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“MODULATION OF T-CELL DERIVED CYTOKINE RESPONSE ASSOCIATED WITH ANTI-INFLAMMATORY POTENTIAL INDUCED BY FRACTIONS OF CURCUMA LONGA AND PTEROCARPUS MARSUPIUM IN MYCOBACTERIUM TUBERCULOSIS INDUCED POLYARTHRITIS”

SYNOPSIS FOR: M. PHARM DISSERTATION

SUBMITTED TO: RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES BENGALURU, KARNATAKA.

SUBMITTED BY: SANJATA MAHAPATRA 1st YEAR M.PHARM

DEPARTMENT OF PHARMACOGNOSY

P.E.S COLLEGE OF PHARMACY

BENGALURU – 560050 (2011-2013) RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA, BENGALURU.

ANNEXURE-II PROFORMA FOR REGISTRATION OF SUBJECT FOR P.G. DISSERTATION

1.0 NAME AND ADDRESS OF THE SANJATA MAHAPATRA CANDIDATE. P.E.S. COLLEGE OF PHARMACY HANUMANTHANAGAR, 50FT. ROAD BENGALOORU—560 050

PERMANENT ADDRESS 6,M.M.FEEDER ROAD FLAT NO-02 1ST FLOOR P.O.-ARIADAHA KOLKATA-57 WEST BENGAL P.E.S. COLLEGE OF PHARMACY 2.0 NAME OF THE INSTITUTION. HANUMANTHANAGAR, 50 FEET ROAD, BENGALOORU – 560050.

3.0 COURSE OF STUDY AND M.PHARM. SUBJECT. PHARMACOGNOSY.

4.0 DATE OF ADMISSION TO 22nd July 2011. COURSE.

5.0 TITLE OF THE TOPIC: “MODULATION OF T-CELL DERIVED CYTOKINE RESPONSE ASSOCIATED WITH ANTI- INFLAMMATORY POTENTIAL INDUCED BY FRACTIONS OF CURCUMA LONGA AND PTEROCARPUS MARSUPIUM IN MYCOBACTERIUM TUBERCULOSIS INDUCED POLYARTHRITIS” 6.0 BRIEF RESUME OF THE INTENDED WORK 6.1) INTRODUCTION:

Herbal drugs are known to posses immunomodulatory properties and generally act by stimulating both specific and nonspecific immunity. About 34 plants are identified as rasayanas in the Indian Ayurvedic system of medicine having various pharmacological properties such as immunostimulant, tonic, neurostimulant, antiaging, antibacterial, antirheumatic, anticancer, adaptogenic and antistress. An entire section of Materia Medica of Ayurveda is devoted to drugs entitled as ‘Rasayana’ used for enhancement of body resistance. Rasayana herbs act as adaptogens, immunomodulators, pro-host probiotics and antimutagenics.

Many plants with potential immunomodulatory activity are reported, some of these have already been undertaken for evaluation of their activities in animals, and also to some extent in humans. A lot more are still to be explored and offer scope for further investigation.1

Immunomodulatory activities were reported in some naturally occurring plants like Clausena excavate, Tridax procubens, Abrus precatorius, Trigonella foenum graecum, Cleome viscose, Rubia cordoifolia etc.2 Immunosuppressants are agents used to suppress the different aspects of immune function.

 Cyclosporin A, Tacrolimus (FK 506), Rapamycin.

 Corticosteroids: Glucocorticoids eg; Prednisolone.

 Cytotoxic agents: Azathioprine, Cyclophosphamide, Mycophenolate mofetil.

 Antilymphocytic globulin (ALG), Antithymocyte globulin (ATG) monoclonal antibodies. Muromonab-CD3 monoclonal antibody, Rho (D) immunoglobulin.

Immunostimulants are agents having the ability to enhance the body’s immune response in a number of disorders.

 Microorganisms: BCG, Muramyl di peptides, Streptococcal components, Klebsiella pneumoniae.

 Peptides: dialyzable leukocyte extract, neuro peptides, thymic factors, tuftsin.

 Cytokines: colony stimulating factors(GM-CSF), Interferons(IFN-α), Interleukins(IL- 2).

 Synthetic compounds: Isoprinosine, levamisole.  Immune globulin.3

6.2 NEED FOR THE STUDY: The impact of the immune system in human disease is enormous. Immunological diseases are growing at epidemic proportions that require aggressive and innovative approaches to the development of new treatments. These diseases include a broad spectrum of autoimmune diseases such as rheumatoid arthritis, diabetes mellitus, systemic lupus erythematosus and multiple scleros; solid tumours and haematologic malignanacies, infectious diseases, asthma and various allergic conditions. Although the pathogenesis of rheumatoid arthritis is largely unknown, it appears to be an autoimmune disease driven primarily by activated T-cells, giving rise to T-cell derived cytokines such as IL-1, IL-4 and TNF (pro-inflammatory cytokines).4

The organisms respond by developing a robust array of receptor mediated sensing and effector mechanism broadly described as innate and adaptive immunity. The two arms of immunity work closely together with the innate immunity being most active early and adaptive immunity becoming progressively dominant over time. Major effectors of innate immunity are complement, granulocytes, monocytes/macrophages, natural killer cells, mast cells and basophils and that of adaptive immunity are B-cells and T-cells. B-cells makes antibodies whereas T-cells function as helper, cytolytic and regulatory(suppressor) cells. This cells are important in normal immune response to infection and tumours but also mediate transplant rejection and autoimmunity.5,6

Medicinal plants are the integral part of humans to combat diseases from the dawn of civilization. Plants are invaluable sources of new drugs. There is an ever-growing interest in investing different species of plants to identify their potential therapeutic applications. In the recent past, scientific studies on plants used in ethno medicine have led to the discovery of many valuable drugs. The current practice of prescribing photochemical to support the immune system or to fight infections is based on centuries old traditions.2

The fractions of the plants Curcuma longa and Pterocarpus marsupium is selected to investigate for its anti-inflammatory and immunomodulatory activity based on the following evidences:  Curcuma longa has many uses such as anti-inflammatory7, anti-pyretic, hepato protective, anti cancer.8,9  Petroleum ether extracts of Curcuma longa rhizome showed significant anti- inflammatory activity in experimental animals without showing any toxicity.the anti-inflammatory activity of turmeric extracts were attributed to curcumin and its analogues.10

 Pterocarpus marsupium has shown anti-oxidant11 and hepatoprotective activities because of rich content of flavanoids.12

 Pterocarpus marsupium also shows anti-hyperglycemic13, anti-hyperlipidaemic and anti-diabetic activity14.

6. 3 OBJECTIVE OF THE STUDY:

1. Extraction and fractionation of Curcuma longa and Pterocarpus marsupium.

2. Evaluation of

- Anti-inflammatory activity of Curcuma longa and Pterocarpus marsupium fractions in experimental animals

- Modulation of T-cell derived cytokines,i.e., IFN-γ (Th1 ) and IL-4 (Th2 ) response induced by the fractions of Curcuma longa and Pterocarpus marsupium PARAMETERS TO BE MEASURED: 1. Paw oedema 2. Percentage of CD4+ and CD8+ T-cell

3. Estimation of Th1 cytokine IFN-γ and Th2 cytokine IL-4

6.4 PLANT PROFILE15: 1. Curcuma longa:

Taxonomy16 : Kingdom – Plantae Division – Magnoliophyta Class – Liliopsida Order – Zingiberales Family – Zingiberacea Genus – Curcuma Species – Curcuma longa

Common names : English name – Turmeric Hindi – haldi English – Indian saffron, turmeric Bengal – haldi Sanskrit – haladi, haridra

Description: A tall herb. Rootstock large, ovoid, with sessile cylindrical tubers orange- coloured inside. Leaves very large, in tufts up to 1.2 m or more long, including the petiole which is about as long as the blade, oblong-lanceolate, tapering to the base. Flowers in autumnal spikes, 10-15 cm long; peduncle 15 cm or more, concealed by the sheathing petiole; flowering bracts pale green; bracts of coma tinged with pink

Habitat - The plant is found in India especially in Bengal, Mumbai and Tamil Nadu were it is cultivated for commercial purpose. It is also found in areas of Mysore and Malabar and is also found growing wild in forests.

Phytoconstituents: • Curcuminoids (1-8%) - Curcumin (75-80%)

- Demethoxy Curcumin(3-5%) - Bisdemethoxy curcumin(15-18%) - Dihydro curcumin • Essential oil (4-6%) - Bisabolene - α-β curcumenes - zingiberene Uses: The rhizome is pungent, bitter, laxative, anthelmentic, tonic, emmolient, improves the complexion, leucoderma, scabies, inflammation, elephantiasis, bolis, bruises, swelling, sprain (Ayurveda) Yunani – Rhizome is bitter, carminative, maturant, diuretic, good for affections of liver and jaundice, scabies, bruises

2. Pterocarpus marsupium17 Taxonomy: Kingdom – Plantae Subkingdom –Tracheobionta Division – Magnoliophyta Class – Magnoliopsida Subclass - Rosidae Order – Fabales Family – Fabaceae Genus – Pterocarpus Species – Pterocarpus marsupium Common names : English – Indian kino tree, Malabar kino tree Hindi – Bijasal, Vijayasar Kanada – hannemara Sanskrit – asanah , bijakah Tamil – vengai Telegu – beddagi

Description : A medium sized to large tree, 15-30 m in height with dark brown or grey bark having shallow cracks exfoliating in thin flakes and exuding a red gummy substance (Gum kino) on injury leaves compound, imparipinnate, leaflets 5-7 coriaceous, oblong, obtuse, emarginated or even bilobed at apex, glabrous on both surfacesmina nervesnumerous, prominent, flowers yellow in terminal panicles, corolla with crisp margins, fruits nearly circular, glabrous, flat winged pods, convexly curved between stipe and style, wings veined, seeds 1-2, convex bony.

Habitat : Throught india in deciduous and evergreen forests.

Chemical constituents : l-epicatechin. Heartwood yields liquiritigennin, isoliquiritigenin, a neutral unidentified component, alkaloid and resin. Also contain a yellow colouring matter and an essential oiland a semi-drying fixed oil. Kino contains a non-glucosidal tannin kinotannic acid, kinoin, kino- red, in addition to small quantities of catechol protocatechuic acid resin, pectin and gallic acid.

Uses : Heartwood is astringent, bitter, acrid, cooling, anti-inflammatory, union promoter, haemostatic, anthelmentic, elephantiasis, leprosy, leucoderma, diarrhoea, dysentery, rheumatoid arthritis, asthma, bronchitis Leaves- boils, sores and skin diseases Flowers – bitter, sweet, cooling, febrifuge in anorexia and fevers Gum – bitter, styptic, antipyretic, anthelmentic , liver tonic used in spasmodic gastralgia, odontalgia, diarrhoea, psoriasis, hepatopathy, wound and ulcers

6.5) REVIEW OF THE LITERATURE :

Reported activities of Curcuma longa : Mukophadhyay et al. (1982) demonstrated the activity of curcumin and other semi-synthetic analogues (sodium curcuminate, diacetyl curcumin,triethyl curcumin and tetrahydro curcumin) in carrageenin-induced rat paw edema and cotton pelletgranuloma models of inflammation in rats. Unnikrishnan and Rao(1995) studied the antioxidative properties of curcumin and its three derivatives (demethoxy curcumin, bisdemethoxy curcumin and diacethyl curcumin). Rasmussen et al. (2000) reported the efficacy of an ethanolic extract from C. longa against Plasmodium falciparum and L. major, which was able to inhibit the in vitro growth of these parasites. Ozaki et al. (2000), examining the action of curcumin on rabbit osteoclast apoptosis, demonstrated that curcumin drastically inhibits bone resorption in parallel with its stimulation of apoptosis in the cells. Huang et al. (1988), studying the effect of curcumin, chlorogenic acid, caffeic acid and ferulic acid on tumor promotion in mouse skin by 12-O-tetradecanoyl-13-acetate (TPA), observed that all these compounds inhibit the epidermal ornithine decarboxilase (ODC) and epidermal DNA synthesis, being curcumin the most efficient. Mazumber et al. (1995) demonstrated that curcumin has an antiviral activity, being a HIV-1 integrase inhibitor (IC50 = 40 μM) and suggested that curcumin analogs may be developed as anti-Aids drugs.8 Neha et al reprted analgesic and antipyretic activities of Curcuma longa rhizome extracts. 9

REPORTED ACTIVITIES OF Pterocarpus marsupium : Manickam et al demonstrated anti-hyperglycemic activity of phenolics of Pterocarpus marsupium Roxb. 13 Maruthupandian and Mohan reported antidiabetic, antihyperlipidaemic and antioxidant activity of Pterocarpus marsupium Roxb. 14 Halagappa et al studied the effect of aqueous extract of Pterocarpus marsupium Roxb. on cytokine TNF-α in type 2 diabetic rats. 18 Mankani et al evaluated hepatoprotective activity of stem bark of Pterocarpus marsupium Roxb.12 Jain et al reported anthelmintic activity of ethanolic extract of Pterocarpus marsupium19 Ramya et al reported antibacterial activity of leaf extracts of Pterocarpus marsupium Roxb.20

Mohammadi et al revealed strong in vitro antioxidant activity in Pterocarpus marsupium extract. 11

7.0 MATERIAL AND METHODS: 7.1) SOURCE OF DATA: The plant material will be procured from authenticated suppliers. Whole experiment is planned to generate data from laboratory studies. Experiment will be performed as described in the standard bibliography, may be obtained from standard journals and text books available within the college or from other pharmacy colleges or from libraries of National Institutes or through internet source.

www.sciencedirect.com www.pubmed.com www.google.co.in

7.2 ) METHODS OF COLLECTION DATA: The whole study is divided into following phases:

Phase 1: Collection of plant material. The whole plant will be procured from authenticated supplier in Bengaluru and it will be authenticated by Taxonomist.

Phase II: Preparations of bio-active fractions 1. Extraction with polar solvent like alcohol. 2. After preliminary extraction the Alcoholic extract will be subjected to TLC and HPTLC studies. 3. The alcoholic extract will be subjected to column chromatography for the fractionation. 4. After fractionation isolating the compound from column chromatography the compound will be subjected to anti-inflammatory and immunomodulatory activities.

Phase III : Acute oral toxicity of bio-active plant extracts (AOT). Acute oral toxicity is carried out as per OECD 423 guidelines. Swiss albino mice (18-20 g) are individually identified and allowed to acclimate to the laboratory condition for 7 days before the start of the study. Only one mouse receives single dose at a particular time. First animal receives a dose of 175 mg/kg and is observed for any toxicity signs, survival or death up to 48 hrs. If the first animal died or appeared moribund, the second animal receives a lower dose (55mg/kg). The dose progression or reduction factor is 3.2 times of the previous dose. If no mortality is observed in the first animal then the second animal receives a higher dose (55 mg/kg). Dosing of the next animal is continued depending on the outcome of the previously dose for a fixed time interval (48 hours). The test is stopped when one of the stopping criteria is observed. 5 reversals occur in any 6 consecutive animals tested. Three consecutive animals died at one dose level. Survived animals are observed for long-term outcomes for a period of 14 days. The acute oral toxicity values are calculated using AOT software (Environmental Protection Agency, USA) based on the short term (48 hours) and long term outcome (14 days)21.

PHARMACOLOGICAL EVALUATION. EXPERIMENTAL DESIGN: 1. Acute studies – a. Carageenan induced oedema in rats b.Dextran induced oedema in rats 2. Chronic studies

A. Acute studies 1.Carrageenan-induced paw oedema in rats Animals : Albino Wister rats of either sex Weight : 160-250 g No. of animals required per group : 05 Group I : Normal control, vehicle ,i.e, normal saline (p.o) + 1%w/v carrageenan Group I : standard drug,i.e, Acetyl salicylic acid 100 mg/ kg.(p.o) + 1%w/v carrageenan Group III : Curcuma longa fraction (low dose # ,p.o ) + 1%w/v carrageenan Group IV : Curcuma longa fraction (medium dose # ,p.o ) + 1%w/v carrageenan Group V : Curcuma longa fraction (high dose # ,p.o) + 1%w/v carrageenan Group VI : Pterocarpus marsupium fraction (low dose # ,p.o) + 1%w/v carrageenan Group VII : Pterocarpus marsupium fraction (medium dose # ,p.o) + 1%w/v carrageenan Group VIII : Pterocarpus marsupium fraction (high dose # ,p.o) + 1%w/v carrageenan # Dose of fractions will be fixed after the outcome of acute oral toxicity test.

Procedure : Male or female Wistar rats with a body weight between 160-250g will be used. The animals are starved overnight. To ensure uniform hydration, the rats receive 5 ml of water by stomach tube (controls) or the test drug dissolved or suspended in the same volume. All the animals will be administered with either vehicle/ bioactive fraction/ Acetyl salicylic acid respectively. Thirty minutes later, the rats are challenged by injection of a freshly prepared 0.1 ml of 1%w/v carrageenan in normal saline injected into the plantar region of the left hind paw. The paw will be marked with ink at the level of the lateral malleolus and immersed in mercury tank of plethysmograph and the paw volume is measured immediately after injection, again at 1, 2, 3 and 4h after challenge. The percent change in paw volume will be compared.22

2. Dextran induced paw eodema in rats (5rats/group) Animals : Albino Wister rats of either sex Weight :160-250 g No. of animals required per group : 05 Group I : Normal control, vehicle ,i.e, normal saline (p.o) + 6% w/v Dextran Group II : standard drug,i.e, Acetyl salicylic acid 100 mg/ kg (p.o) + 6% w/v Dextran Group III : Curcuma longa fraction (low dose # , p.o) + 6% w/v Dextran Group IV : Curcuma longa fraction (medium dose # , p.o) + 6% w/v Dextran Group V : Curcuma longa fraction (high dose # , p.o) + 6% w/v Dextran Group VI : Pterocarpus marsupium fraction (low dose # , p.o) + 6% w/v Dextran Group VII : Pterocarpus marsupium fraction (medium dose # , p.o) + 6% w/v Dextran Group VIII : Pterocarpus marsupium fraction (high dose # , p.o) + 6% w/v Dextran

# Dose of fractions will be fixed after the outcome of acute oral toxicity test.

Procedure : Male or female Wistar rats with a body weight between 160-250g will be used. All the animals will be administered with either vehicle/ bioactive fraction/ Acetyl salicylic acid respectively. Thirty minutes later, the rats are challenged by injection of a freshly prepared 0.1 ml of 6%w/v Dextran in normal saline injected into the plantar region of the left hind paw. The paw will be marked with ink at the level of the lateral malleolus and immersed in mercury tank of plethysmograph and the paw volume is measured immediately after injection, and again at 1, 2, 3 and eventually 4h. The percent change in paw volume will be compared.23 B. Chronic studies Animals : Male Albino rats Weight : 120-160 g No. of animals required per group : 05 Group I : Normal control, treatment with vehicle ,i.e, normal saline (p.o) Group II : treatment with standard drug ,i.e, Prednisolone (p.o) Group III : treatment with Curcuma longa fraction (low dose # , p.o) Group IV : treatment with Curcuma longa fraction (medium dose # , p.o) Group V : treatment with Curcuma longa fraction (high dose # , p.o) Group VI : treatment with Pterocarpus marsupium fraction (low dose # , p.o) Group VII : treatment with Pterocarpus marsupium fraction (medium dose # , p.o) Group VIII : treatment with Pterocarpus marsupium fraction (high dose # , p.o)

# Dose of fractions will be fixed after the outcome of acute oral toxicity test.

Procedure : Graded doses of the plant fractions were administered orally (p.o.) once a day for the duration of the experiment. Adjuvant-induced developing arthritis in rats : Chronic arthritis in rats was induced by the injection of 0.05 mL of (0.5% w/v) suspension of killed Mycobacterium tuberculosis, homogenized in liquid paraffin in the left hind foot. The volume of the paw was measured on alternative days and the percentage inhibition was determined on day 13.24,25

Immunophenotyping : Flow cytometry enables the characterization of cells and subcellular organelles on the basis of size and granularity and a number of different parameters defined by fluroscent probes. The arthritic animals were bled on day 14 from the retro-orbital plexus to carry out immune-phenotyping of different T-cell receptors. Specific molecules present on the cell surface define the lymphocytes functional state and capabilities. FITC-labelled anti-mouse CD4 and PE-labelled CD8 monoclonal antibodies were used to determine the percentage of CD4+ and CD8+ T-cells in control and treated group of animals. FITC-labelled CD4 and PE- labelled CD8 monoclonal antibodies were added directly to 100 μL of whole blood . tubes were incubated in the dark for 30 min at room temperature. Subsequently, 1XFACS lysing solution was added at room temperature with gentle mixing followed by incubation for 10 min. the samples were centrifuged at 300-400 X g, the supernatant aspirated and the sample given 3 washings with phosphate buffer saline (pH 7.4) . The resulting stained cell pellet was resuspended in 500 μL of phosphate buffer saline and was run on a flow cytometer. Analysis was done directly on a flow cytometer using Cell Quest Pro software (BD Biosciences, USA).26

Estimation of IFN-γ (Th1 ) and IL-4 (Th2 ) cytokine : The animals were bled retroorbitally and blood was collected in EDTA- coated tubes for the estimation of Th1 cytokine IFN-γ and

Th2 cytokine IL-4. FITC-labelled anti-mouse CD4+ monoclonal antibody and PE-labelled anti- mouse IFN-γ monoclonal antibodies were used in one set and FITC-labelled anti-mouse CD4+ monoclonal antibody along with PE-labelled anti-mouse IL-4 monoclonal antibody was used in another set of experiments. Analysis was done on a flow cytometer. 26,27 . 7.5) Statistical analysis: Results will be expressed as mean ± SEM and data will be subjected to statistical analysis by one-way ANOVA, followed by Dunett’s test.

7.6) Does the study required any investigation to be conducted on patients or animals?

Yes, the entire experimental models require usage of laboratory animals.

7.7) Has ethical clearance been obtained from your institution in 7.3? YES

REFERENCES :

1. Ghaisas MM, Shaikh SA, Despande AD. Evaluation of the immunomodulatory activity of ethanolic extract of the stem bark of Bauhinia variegata Linn. Int J of Green Pharmacy. 2009;27:70-4 2. Vrushali D, Madhavi I. Immunostimulatory activity of Amoora rohituka and Azadirachta indica. Adv Pharmacol Toxicol. 2006;7:5-12. 3. Seth SD. Text book of Pharmacology. 2nd ed. Noida: Reed Elsevier India Pvt. Ltd; 2005. 4. Hardman JG, Limbird LE, Goodman Gilman A. Goodman & Gilman’s The Pharmacological Basis of Therapeutics. 10th ed. New York: Mc Graw Hill; 2001:1463-4 5. Janeway CA, Travers P, Walport M, and Capra JD, eds. Immunobiology: The Immune System in Health and Disease. 4th ed. London: Current Biology Publications; 1999 6. Paul WE, ed. Fundamental Immunology. 4th ed. Philadelphia: Lippincott-Raven; 1997 7. Sengupta M, Sharma GD, Chakraborty B. Hepatoprotective and immunomodulatory properties of aqueous extract of Curcuma longa in carbon tetra chloride intoxicated

Swiss albino mice. Asian Pacific Journal of Tropical Biomedicine. 2011:193-9 8. Araújo CAC, Leon LL. Biological Activities of Curcuma longa L. Mem Inst Oswaldo Cruz. Rio de Janeiro. 2001;96(5): 723-8 9. S Neha, GD Ranvir, CR Jangade. Analgesic and antipyretic activity of Curcuma longa rhizome extract in wister rats. Veterinary world. 2(8):304-6 10. http://www.himalayahealthcare.com/herbfinder/h_curcum.htm

11. Mohammadi M, Khole S, Devasagayam TPA, Ghaskadbi SS. Pterocarpus marsupium extract reveals strong in vitro antioxidant activity. Drug Discov Ther. 2009; 3(4):151- 61.

12. Mankani KL, Krishna V, Manjunatha BK, Vidya SM, Jagadeesh Singh SD, Manohara YN,Anees-Ur Raheman, Avinash KR. Evaluation of hepatoprotective activity of stem bark of Pterocarpus marsupium Roxb. Indian J Pharmacol. June 2005;37(3):165-8 13. Manickam M, Ramanathan M, Farboodniay Jahromi MA, Chansouria JPN, and Ray AB. Antihyperglycemic Activity of Phenolics from Pterocarpus marsupium. J. Nat. Prod. 1997;60 (6):609–10 14. Maruthupandian A and Mohan VR. Antidiabetic, Antihyperlipidaemic and Antioxidant activity of Pterocarpus marsupium Roxb. in alloxan induced diabetic rats. International Journal of PharmTech Research. 2011;3:1681-7 15. Kiritikar K. Basu B. Indian Medicinal Plants. 1999;2:2423-25 16. Curcuma longa information from NPGS/GRIN". www.ars-grin.gov 17. Prajapati ND, Purohit SS, Sharma AK, Kumar T. A Handbook of Medicinal plants. A complete source book. 1st ed. Jodhpur:Agrobios; 2003:429 18. Halagappa K, Girish HN, Srinivasan BP. The study of aqueous extract of Pterocarpus marsupium Roxb. on cytokine TNF-α in type 2 diabetic rats. Indian J Pharmacol 2010;42:392-6 19. Jain D, Maheshwari D, Somani R. Anthelmintic activity of ethanolic extract of Pterocarpus marsupium. Journal of advances in drug research. 2011;1(2) . 20. Ramya S, Kalayansundaram M, Kalaivani T and Jayakumararaj R. Phytochemical Screening and Antibacterial Activity of Leaf Extracts of Pterocarpus marsupium Roxb. (Fabaceae). Ethnobotanical Leaflets. 2008;12:1029-34. 21. 423 Acute Oral toxicity- Acute Toxic Class Method( updated guidelines, adopted 20th December,2001), www.oecd.org/document/55/0,3343,en_2649_34377_2349687_1_1_1_1,00.html-35k

22. Shenoy S, Shwetha K, Prabhu K, Maradi R, Bairy KL, Shanbhag T. Evaluation of antiinflammatory activity of Tephrosia purpurea in rats. Asian Pacific Journal of Tropical Medicine. 2010;3(3):193-5.

23. Sharma US, Sharma UK, Sutar N, Singh A, Shukla DK. Anti-inflammatory activity of Cordia dichotoma forst f. seeds extracts. International Journal of Pharmaceuticals Analysis. 2010;2(1):1-4.

24. Bani S, Chand D, Suri KA, Suri OP, Sharma OP. Anti-inflammatory Effects of an Ethyl Acetate Extract of Euphorbia royleana. Phytotherapy Research. 1996;18:285-91 25. Newbould BB. Chemotherapy of arthritis induced in rats by mycobacterial adjuvant. Br. J. Pharmacol. 1963;21:127-36 26. Pandey A, Kour K, Bani S, Suri KA, Satti NK, Sharma P and Qazi GN. Amelioration of Adjuvant Induced Arthritis by Apocynin. Phytotherapy Research. 2009;23:1462-68 27. Bani S, Kaul A, Khan B, Ahmad SF, Suri KA, Satti NK. 2005.Immunosuppressive properties of an ethyl acetate fraction from Euphorbia royleana. J Ethnopharmacol 99: 185–92 10.1 NAME OF THE CANDIDATE SANJATA MAHAPATRA

10.2 SIGNATURE OF THE CANDIDATE (SANJATA MAHAPATRA)

11.1 REMARKS OF THE GUIDE

11.2 Mrs. K.Girija. NAME AND DESIGNATION OF THE ASSISTANT PROFESSOR GUIDE DEPT. OF PHARMACOGNOSY P.E.S. COLLEGE OF PHARMACY SIGNATURE 11.3

11.4 HEAD OF THE DEPARTMENT Dr.K.Lakshman DEPT. OF PHARMACOGNOSY P.E.S. COLLEGE OF PHARMACY 11.5 SIGNATURE

11.6 REMARKS OF CO-GUIDE

11.7 NAME AND DESIGNATION OF CO- R.Srinath GUIDE HOD & ASSISTANT PROFESSOR DEPT. OF PHARMACOLOGY P.E.S. COLLEGE OF PHARMACY SIGNATURE 11.8

REMARKS OF THE PRINCIPAL 12.1

12.2 SIGNATURE Prof. Dr. S.Mohan PRINCIPAL AND DIRECTOR P.E.S. COLLEGE OF PHARMACY BENGALOORU -560 050

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