IN-VITRO ANTIOXIDANT AND IMMUNOMODULATORY ACTIVITY OF QUERCETIN-PHOSPHOLIPID PHYTOSOMES

M. Pharm Dissertation Protocol Submitted to

RajivGandhiUniversity of Health Sciences, Karnataka

Bangalore – 560041

By

Gururaj Dixit.

B.Pharm.

Under the Guidance of

Dr. P. V. Habbu.

M. Pharm., Ph. D.

Professor

Post Graduate Department of Pharmacognosy,

SET’S College of Pharmacy,

S.R.Nagar, Dharwad,

KARNATAKA - 580002.

RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES,

KARNATAKA, BANGALORE.

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1 / NAME OF THE CANDIDATE AND ADDRESS / GURURAJ DIXIT
SET’S COLLEGE OF PHARMACY
S.R.Nagar, Dharwad,
Karnataka – 580002.
2 / NAME OF THE INSTITUTION / SET’S COLLEGE OF PHARMACY
S.R.Nagar, Dharwad,
Karnataka – 580002.
3 / COURSE OF STUDY AND SUBJECT / MASTER OF PHARMACY IN PHARMACOGNOSY
4 / DATE OF THE ADMISSION / JUNE 2010
5 /

TITLE OF THE TOPIC

“IN-VITRO ANTIOXIDANT AND IMMUNOMODULATORY ACTIVITY OF QUERCETIN –PHOSPHOLIPID PHYTOSOMES”
6 / BRIEF RESUME OF THE INTENDED WORK
6.1 / Need for study
During the last century chemical and pharmacological studies have been performed on a lot of plant extracts in order to know their chemical composition and confirm the indications of traditional medicine. Development of valuable drug delivery system from natural resources is very much necessary because of the beneficial role of herbal drug in the management of various diseases. Continuing research is very much essential to explore the therapeutic efficacy of the natural molecules as well as to develop proper delivery system for enhancement of therapeutic potential of those molecules 1.
Improved bioavailability and faster actions of herbal extracts/constituents, especially herbal lipophilic constituents can be achieved by novel drug delivery systems such as Phytosomes. The term “phyto” means plant while “some” means cell like. It is also known as herbosomes. Phytosomes are created when the standardized extract/active ingredient of herb are bound to the phospholipids on a molecular level2.
Recent research shows improved absorption and bioavailability with phytosomes as compared to the conventional phytomolecules or botanical extracts. Most of the bioactive constituents are flavonoids e.g. anthocynidines from bilberry, catechins from green tea, silymarin from milk thistle etc. Specifically, the choline head of the phosphatidylcholine molecule binds to these compounds while the fat-soluble phosphatidyl portion comprising the body and tail then envelopes the choline-bound material. The result is a little micro sphere or cell is produced 3.
Keeping in view the advantages of phytosomes it has become nessary to apply the phytosomes technology for standardised extracts and phytoconsttuents to improve the bioavailabilty and better clinical efficasy.
6.2 / Review of literature
Phytosome technology has been successfully adopted for various standardized extracts/phytocondtituents. Previous literature revealed the preparation, characterization and efficacy of various phytosomes of Silybin4, Curcumin5, Bioflavonoids6, Gingko biloba7, FarnesiferolC8, Boswellic acid 9.
Qerecetin belongs to the flavonoids family and consists of 3 rings and 5 hydroxyl groups. Querecetin is also a building block for other flavonoids. Quercetinoccurs in food as an aglycon ( attached to a sugar molecule) .Only a small amount of the ingested quercetin will get absorbed in the blood.Quercetin is found in many common foods , including apple, onion, nuts, berries, cauliflower, and cabbage10.
Quercetinhas many therapeutic uses such as atherosclerosis, antioxidant11, anti cancer12, allergy and anflammation13, immunomodulatory effects14.
To our best knowledge no scientific data regarding the Preparation of Quercetin-phospholipid complex (phytosome) in literature is available, therefore present study was under taken with following objectives.
6.3 / Objectives of the study
To prepare phytosome complex of quercetin-phospholipid .
To study its physicochemical parameters.
To Evaluate the quercetin-phospholipidphytosome for in-vitro anti oxidant and immunomodulatory activities.
7 / MATERIALS AND METHODS
7.1 / Sources of data
. International Journal of Pharmaceutics
. Science Direct
. Journal of Pharmaceutical sciences
. Ethnopharmacology Journal
. Psychopharmacology
. Indian Journal of Exp Biology
. Food Chemical Toxicology
. Planta Med
. Pubmed
. Fitoterapia
. Word Wide Web
. J-Gate@ Helinet etc
7.2 / Method of collection of Data
The sources of data are from the laboratory experiments, which involve preparation and characterization of phytosomes and evaluation of in-vitro antioxidant and immunomodulatory activities on experimental models
7.3 / Experimental Techniques
1) Preparation of Quercetin-phospholipid complex.
As per the earlier reported method, Phytosomes will be prepared with 1:1 ratio of drug to Phospholipid with anhydrous ethanol, evaporating under pressure, the dried residue will be gathered and placed in desiccators overnight, then powdered and stored5.
2) Determination of Querecetin content in Phospholipid complex by HPLC15.
3) Characterization of phytosomes5.
a)Microscopic view of the complex
b)Scanning electron microscopy
c)Differential Scanning of complex
In-vitro free radical scavenging activity16,17,18
As per reported studies, the following parameters will be studied.
A. Hydroxyl radical scavenging activity.
B. 2,2-diphenyl-1-picrylhydrazyl [DPPH] Scavenging activity.
C. Reducing power.
In-vitroimmunomodulatory activity19,20,21
1. QUALITATIVE NITROBLUETETRAZOLIUM TEST
A suspension a suspension of leucocytes (5x106/ml) in 0.5ml PBS solution in 7 tubes.0.1 ml solution (control) and 0.1ml of endotoxin activated plasma(standard) is added to the 1st and 2nd test tube respectively and the other 5 tubes added 0.1 ml of different concentrations (5.10,20,50 and 100 mcg/ml) of test samples.0.2ml of freshly prepared 0.15% NBT solution was added to each test tube and incubated at 370C for 20 min. centrifuge at 400 g for 3-4 min to discard the supernatant. The cells were resuspended in the small volume of PBS solution. A thin film was made with the drop on slide, dried, fixed by heating, counterstained with dilute carbon-fuchsin for 15 sec. the slide was washed under tap water,dried and focused under 100 x oil emersion objective, 200 neutrophils were counted to determine the percentage of NBT-positive cells containing blue deposits granules or course lumps.
2. PHAGOCYTOSIS of killed Candida Albicans
Human blood (0.2ml) was obtained by finger prick method on a sterile glass slide and incubated prick method on a sterile glass slide and incubated at 370C for 25 min to allow clotting. The blood clot was removed very gently and slide was drained slowly with sterile normal saline, taking care not to wash the adhered neutrophils (invisible).the slide consisting of PolymorphnuclearNeutrophils (PMNs) was flooded with predetermined concentration of test sample and incubated at 370C for 15 min .The PMNs were covered with Candida Albicanssuspension and incubated at 370C for 1 hour. The slide was drained, fixed with methanol and stained with Giemsa stained. The mean numbers of phagocytosed cells on the slide were determined microscopically for 100 granulocytes using morphological criteria. This number was taken as the phagocytic index (PI) and was compared with the basal (PI) of controls. This procedure was repeated for different concentrations.
3. CANDIDACIDAL ASSAY
Duplicate set of tubes were taken and t to it 0.25 ml of MEM,0.15ml of WBC suspension and 0.25 ml of candida cell suspension was added. Each tube is filled with diluted extract.Control tube was set up with 0.25ml of pooled normal human serum instead of extract.Tubes are incubated at 370c for 30min, 0.25ml of 2.5% sodium deoxycholate and 4ml of 0.01% methylene blue were added to each tube. Tubes are centrifuged to discard the supernatant solution and one drop at the bottom of the tube is retained. one drop of the above solution were added to clean glass slide and covered with coverslip and observed with coverslip and observed under 40X lens microscope. Number of dead cells were counted and expressed in percentage. The viable cells do not take up methylene blue and remain unstained; the dead cells take-up the stain and appear blue in color.
NEUTROPHILS LOCOMOTION AND CHEMOTAXIS
Neutrophils cell suspension was prepared in phosphate buffer salinesolution (PBS) at about 106 cells /ml. the lower compartment of chemotactic chamber was filled with appropriate chemotactic reagents pre adjusted to the pH of 7.2.the upper compartment was placed into the lower compartment and incubated at 370C for 180 min. The upper compartment was removed and inverted to empty the fluid. The lower surface of the filter was fixed with 70% ethanol for 2 min and then stained with haematoxylin dye for 5 min.the fixed filters were observed under microscope using 100x lens and number of neutrophil cells reached to the lower surface was counted.
7.3 / Does the study require any investigation or interventions to be Conducted on patients or other humans or animals?If so please describe briefly.
NO.
7.4 / Has ethical clearance been obtained from your institution in case of 7.3?
NA
8 / REFERENCES
1. Murry MT. Phytosome Herbal Support, Herbal Phytosomes natural factors Nutritional Products Ltd. Mind Publishers; 2001.
2. Dang Yi, 2000, UPC code-0300540111783, New product concept.
3. Mascarella S., 1993, Therapeutic and antilipoperoxidant effects of silybin-phosphatidylcholine complex in chronic liver disease, Preliminary results. CurrTher.Res 1993;53(1):98-102.
4. YanyuX ,Yunmei S, Zhipeng C, Quineng P. The preparation of silybin-phospholipid complex and the study on its pharmacokinetics in rats.Int J Pharm 2006;307(1):77-82(s).
5. MaitiKet, MukherjeeK,Gantait A, SahaBP,Mukherjee PK. Curcumin-phospholipidcomplex:therapeutic evaluation and pharmacokinetic study in rats. Int J Pharm 2007;330(1-2):55-163(s).
6. Franco PG, Bombardelli, Ezio. Complex compound of bioflavonoids with phospholipidcomplex,their preparation and use and pharmaceutical and cosmetic composition contaioning them. US patent EPTO no EPO275005,1988;6-14.
7. Naik SR, Panda VS. Antioxidant and hepatoprotectiveeffects of Ginkgo bilobaphytosomes carbon tetra chloride- induced liver damage in rodents.Liver Int;2007 Apr;27(3):393-9.
8. Mashinchian O, Salehi R, Dehghan G, Aganejad A, Davaran S, Omidi Y. Novel thermosensitive poly(N-isopropylacrylamide-co-vinylpyrrolidone-co-methacrylic acid) nanosystems for delivery of natural products. Inter J Drug Deliv2002;(2):278-86.
9. Sharma A, Gupta NK, Dixit VK. Complexation with phosphatidylcholine as astrategy for absorption enhancement of boswellic acid. Drug Deliv2010 ;17(8):587-95.
10. Kaushal GP, Sekhon BS, Bhatia IS. Direct spectorscopic determination of quercetin with VO+. MicrchimicaActa 1979;71:365-70.

11. ATerao J, Kawai Y, Murota K . Vegetable flavonoids and cardiovascular disease.Asia Pac j Clin Nutr.2008;17suppl 1:291-3.

12. Murakami a, ashidaH,Terao J. Multitargeted cancer prevention by quercetin. Cancer Lett 2008;269(2):315-25.

13. Shaik YB, Castellani ML, PerrellaA.Role of quercetin in allergy and inflammation.Alternative Medicine Review;2008.
14. Sternberg Z,Chanda K, Liberman A, Hojnacki D, DarkeA,ZamboniP,et al. Quercetin and interferon-beta modulate immune response(s) in peripheral blood mononuclear cells isolated from multiple sclerosis patients. J.Neuroimmnol. 2008 Dec 15;205(1-2);41-43.
15.Biosutto L, Marotta E, Gabisa S, Zoratti M, Paradisi C. Determination of querecetin and resveratol in whole blood implications for bioavailability studies. Molecules 2010;15(9);6570-9.
16. Narla RS, Rao MNA. Scavenging of free radicals and inhibition of lipid
peroxidation by 3-phenyl syndone. J Pharm Pharmacol 1995;47:623–25.
17. Kunchandy E, Rao MNA. Effect of curcumin on hydroxyl radical
generation through Fenton reaction. Int J Pharm1989;57:173-76.
18. Mahmood RM, Soheila M, Said A. Scavenging and Reducing power of
Salvia Mirzayaniisubfractions. Molecules 2008;13:2804-13.
19. R.P. Stakes and R.A.Thompson, Techniques in Clinical Immunology,Blackwell Scientific Publication, Oxford, Edinburg, 2ndedn., 1981; 273-276.
20. A.Julia, Metcalf, John andI.Gallin. Laboratory Mannual of Neutrophil Function Test Raven Press Publishers, New York, 1986; 60-63.
21. H.C. Gooi and H. Chapel, Clinical Immunology-A Practical Approach,Department of Immunology, John Radcliffe Hospital, Oxford, U.K, Oxford University Press, New York, 1990; 51-54.

9

/

SIGNATURE OF THE CANDIDATE

10 / REMARKS OF THE GUIDE / The above mentioned information and literature has been extensively investigated, verified and was found to be correct. The present study will be carried out under my supervision and guidance.
11 / NAME AND DESIGNATION OF GUIDE

SIGNATURE

/ Dr. P. V. HABBU,M. Pharm., Ph.D.,
Professor and Head,
Post Graduate Department of Pharmacognosy,
SET’s College of Pharmacy, S.R.Nagar,
Dharwad. Karnataka – 580002.
Mobile No.: +91-9448224894
E-mail:

12

/

NAME AND DESIGNATION OF CO – GUIDE

SIGNATURE / Mrs.SMITA.D.MADAGUNDIM.Pharm.,
Lecturer,
Poat Graduate Department of Pharmacognosy,
SET’s College of Pharmacy,S.R. Nagar,
Dharwad. Karnataka – 580002.
E-mail:
Mobile No: 9986033439
13 / NAME AND DESIGNATION OF HOD
SIGNATURE / Dr. P. V. HABBU,M. Pharm., Ph.D.,
Professor & HOD
Post Graduate Department of Pharmacognosy,
SET’s College of Pharmacy, S.R.Nagar,
Dharwad. Karnataka – 580 002.
Mobile No.: +91 – 9448224894
E-mail:

14

/

REMARKS OF PRINCIPAL

/ The above mentioned information is correct and I recommend the same for approval.
15 / NAME OF THE PRINCIPAL
SIGNATURE / Dr. V. H. KULKARNI, M. Pharm., Ph.D.,
Principal, SET’s College of Pharmacy,
S.R.Nagar, Dharwad. Karnataka – 580002.
Mobile No.: +91 – 9448357804
E-mail: