogenic
glycosides
Present
-
-
+
+
Tannins
Present
++
+
+++
+
Cardiac
glycoside
Present
-
-
+
+
4.1.2
Results for extraction
The extraction yields obtained are presented in table 4.1.2 below
Table 4.1.2 Extraction yields
METHOD
cold maceration
EXTRACT water extract
WEIGHT/g
4.9651
% YIELD
9.93
ethanol extract
3.2614
6.52
soxhlet extraction
water extract
12.2312
24.46
ethanol extract
5.8434
11.69
4.1.3 Results for thin layer chromatography
The results obtained for the thin layer chromatography conducted are presented in table 4.1.3
below. The Solvent system used is ethanol: water: acetone in the ratio of 6:3:1 and the solvent
front was 8.4cm.
Table 4.1.3: Results for thin layer chromatography
SEPARATIONS
AVERAGE SAMPLE
FRONT/cm
water
ethanol
Rf VALUE
water
ethanol
A
0.5
0.4
0.06
0.05
B
3.2
3.2
0.38
0.38
C
5.0
5.1
0.60
0.61
D
7.4
7.4
0.88
0.88
E
8.1
8.2
0.96
0.98
4.1.4 Results for antibiotic activity tests
The results obtained for antimicrobial activity tests with both extracts and ciprofloxacin are
presented in table 4.1.4a, table 4.1.4b and table 4.1.4c. The results have also been presented
graphically in figures 4.1a, 4.1b, 4.2a, 4.2b, 4.3a and 4.3b. The Minimum Inhibitory
Concentrations obtained from the graphs are further presented in tables 4.1.5a and 4.1.5b.
Table 4.1.4a: Antibiotic activity test results for ethanol extract
ZONE OF GROWTH INHIBITION/mm AT DIFFERENT
TEST ORGANISM
EXTRACT CONCENTRATIONS
25%w/v
15%w/v
10%w/v
5%w/v
E faecalis
27.0
25.5
24.0
22.5
Ps aeruginosa
25.0
23.5
21.0
20.0
Pr vulgaris
28.0
26.0
22.0
20.0
Staph aureus
22.0
21.5
19.0
18.0
B subtilis
29.0
25.5
22.5
20.0
E coli
18.0
17.0
15.0
12.5
B. thuringiensis
28.0
27.0
24.0
21.5
Salmonella typhi
26.5
23.0
21.0
16.0
N. gonorrhoeae
22.0
20.0
14.0
11.0
Table 4.1.4b: Antibiotic activity test results for water extract
ZONE OF GROWTH INHIBITION/mm AT DIFFERENT
TEST ORGANISM
EXTRACT CONCENTRATIONS
50%w/v
30%w/v
20%w/v
10%w/v
E faecalis
28.0
26.0
23.5
21.0
Ps aeruginosa
24.5
23.5
20.0
19.0
Pr vulgaris
28.0
25.5
22.0
20.0
Staph aureus
23.0
21.5
19.0
17.0
B subtilis
28.0
25.0
23.0
19.5
E coli
21.5
17.5
15.0
14.0
B thuringiensis
28.0
25.0
22.5
21.0
Salmonella typhi
25.0
24.0
21.5
14.0
Neisseria gonorrhoeae
25.0
21.0
16.0
12.0
Table 4.1.4c: Antibiotic activity test results for ciprofloxacin
TEST ORGANISM
ZONE OF GROWTH INHIBITION/mm AT DIFFERENT
CIPROFLOXACIN CONC.
7%w/v
5%w/v
3%w/v
1%w/v
E. faecalis
35.0
30.0
26.0
24.0
Pr vulgaris
30.5
26.0
22.0
20.0
Staph aureus
20.0
17.0
15.5
13.0
B subtilis
25.0
22.0
17.5
14.0
E coli
24.5
23.0
19.0
18.5
B. thuringiensis
21.5
16.5
14.0
12.5
S. taphyi
36.0
32.5
28.0
26.5
Ps. Aeruginosa
20.0
17.0
15.0
14.0
Neisseria gonorrhoeae
34.5
30.0
28.5
25.5
Figure 4.1.4a: Plots of Zone of Growth Inhibition against Log Concentration of Ethanol Extract
Zone of Growth 30
Inhibition/mm
25
E. faecalis
Ps. aeruginosa
Pr. vulgaris
Staph aureus
B subtilis
20
15
2
R = 0.9884
2
R2 = 0.9064
R = 0.9347
2
R = 0.9480
2
R = 0.9716
10
5
-3
-2.5
-2
-1.5
-1
-0.5
0
0
0.5
1
1.5
Log [Ethanol extract]
Figure 4.1.4b: Plots of Zone of Growth Inhibition against Log Concentration of Ethanol Extract
Zone of Growth 30
Inhibition/mm
25
E. coli
B. thuringiensis
S. typhi
N. gonorrhoeae
20
15
2
R = 0.9617
R2 = 0.9796
10
R2 = 0.9961
R2 = 0.9374
5
0
-2
-1
0
1
2
Log [Ethanol extract]
Figure 4.1.4c: Plots of Zone of Growth Inhibition against Log Concentration of Water Extract
Zone of Growth 30
Inhibition/mm
25
E. faecalis
Ps. aeruginosa
Pr. Vulgaris
20
Staph aureus
2
R 2 = 0.8957
R = 0.9899
2
R = 0.9884
2
R = 0.9593
15
10
5
-1.5
-1
-0.5
0
0.5
1
1.5
2
Log [Water extract]
Figure 4.1.4d: Plots of Zone of Growth Inhibition against Log Concentration of Water Extract
B. subtilis
Zone of Growth 30
Inhibition/mm
E. coli
25
B. thuringiensis
20
S. typhi
15
N. gonorrhoeae
10
2
R = 0.9438
2
R = 0.9984
2
R = 0.8840
2
5
-1
-0.5
R = 0.9068
2
R = 0.9797
0
0.5
1
1.5
2
Log [Water extract]
Figure 4.1.4e: Plots of Zone of Growth Inhibition against Log Concentration of Ciprofloxacin
E. faecalis
Ps. aeruginosa
Pr. vulgaris
Staph aureus
B subtilis
Zone of Growth35
Inhibition/mm 30
25
20
2
R = 0.9834
15
2
R = 0.9341
R2
10
= 0.9778
5
2
R = 0.8160
2
R = 0.9064
0
-0.8
-0.5
-0.2
0.1
0.4
0.7
1
Log [Ciprofloxacin]
Figure 4.1.4f: Plots of Zone of Growth Inhibition against Log Concentration of Ciprofloxacin
Zone of Growth
35
Inhibition/mm
30
E. coli
B. thuringiensis
S. typhi
25
N. gonorrhoeae
20
R2 = 0.9747
R2 = 0.9245
15
R2 = 0.8724
10
2
R
= 0.9483
5
-1.2
-0.9
-0.6
-0.3
0
0.3
0.6
0.9
Log[Ciprofloxacin]
Table 4.1.4e: Minimum Inhibitory Concentrations (MIC) and Ratios
TEST ORGANISM
MIC/ %w/v
Ethanol Extract Water Extract
MIC RATIO
Water Extract: Ethanol Extract
B. Subtilis
0.1682
0.2622
1.5589
Pr. vulgaris
0.1204
0.2233
1.8547
0.01
0.0652
6.5200
Staph aureus
0.0066
0.1239
18.7727
E. faecalis
0.0020
0.0898
44.9000
N. gonorrhoeae
1.2083
2.4848
2.0564
S. typhi
0.3882
1.1431
2.9446
E. coli
0.1328
0.5853
4.4074
B. thuringiensis
0.0311
0.0948
3.0482
Ps. aeruginosa
Table 4.1.4f: Minimum Inhibitory Concentrations and Ratios
MIC/ %w/v
TEST ORGANISM
Cipro.
MIC RATIO
Cipro :Ethanol Extract
Cipro: Water Extract
B. Subtilis
0.7335
4.3609
2.7975
Pr. vulgaris
0.3513
2.9178
1.5732
Ps. aeruginosa
0.2123
21.2300
3.2561
Staph aureus
0.3380
51.2121
2.7280
E. faecalis
0.2294
114.7000
2.5546
N. gonorrhoeae
0.0873
0.0723
0.0351
S. typhi
0.1266
0.3261
0.1108
E. coli
0.1119
0.8426
0.1912
B. thuringiensis
0.6090
19.5820
6.4241
4.1.5 Results for antioxidant activity tests
The results of the antioxidant activity tests are detailed in tables 4.1.5a, 4.1.5b, 4.1.5c
Table 4.1.5a: Total Phenolic Content test results
DETERMINATION
TANNIC ACID
S. GRATUS
Concentration in mg/ml
0.0100 0.0300 0.1000 0.3000 0.1000 0.3000 1.0000 3.0000
Mean Absorbance
0.0125 0.0477 0.1261 0.3882 0.0251 0.0751 0.2620 0.7862
Total Phenolic Content in
mg TAE/ml
0.0216 0.0606 0.2064 0.6153
Figure 4.1.5a(A): A plot of Absorbance of tannic acid against concentration of tannic acid
A
2
R = 0.9970
Mean 0.5
Absorbance
0.4
0.3
0.2
0.1
0
0
0.1
0.2
0.3
0.4
[Tannic Acid]/ mg/mL
Figure 4.1.5a(B): A plot of the total phenolic content present in S. gratus expressed as tannic
acid equivalent (TAE) against concentration of S. gratus.
B
Total
0.8
Phenolic
Content/
mgTAEmL-1
0.6
2
R =1
0.4
0.2
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
[S. gratus]/mg/mL
Table 4.1.5b: Total antioxidant capacity test results
DETERMINATION
ASCORBIC ACID
S. GRATUS
Concentration in mg/ml
0.0100
0.0300
0.1000
0.3000
0.1000
0.3000
1.0000
3.0000
Mean Absornbance
0.0214
0.0642
0.2241
0.7422
0.0121
0.0486
0.0593
0.2371
0.0074
0.0174
0.0548
0.1596
Total Antioxidant Capacity in mg
TAE/ml
Figure 4.1.5b (A): A plot of the Absorbance of PMoV4MoVI8O407−(formed in ascorbic acid
solutions) against concentration of ascorbic acid.
A
0.8
Mean
Absorbance of
PMoV4MoVI8O407−
0.6
2
R = 0.9991
0.4
0.2
0
0
0.1
0.2
0.3
0.4
[Ascorbic Acid]/mg/mL
Figure 4.1.5b(B) A plot of the total antioxidant capacity (TAC, expressed as Ascorbic acid
equivalent - AAE) of S. gratus against concentration of S. gratus
B
0.20
Total Antioxidant
Capacity (TAC)/
0.15
mg AAE/mL
2
R =1
0.10
0.05
0.00
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
[S gratus]/mgmL-1
Figure 4.1.5c: A plot of total phenolic content (TPC) expressed as tannic acid equivalents (TAE)
against total antioxidant capacity (TAC) expressed as ascorbic acid equivalents (AAE) of S.
gratus.
0.70
Total Phenolic
Content/
0.60
mgTAE/mL
0.50
R2 = 1
0.40
0.30
0.20
0.10
0.00
0.00
0.05
0.10
0.15
0.20
Total Antioxidant Capacity/mgAAE/mL
Table 4.1.5c: Reducing Power test results for S. gratus and n-propylgallate
S GRATUS
n-PROPYL GALLATE
Log conc.
Mean absorbance of Fe2+
Log conc. Mean absorbance of Fe2+
-1.5228
0.0234
-3.0000
0.0375
-1.0000
0.0656
-2.5229
0.0656
-0.5229
0.1313
-2.0000
0.3125
0.0000
0.4875
-1.5228
0.6750
0.3010
0.5123
-1.2218
0.7522
0.4771
0.5233
-1.0458
0.7530
0.6021
0.5235
-0.9208
0.7540
Figure 4.1.5d: Reducing power of S. gratus compared to n-propyl gallate
0.8
S gratus
n-propyl gallate
Mean
Absorbance
of Fe2+
0.7
0.6
EC50 =0.0126
mgmL-1
EC50 =0.3981
mgmL-1
0.5
0.4
0.3
0.2
0.1
0
-4
-3
-3
-2
Log [Drug]
-2
-1
-1
0
-0.1
4.1.6 Results for the IR spectrophotometry
The conditions for the ethanol extract IR spectrum are:
Resolution: 4.0 cm-1
No. of scan: 40
Detector speed: 2.8mms-1
Remarks: Ethanol extract: KBr disc
Similarly, the conditions for the water extract IR spectrum are:
Resolution: 4.0 cm-1
No. of scan: 30
Detector speed: 2.8 mms-1
Remarks: Water extract: KBr disc
The IR spectra are depicted in figures 4.1.6a and 4.1.6b
1
1
Figure 4.1.6a: IR spectrum of hot ethanol extract of S. gratus
Figure 4.1.6b: IR spectrum of hot water extract of S. gratus
4.2 GENERAL DISCUSSIONS
There was an immediate change to dark coloration upon the addition of water to the S. gratus
sample. However, the addition of ethanol to the sample slightly changed the colour of ethanol.
When subjected to heat (soxhlet extraction), both solvents intensified in colour with that of water
forming foam in the process of boiling. As a result, maceration yielded low percentage of
extract; 9.93% (water extract) and 6.52% (ethanol extract). Soxhlet extraction on the other hand
yielded relatively high percentage of extract; 24.46% (water extract) and 11.69% (ethanol
extract).
For cold maceration, the water extract tested positive for saponins, alkaloids and tannins
whereas the ethanol extract tested positive for flavonoids, alkaloids and tannins. Both the water
and ethanol extracts from soxhlet extraction contained saponins, flavonoids, alkaloids,
anthraquinone glycosides cyanogenetic glycosides, tannins and cardiac glycosides. Except for
tannins and anthraquinone glycosides, the phytochemical test results are in consonant with
earlier research by Ojiako et al, 2009 on a related species, Strophanthus hispidus. However, the
intensity of coloration for the tests indicated that the water extract contained more tannins
whereas the ethanol extract contained more flavonoids. These differences are obviously due to
solubility differences. Tannins, though organic compounds are more polar because they are
polyphenolic making them more soluble in water. Flavonoids are also phenolic compounds but
the fact that the ethanol could extract more flavonoids than the water indicates that the
flavonoids present in S. gratus are less polar making them more soluble in ethanol than in water.
The percentage yields indicate that the water was able to extract about two times as ethanol
for both cold maceration and soxhlet extraction. Since the phytochemical tests indicate that the
water is able to extract more tannins than the ethanol, the differences in yields are more likely to
be due to the relative quantities of tannins extracted by the solvents. Even though, the yields
were different due to variations in the relative quantities of different phytochemical groups
extracted, the phytochemical groups extracted for the soxhlet extraction were the same. This is
evidenced by the TLC results – resolved components had similar Rf values (Table 4.1.3). This is
further supported by the similar IR profiles of the two extracts (Fig. 4.1.6a and 4.1.6b). The main
differences in the IR profiles occur at 2927.7 cm-1 where the ethanol extract had a more intense
absorption than the water extract. Such peaks are usually due to aliphatic hydrogens and are not
of much importance in antimicrobial activity. However, it indicates that the ethanol is able to
extract more non-polar components than the water.
Since the extracts contain several phytochemicals, not much information can be deduced
from the IR as there could be several overlapping peaks. However, some observations and
deductions may be made. The broad peaks near 3500cm-1 are characteristic of hydroxy
functional groups (Williamson et al, 2007) and this confirms that the extracts contain phenolic
compounds (tannins and flavonoids). The peaks occurring around 1640cm-1 may be due to
aromatic C=C since peaks between 1660 – 1600cm-1 are usually due to C=C bonds (Williamson
et al, 2007) and this confirms that the extracts contain tannins and flavonoids which are phenolic
compounds having C=C bonds.
4.3 ANTIMICROBIAL ACTIVITY
The antimicrobial activity tests results show that both the water extract and ethanolic
extract of the stem of S. gratus possess antimicrobial activity against all the test organisms used
(Table 4.1.4a, b and c). Many plants do possess antimicrobial activity (Evans, 1996). It is
therefore not strange that these extracts also possess some antimicrobial activity. The Minimum
Inhibitory Concentration (MIC) of a drug is the least concentration that can inhibit the growth of
a particular test organism (Sawer, 2002). Based on the experimental MICs (Table 4.1.4e, and f),
the ethanol extract was most potent against E. faecalis (MIC = 0.002) and least potent against
Neisseria gonorrheoae (MIC=1.2083). The order of increasing potency against the test
organisms for the ethanolic extract is in the order of N. gonorrheoae < S. typhi < B. subtilis < E.
coli < Pr. vulgaris < B. thuringiensis < Ps. aeruginosa < Staph. aureus < E. faecalis. The water
extract was however most potent against Ps. aeruginosa (MIC = 0.0652) and least potent against
N. gonorrheoae (MIC = 2.4848). The order of increasing potency against the test organisms for
the water extract is in the order of N gonorrheoae < S typhi < E coli < B subtilis < Pr vulgaris <
Staph aureus < B thuriengiensis < E faecalis < Ps aeruginosa.
The orders of potency of the extracts against the test organisms were not the same. This is
an indication that the compositions of the extracts are not the same. Earlier, qualitative tests done
on the extracts indicate that some of the phytochemicals are more soluble in water than alcohol
and vice versa. This is probably the major cause of the different orders of potency. Strikingly,
both extracts recorded the least potency against Neisseria gonorrhoeae, the organism which
causes gonorrhea, the disease the decoction of S. gratus stem is mostly used to treat in Ghana.
The extracts rather show more in vitro activity towards other common disease causing organisms
such as Pseudomonas aeruginosa and Staph aureus. Nonetheless, in vitro and in vivo results can
be totally different. Some drugs when taken orally are activated or deactivated by the liver in a
process called first pass metabolism (Shargel et al, 2007). Even though, the in vitro activities
show that the extracts are less potent towards Neisseria gonorrheoae and more potent against the
other test organisms, but in vivo activity may prove otherwise.
A two-factor ANOVA without replication (appendix 1) indicates that there were no
significant differences between the mean MICs for the various extracts and that the
susceptibilities of the organisms to the various extracts were similar. This was so because the
MICs for three organisms (namely Neisseria, E coli and S typhi) deviated from the normal
pattern of MICs whereby the MICs of ethanol extract were lesser than those of the water extract
which were also lesser than those of ciprofloxacin (Table 4.1.4e). The deviation is clearly seen
in an exploratory plot (appendix 2) of individual MICs for the various organisms from the
different extracts. The two-factor ANOVA without replication is therefore misleading as a
statistical tool in this analysis without considering other statistical analysis.
A paired t-test between the ethanol extract and the water extract (appendix 3) indicates
that there was significant differences (tcal>tcrit) between the MICs for the two extracts and the
differences are not just attributable to random errors. However, the differences are likely to be
due to the variable susceptibilities of the organisms to the extracts. The exploratory plots for both
extracts followed similar patterns and that also accounts for the high correlation (R 2 = 0.9666)
between the MICs for the water and ethanol extracts. Except for the huge deviation of N.
gonnorrhoeae, E. coli and S. typhi, the other paired t-tests would have also followed a similar
pattern. As a result of the deviations, the correlation of MICs between ethanol extract or water
extract and ciprofloxacin (appendix 4) were very poor (R2<0.3). Since an antibiotic is not
intended to be effective against all pathogenic organisms, it is very expedient to analyse the
susceptibility of the organisms to the extracts and ciprofloxacin, organism by organism.
The ratios of the Minimum Inhibitory Concentration of the aqueous extract to the
Minimum Inhibitory Concentrations of the ethanol extract for the various test organisms ranged
from 1.5 to 45 (Table 4.1.4e). Therefore, for any test organism used, the Minimum Inhibitory
Concentration of the water extract was higher than the Minimum Inhibitory Concentration of the
ethanol extract. Since the lower the MIC, the more potent a test drug against a test organism, the
water extract can be said to be 1.5 to 45 times less potent than the ethanol extract against the
various test organisms used. Once again, these are in vitro tests and in vivo tests may sometimes
prove otherwise.
Flavonoids and tannins are known to exhibit antimicrobial activity (Bhat et al, 2005). The
alkaloids and steroids usually exhibit metabolic activity and in some cases alkaloids exhibit
antiprotozoa activity (e.g quinine for the treatment of malaria). The anthraquinones and
anthracene glycosides may exhibit antimicrobial activity but they are widely known for their
purgative actions (Evans, 1989). It was deduced from the qualitative phytochemical screening
that the ethanol extract contained a higher concentration of flavonoids than the water extract,
while the water extract contained a higher concentration of tannins than the ethanol extract.
These were the major differences in the phytochemical screening tests. Therefore, the
antimicrobial activities of the two extracts may be mainly due to the tannins and flavonoids
present. The higher activity of the ethanol extract is most likely due to the higher concentration
of flavonoids since that was the major difference between the water and ethanol extracts. Also,
the favonoids in the stem of S. gratus exhibit more profound antimicrobial activity than the
tannins against the test organisms because the ethanol extract depicted higher in vitro
antimicrobial activity.
The MICs revealed that both extracts were least potent against Neisseria gonorrheae, the
organism whose infections folks use the decoctions of the stem extract to treat (Burkill, 1985).
The folkloric use of S. gratus for gonorrhea treatment is therefore not wrong but folks use water
as the solvent for extraction. Meanwhile, the tests have proved that the water extract is two times
less potent than the ethanol extract against Neisseria gonorrheae. Therefore, it would be better if
folks used ethanol for the extraction.
Apart from its use in gonorrhea, the plant is used traditionally in the treatment of wounds
and constipation (Burkill, 1985). The usefulness of S gratus in wound treatment may result from
its antimicrobial actions and also its antioxidant actions. The antimicrobial action would assist in
the prevention of wound sepsis and the antioxidant would help in the prevention of cell
degeneration in the wound. The plant’s ability to treat constipation must be due to the presence
of anthraquinone glycosides. Anthraquinone and its glycosides are known to induce peristalsis of
the gastrointestinal tract (Evans, 1989).
Before any drug would be accepted for use, pharmacological and toxicological (safety)
tests must be carried out (Shargel et al, 2007). In both tests, the results are usually compared with
a drug or drugs already in use for indications of interest and whose pharmacological and
toxicological profiles are well-known. When the MIC of the extracts were compared with those
of Ciprofloxacin under similar conditions, it was realized that Ciprofloxacin was 4 to 115 times
less potent than the ethanol extract and 1.5 to 6.5 times less potent than the water extract against
all the test organisms with the exception of Neisseria gonorrheae, S. typhi and E. coli. For these
three organisms, the ethanol extract was 1.2 to 14 times less potent and the water extract 5 to 28
times less potent than ciprofloxacin. So based on MICs in vitro, ciprofloxacin is a better
antimicrobial agent than S gratus for gonorrhea, typhoid fever and E coli infections. Therefore
the extracts cannot be better than ciprofloxacin in the treatment of the named infections. The side
effects of ciprofloxacin (nausea, constipation, abdominal pains etc) are tolerable within
therapeutic doses (BNF, 2009). The toxicology of the extracts must be studied to ascertain their
effects and compared with the conventional antibiotics. It could be that the extracts have less side
effects and are more potent when used in vivo, in which their use may be further considered.
4.4 ANTIOXIDANT ACTIVITY
The phenol content of tannic acid increased with increasing concentration (Table 4.1.5a).
The extract also showed a concentration dependent increase in phenolic content expressed as
tannic acid equivalent (Table 4.1.5b). The total antioxidant capacity of ascorbic acid increased
with increasing concentration. The extract also depicted a concentration dependent increase in
total antioxidant capacity expressed as ascorbic acid equivalent. This implies that, the higher the
concentration the better the capacity of the extract to reduce Mo(VI) to Mo(V). The antioxidant
capacity of S. gratus (TAC) was strongly dependent on the total phenolic contents (TPC) as
revealed by the high correlation between TPC and TAC (Fig. 4.1.5b and 4.1.5c). The extract and
the reference antioxidant n-propyl gallate increasingly reduced Fe3+ to Fe2+ with increasing
concentration. This resulted in concentration dependent increase in absorbance. From the EC50
values, S. gratus was found to be about 31 fold less potent than n-propyl gallate, the reference
antioxidant (Table 4.1.5c and Fig 4.1.5d). The detection of phenols in the S. gratus extract was a
preliminary evidence of its possible antioxidant activity. Phenolic compounds commonly
responsible for antioxidant effects are tannins and flavonoids. S gratus contains both groups of
antioxidant compounds.
The antioxidant study indicates that the ethanolic extract of Strophanthus gratus
possesses antioxidant effects. This property may augment the use of the plant for its
antimicrobial effects as in vivo bacteria activity may lead to the production of oxidants (Salawu
et al, 2006).
CHAPTER FIVE
5
CONCLUSIONS AND RECOMMENDATIONS
This section has two subsections namely conclusions and recommendations.
5.1
CONCLUSIONS
The aqueous and ethanolic stem extracts of Strophanthus gratus possess antimicrobial
activities against N. gonorrheoae, S. typhi, B. subtilis, E. coli, Pr. vulgaris , B. thuringiensis, Ps.
aeruginosa,, Staph. aureus and E. faecalis. The ethanolic extract was more active than the
aqueous extract against the test organisms. When the activities of the extracts were compared
with those of ciprofloxacin under the same in vitro experimental conditions, it was discovered
that the extracts were more potent than ciprofloxacin with the exceptions of N. gonorrhoeae, S.
typhi and E. coli. The ethanolic extract of S. gratus has antioxidant properties that may augment
its antimicrobial properties.
5.2
RECOMMENDATIONS
The experiment focused on only in vitro tests. Since in vitro results may not reflect in
vivo results, it is recommended that later works be extended to that area. The work did not also
reach separation of groups of phytoconstituents and individual phytoconstituents. It is
recommended that later works also consider such areas to clearly ascertain which
phytoconstituents are responsible for the antimicrobial activities. Later works may also focus on
toxicological
studies
to
determine
the
safety
of
the
use
of
extracts
in
man.
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Appendix 1: Anova: Two-Factor Without Replication
Test Organism
B. Subtilis
Pr. vulgaris
Ps. aeruginosa
Staph aureus
E. faecalis
N. gonorrhoeae
S. typhi
E. coli
B. thuringiensis
Ethanol
Extract
0.1682
0.1204
0.01
0.0066
0.002
1.2083
0.3882
0.1328
0.0311
Water
Extract
0.2622
0.2233
0.0652
0.1239
0.0898
2.4848
1.1431
0.5853
0.0948
Cipro.
0.7335
0.3513
0.2123
0.338
0.2294
0.0873
0.1266
0.1119
0.609
Anova: Two-Factor Without Replication
SUMMARY
B. Subtilis
Pr. vulgaris
Ps. aeruginosa
Staph aureus
E. faecalis
N. gonorrhoeae
S. typhi
E. coli
B. thuringiensis
Ethanol Extract
Water Extract
Cipro.
Count
3
3
3
3
3
3
3
3
3
Sum
1.1639
0.695
0.2875
0.4685
0.3212
3.7804
1.6579
0.83
0.7349
Average
0.387966667
0.231666667
0.095833333
0.156166667
0.107066667
1.260133333
0.552633333
0.276666667
0.244966667
Variance
0.091753963
0.013381203
0.010935123
0.028237343
0.013151293
1.439016583
0.278596803
0.071550103
0.100404623
9
9
9
2.0676
5.0724
2.7993
0.229733333
0.5636
0.311033333
0.149653688
0.639993145
0.05121868
df
MS
0.397308713
0.272799843
0.2217784
F
1.791467132
1.230055965
ANOVA
Source of
Variation
Rows
Columns
Error
SS
3.178469707
0.545599687
3.548454393
Total
7.272523787
8
2
16
26
P-value
0.152724869
0.318482241
F crit
2.5910962
3.6337235
Appendix 2: MIC Exploratory plots
MIC PLOTS - EXPLORATORY
0
1
0
B. Subtilis
2
3
4
Ps. aeruginosa
Pr. vulgaris
5
6
E. faecalis
7
8
9
10
B. thuringiensis
Staph aureus
0.5
E. coli
Ethanol Extract
1
Water Extract
S. typhii
1.5
2
2.5
3
N. gonorrhoeae
Cipro.
Appendix 3: t-Tests
t-Test: Paired Two Sample for Means
Mean
Variance
Observations
Pearson Correlation
Hypothesized Mean Difference
df
t Stat
P(T<=t) one-tail
t Critical one-tail
P(T<=t) two-tail
t Critical two-tail
Ethanol Extract
0.229733333
0.149653688
9
0.983163506
0
8
-2.353550478
0.023211625
1.859548033
0.046423249
2.306004133
t-Test: Paired Two Sample for Means
Ethanol Extract
Mean
0.229733333
Variance
0.149653688
Observations
9
Pearson Correlation
-0.405734757
Hypothesized Mean Difference
0
df
8
t Stat
-0.467728434
P(T<=t) one-tail
0.326227909
t Critical one-tail
1.859548033
P(T<=t) two-tail
0.652455818
t Critical two-tail
2.306004133
Water Extract
0.5636
0.639993145
9
Cipro.
0.311033333
0.05121868
9
t-Test: Paired Two Sample for Means
Mean
Variance
Observations
Pearson Correlation
Hypothesized Mean Difference
df
t Stat
P(T<=t) one-tail
t Critical one-tail
P(T<=t) two-tail
t Critical two-tail
Water Extract
0.5636
0.639993145
9
-0.514859662
0
8
0.808794351
0.221010183
1.859548033
0.442020365
2.306004133
Cipro.
0.311033333
0.05121868
9
Appendix 4: Correlation of MICs for between test drugs
Ethanol extract against water extract
3
Ethanol
Extract 2.5
y = 2.0332x + 0.0965
2
R = 0.9666
2
1.5
Water Extract
1
0.5
0
0
0.5
1
1.5
Water Extract
ciprofloxacin against water extract
0.8
0.7
0.6
0.5
Cipro. 0.4
0.3
0.2
0.1
0
y = -0.1457x + 0.3931
2
R = 0.2651
0
0.5
1
1.5
2
2.5
Water extract
3
Appendix 4: Correlation of MICs for between test drugs
Ethanol
Extract
0.8
Ethanol Extract against Ciprofloxacin
0.7
y = -0.2374x + 0.3656
2
R = 0.1646
0.6
0.5
0.4
0.3
0.2
0.1
0
0
0.5
1
1.5
Ciprofloxacin