“DESIGN AND CHARACTERISATION OF PHYTOSOMAL
NANO CARRIERS FOR ENHANCED RUTIN DELIVERY”
MASTER OF PHARMACY DISSERTATION PROTOCOL
SUBMITTED TO THE
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA,
BANGALORE.
BY
Mr. RAVI G.S
M.Pharm-I
Under The Guidance of
Mr.VIRESH K.CHANDUR .M.Pharm.
DEPARTMENT OF PHARMACEUTICS.
SRINIVAS COLLEGE OF PHARMACY, VALACHIL, MANGALORE – 574143
2013-2015
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
BANGALORE, KARNATAKA
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION
1.
Name of the Candidate and
Address:
2.
Name of the Institution:
3.
Course of Study and Subject:
4.
Date of Admission:
5.
Title of the Project:
RAVI G.S
1stYEAR M.PHARM,
DEPT. OF PHARMACEUTICS,
SRINIVAS COLLEGE OF PHARMACY,
VALACHIL, MANGALORE-574143.
SRINIVAS COLLEGE OF
PHARMACY,
VALACHIL, FARANGIPETE POST,
MANGALORE-574143.
MASTER OF PHARMACY
(PHARMACEUTICS)
25-07-2013
“DESIGN AND CHARACTERISATION OF PHYTOSOMAL NANO
CARRIERS FOR ENHANCED RUTIN DELIVERY”
6.
Brief Resume of the intended work:
6.1
Need of the study:
Flavonoids:

Phytopharmaceuticals stand out as recent promising candidates for the
treatment of chronic diseases like cancer. Fewer side effects and lower
phytochemical costs from natural resources open new avenues for the
treatment of various diseases. Flavonoids like quercetin, curcumin, luteolin,
diosmin and rutin are phenolic compounds possess strong antioxidant activity1
as well as other interesting potential effects including anti-inflammatory2,
anti-cancer3, and anti-ulcer4 activities.

Unfortunately, despite the wide therapeutic potential of flavonoids, their
phenolic nature renders them polar but poorly water soluble, and scarce
absorption is a reported consequence of these features5. These aspects
constitute a handicap against the widespread use of flavonoids in the pharmaceutical field. These are characterized by poor solubility in water and most
organic solvents as well. Poor drug dissolution is responsible for its poor
bioavailability and high inter-subject variation following oral administration.

Low drug solubility results in reduced amounts of drug absorbed, paving the
way for a high influence of exogenous factors, such as diet and dosage
regimen. Therefore, a large standard dose is usually required for oral dosage
regimens6, so it is necessary to formulate flavonoids in the form of
phytosomes to overcome the poor absorption and bioavailability7, 8.
Rutin:

Rutin, also called rutoside, quercetin-3-rutinoside and sophorin, is a citrus
flavonoid glycoside. It is a glycoside between the flavonol quercetin and the
disaccharide rutinose9 having antioxidant10, anti-inflammatory11,12 properties.
The usual oral dosage regimen is 200 to 600 mg once or twice per day13. The
most commonly occurring adverse side effects during treatment with rutin
preparations are headache, flushing, rashes, or stomach upset etc.
Phosphatidylcholine:

Phosphatidylcholines (PC) are a class of phospholipids that incorporate
choline as a headgroup. They are a major component of biological membranes
and can be easily obtained from a variety of readily available sources, such as
egg yolk or soybeans, from which they are mechanically or chemically
extracted using hexane. They are also a member of the lecithin group of
yellow-brownish fatty substances occurring in animal and plant tissues. The
phospholipid is composed of a choline head group and glycerophosphoric
acid, with a variety of fatty acids, one being a saturated fatty acid and another
being an unsaturated fatty acid14.
Phytosomes:

Hydrophilic phytoconstituents can be complexed with clinically useful
nutrients such as phospholipids to convert them into lipid soluble complexes.
Such complexes can be used to prepare liposome-like vesicles called as
phytosomes15, 16. In phytosomes, the complexation of phospholipids and water
soluble active plant components involve chemical bond formation and
therefore more stable. Whereas in liposomes no chemical bond is formed;
phosphatidylcholine
molecules
simply
surround
the
water-soluble
components15. The phytosomes substantially improve the bioavailability of
these hydrophilic active components. Some of the phospholipids that are
reported
for
phytosome
preparation
include
soy
phospholipids,
phosphodidylcholine etc. Phytosomes can easily cross the lipid membranes
and are reported to increase the bioavailability of poorly lipid soluble plant
based drugs by increasing the absorption in gastrointestinal tract.
Advantages of phytosomes are:
1. They enhance the absorption of lipid insoluble polar phytoconstituents and
shows better bio availability, hence they have significantly greater therapeutic
benefit.
2. They improve the absorption of active constituent(s) which further reduce its
dose requirement.
3. Phosphatidylcholine used in preparation of phytosomes, besides acting as a
carrier also acts as a hepatoprotective, hence giving the synergistic effect
when the hepatoprotective substances are employed.
4. Chemical bonds are formed between phosphatidylcholine molecules which
added nutritional benefit of phospholipids.
5. They have appreciable drug entrapment.
6. Significantly greater clinical benefit.
7. Assured delivery to the tissue.
6.2
Review of literature:

Maiti et al., developed the phytosomes of curcumin (flavonoid from turmeric,
Curcumalonga linn) and naringenin (flavonoid from grape, Vitis vinifera) in
two different studies. The antioxidant activity of complex was significantly
higher than pure curcumin in all dose levels tested. In the other study the
developed phytosome of naringenin produced better antioxidant activity than
the free compound with a prolonged duration of action, which may be due to
decrease in the rapid elimination of the molecule from the body17,18.

Mukerjee et al., (2008) developed a novel hesperetin phytosome by
complexing hesperetin with hydrogenated phosphatidyl choline. This complex
was then evaluated
for antioxidant activity in CCl4 intoxicated rats along
with pharmacokinetic studies. It was found that the phytosome had a
sustained release property for over 24h and
enhanced antioxidant activity.
Pharmacokinetic study revealed that the phytosome had higher relative
bioavailability than that of parent molecule at the same dose level19.

Yanyu et al., (2006) prepared the silymarin phytosome and studied its
pharmacokinetics in rats. In the study the bioavailability of silybin in rats was
increased
remarkably
after
oral
administration
of
prepared
silybinphospholipid complex due to an impressive improvement of the
lipophilic property of silybin-phospholipid complex and improvement of the
biological effect of silybin20.

Ravarotto et al., (2004) reported silymarin phytosome show better
antihepatotoxic activity than silymarin alone and can provide protection
against the toxic effects of aflatoxin B1 on performance of broiler chicks21.

Bombardelli et al., (1991) reported Silymarin phytosomes, in which Silymarin
(A standardized mixture of
flavanolignansextracted from the fruitsof S.
marianum) was complexed with phospholipids. Phytosomes showed much
higher specific activity and a longer lasting action than the single components,
with respect to percent reduction of odema, inhibition of myeloperoxidase
activity, antioxidant and free radical scavenging properties22.

Maiti et al., (2005) developed the quercetin–phospholipids complex by a
simple and reproducible method and also showed that the formulation exerted
better therapeutic efficacy than the molecule in rat liver injury induced by
carbon tetrachloride23.

Barzaghi et al., (1990) conducted a human study designed to assess the
absorption of silybin when directly bound to phosphatadylcholine. Plasma
silybin levels were determined after administration of single oral doses of
silybin phytosome and a similar amount of silybin from milk thistle in healthy
volunteers. The results indicated that the absorption of silybin from silybin
phytosome is approximately seven times greater compared to the absorption
of silybin from regular milkthistle extract (70-80 % silymarin content)24.

Moscarella et al., (1993) investigated in one study of 232 patients with
chronic hepatitis (viral, alcohol or drug induced) treated with silybin
phytosome at a dose of 120 mg either twice daily or thrice daily for up to 120
days, liver function returned to normal faster in patients taking silybin
phytosome compared to a group of controls (49 treated with commercially
available silymarin, 117 untreated or given placebo)25.

Grange et al., (1999) conducted a series of studies on silymarin phytosome,
containing a standardized extract from the seeds of S. marianum, administered
orally and found that it could protect the fetus from maternally ingested
ethanol26. Grape seed phytosom is composed of oligomeric polyphenols
(grape proanthocyanidins or procyanidins from grape seed extract, Vitis
vinifera) of varying molecular size, complexed with phospholipids. The main
properties of procyanidin flavonoids of grape seed are an increase in total
antioxidant capacity and stimulation of physiological antioxidant defences of
plasma, protection against ischemia/reperfusion induced damages in the heart,
protective effects against atherosclerosis thereby offering marked
protection
for the cardiovascular system and other organs through a network of
mechanisms that extend beyond their great antioxidant potency27. Green tea
extract generally contains a totally standardized polyphenolic fraction (not
less than 66.5%, containing epigallocatechin and its derivatives ) obtained
from green tea leaves (Thea sinensis) and mainly characterized by the
presence of epigallocatechin 3-O-gallate, the key compound. These
compounds are potent modulators of several biochemical processes linked to
the breakdown of homeostasis in major chronic-degenerative diseases
following oral administration. Over the study period of 6 hours the plasma
concentration of total flavonoids was more than doubled when coming from
the phytosomal versus the nonphytosomal extract. Antioxidant capacity was
measured as TRAP (Total Radical-trapping Antioxidant Parameter). The peak
antioxidant effect was a 20% enhancement and it showed that the phytosome
formulation had about double the total antioxidant effect28.
6.3
Objectives of the study:
1. To study drug and carrier compatibility by FTIR.
2. Preparation of phytosomes of rutin.
3. Characterization of the prepared phytosomes of rutin.
4. In-vitro evaluation of the prepared phytosomes of rutin.
5. Stability studies of the selected formulations as per ICH guidelines.
7.
Materials and Methods:
7.1
Materials:
1. Drug: Rutin.
2. Phospholipid: Soybean phosphatidylcholine.
3. Other: Dimethyl sulphoxide(DMSO), T-butylalcohol, Sodium dodecyl sulphate, nhexane, dehydrated Ethanol, Chloroform and all other reagents and chemicals are
of analytical grade.
7.2
Methods:
I.
Formulation of phytosomes:
Phytosomes of rutin are prepared by either of the following methods:
II.

Solvent evaporation method.

Salting out method.
Evaluation studies
a. Compatibility study by FTIR.
b. Particle size and zeta potential by DLS.
c. Surface morphology by SEM.
d. Drug content.
e. Solubility studies.
f. In-vitro drug release.
g. In-vitro self phytosomal stability.
7.3
Source of data:
Review of literature from
a) Journals such as
 Journal of Chemical and Pharmaceutical Research.
 International Journal of Biomedical Research.
 Journal of Pharmacy Research.
 Scholars Research Library.
 Current Research and Information on Pharmaceutical Sciences.
 International Journal of PharmTech Research.
 International Research Journal of pharmacy.
 American Journal of PharmTech Research.
 International Journal of Pharma and Bio Sciences.
 International Journal of Pharmaceutical Innovations.
b) Internet Browsing
7.4
Method of Collection of Data:

Data on drug and Excipients will be collected from the drug information
centre, Patents, Reference books, Text books, catalogues etc.

Data will be collected from the prepared formulations, in-vitro dissolution
studies and stability studies as per ICH Guidelines.
7.5
Does the study require any investigations or interventions to be conducted on
patients or other humans or animals? If so, please describe briefly.
- Not applicable
7.6
Has ethical clearance been obtained from your institution in case of 7.3?
-
8.
Not applicable
List of references:
1. Kandaswami C, Middleton E. Free radical scavenging and antioxidant activity
of plant flavonoids. Adv Exp Med Biol. 1994; 366:351–76.
2. Di Perri T, Auteri A. Action of S 5682 on the complement system. In vitro
and in vivo study. Int Angiol. 1988; 7(Suppl 2):11–5.
3. Dung TD, Day CH, Binh TV. PP2A mediates diosmin p53 activation to block
HA22T cell proliferation and tumor growth in xenografted nude mice through
PI3K-Akt-MDM2
signaling
suppression.
Food
Chem
Toxicol.
2012;50(5):1802–10.
4. Izzo AA, Carlo GD, Mascolo N, Capasso F, Autore G. Antiulcer effect of
flavonoids. Role of endogenous PAF. Phytother Res. 1994;8: 179–81.
5. Havsteen BH. The biochemistry and medical significance of the flavonoids.
Pharmacol Ther. 2002;96(2–3):67–202.
6. Garner RC. Comparison of the absorption of micronized (Daflon 500 mg) and
nonmicronized 14C-diosmin tablets after oral administration to healthy
volunteers by accelerator mass spectrometry and liquid scintillation counting.
J Pharm Sci. 2002;91(1):32–40.
7. Priprem A, Watanatorn J, Sutthiparinyanont S, Phachonpai W, Muchimapura
S. Anxiety and cognitive effects of quercetin liposomes in rats.
Nanomedicine. 2008;4(1):70–8.
8. Mukerjee A, Vishwanatha JK. Formulation, characterization and evaluation of
curcumin-loaded PLGA nanospheres for cancer therapy. Anticancer Res.
2009;29(10):3867–75.
9. Lucci, Mazzafera. Rutin synthase in fava d anta: Purification and influence of
stressors. Canadian journal of plant science 89 (5): 895–902.
10. Antioxidant and Antibacterial Properties of Mesembryanthemum crystallinum
and Carpobrotus edulis Extracts. Bouftira Ibtissem, Chedly Abdelly and
Souad Sfar, Advances in Chemical Engineering and Science, 2012;2 (3): 35965,
11. Guardia. Anti-inflammatory properties of plant flavonoids. Effects of rutin,
quercetin and hesperidin on adjuvant arthritis in rat. Farmaco. 2001; 56 (9):
683–87.
12. Chan Hun Jung, Cho, Chul Hyung Kim, Chang Jong. Anti-asthmatic action of
quercetin and rutin in conscious guinea-pigs challenged with aerosolized
ovalbumin. Arch. Pharmacal Research. 2007; 30 (12): 1599–607.
13. Pasillas M. Drugs & Supplements, Drugs & Supple-ments Q-R, Rutin Side
Effects, The Effects of Rutin on Pregnancy. 2012; 1(12): 431-35
14. Phosphatidylcholine [Internet] 2013 [cited 2013 Nov 8]. Available from:
http://en.wikipedia.org/wiki/Phosphatidylcholine.
15. Awasthi R, Kulkarni GT, Pawar VK. Phytosomes: an approach to increase
bioavailability of plant extracts. Int J Pharm Pharm Sci. 2011; 3(2): 1-3.
16. Chauhan NS, Gowthamarajan K, Gopalakrishna B. Phytosomes: potential
phyto-phospholipid carriers for herbal drug delivery. J Pharm Res. 2009; 2(7):
1267-70.
17. Maiti K, Mukherjee K, Gantait A, Saha BP, Mukherjee PK. Enhanced
Therapeutic Potential of Naringenin-Phospholipid Complex in Rats, J pharm
Pharmacol,2006;58(9): 1227-33.
18. Maiti K, Mukherjee K, Gantait A,Saha BP, Mukherjee PK. CurcuminPhospholipid
Complex:
Preparation,
Therapeutic
Evaluation
and
Pharmacokinetics Study in Rats, Int J Pharm,2007;330(1-2): 155-63.
19. Mukherjee K, Maiti K, Venkatesh M, Mukherjee PK. Phytosome of
th
Hesperetin, A Value Added Formulation with Phytomolecules. 60 Indian
Pharmaceutical Congress; New Delhi, 2008; 287.
20. Yanyu X, Yunmei S, Zhipeng C, Quineng P. The Preparation of SilybinPhospholipid Complex and the Study on Its Pharmacokinetics in Rats. Int J
Pharm. 2006; 307:77-82.
21. Ravarotto L. Eficacy of
Silymarin-Phospholipid Complex in Reducing
the Toxicity of Aflatoxin B1in Broiler Chicks. Poult Sci. 2004; 83:1839-43.
22. Bombardelli E, Spelta M, Loggia Della R, Sosa S, Tubaro A. Aging
Skin: Protective Effect of Silymarin- Phytosome. Fitoterapia. 1991; 62:11522.
23. Maiti K, Mukherjee K, Gantait A, Ahamed HN, Saha BP,
Enhanced Therapeutic
Benefit
Mukherjee PK.
of Quercetin–Phospholipid Complex In
Carbon Tetrachloride Induced Acute Liver Injury In Rats: A Comparative
Study. Iran J Pharmacol Ther. 2005; 4:84–90.
24. Barzaghi N, Crema F, Gatti G, Pifferi G, Perucca E. Pharmacokinetic Studies
on Idb 1016, A Silybin Phosphatidylcholine Complex In Healthy Human
Subjects. Eur J Drug Metab Pharmacokinet.1990; 15:333-38.
25. Moscarella S. Therapeutic and
Antilipoperoxidant
Effects of Silybin
Phosphatidylcholine Complex in Chronic Liver Disease: Preliminary Results.
Curr Ther Res. 1993; 53:98-102.46.
26. Guangxi Z, Hongxiang L, Dianzhou B et al, Interaction of puerarin with
phospholipid in solid dispersion, J.Chin.Pharm.Sci.,72(1), 2003; 36-40.
27. Ying L. Comparison of pharmacokinetics between puerarin and its
phospholipid complex in beagle dogs in vivo, Chinese herbal medicine, 37(5),
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28. Phospholipids: The Vital Lipids [online]. 2010 [cited 2010 Mar 26]. Available
from: http://www.phospholipidsonline.com
9. Signature of the candidate
(RAVI G.S)
10.
Remarks of the Guide
11.
11.1 Name and Designation of the
Guide
The work, which is assigned to
Mr.RAVI G.S is under my guidance.
Mr. Viresh K. Chandur
Assistant Professor,
Department of Pharmaceutics,
Srinivas College of Pharmacy,
Valachil, Mangalore- 574143.
11.2 Signature
11.3 Name and Designation of the
Co-Guide
_____
11.4 Signature
_____
11.5 Head of the Department
Dr. A. R. SHABARAYA M.Pharm. MBA, Ph.D.
Principal and Director,
Department of Pharmaceutics,
Srinivas College of Pharmacy,
Valachil, Mangalore- 574143
11.6 Signature
12.
12.1 Remarks of the Principal
12.2 Signature
Recommended and forwarded for
favourable consideration.