“Evaluation of immunomodulatory and anti-oxidant activity ...



“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

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SUBMITTED TO:

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

BENGALURU, KARNATAKA.

SUBMITTED BY:

SANJATA MAHAPATRA

1st YEAR M.PHARM

DEPARTMENT OF PHARMACOGNOSY

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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 CANDIDATE. |SANJATA MAHAPATRA |

| | |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 SUBJECT. |M.PHARM. |

| | |PHARMACOGNOSY. |

| | | |

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

| | |

|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: |

| |Paw oedema |

| |Percentage of CD4+ and CD8+ T-cell |

| |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. |

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| |google.co.in |

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| | |

| |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 |

| |Extraction with polar solvent like alcohol. |

| |After preliminary extraction the Alcoholic extract will be subjected to TLC and HPTLC studies. |

| |The alcoholic extract will be subjected to column chromatography for the fractionation. |

| |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: |

| |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. |

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| |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. |

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

| |YES |

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| |REFERENCES : |

| | |

| |Ghaisas MM, Shaikh SA, Despande AD. Evaluation of the immunomodulatory activity of ethanolic extract of the stem bark of Bauhinia variegata|

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| |Seth SD. Text book of Pharmacology. 2nd ed. Noida: Reed Elsevier India Pvt. Ltd; 2005. |

| |Hardman JG, Limbird LE, Goodman Gilman A. Goodman & Gilman’s The |

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| |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 |

| |Araújo CAC, Leon LL. Biological Activities of Curcuma longa L. Mem Inst Oswaldo Cruz. Rio de Janeiro. 2001;96(5): 723-8 |

| |S Neha, GD Ranvir, CR Jangade. Analgesic and antipyretic activity of Curcuma longa rhizome extract in wister rats. Veterinary world. |

| |2(8):304-6 |

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| |Mohammadi M, Khole S, Devasagayam TPA, Ghaskadbi SS. Pterocarpus marsupium extract reveals strong in vitro antioxidant activity. Drug |

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| | |

| |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 |

| |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 |

| |Maruthupandian A and Mohan VR. Antidiabetic, Antihyperlipidaemic |

| |and Antioxidant activity of Pterocarpus marsupium Roxb. in alloxan induced diabetic rats. International Journal of PharmTech Research. |

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| |Kiritikar K. Basu B. Indian Medicinal Plants. 1999;2:2423-25 |

| |Curcuma longa information from NPGS/GRIN". ars- |

| |Prajapati ND, Purohit SS, Sharma AK, Kumar T. A Handbook of Medicinal plants. A complete source book. 1st ed. Jodhpur:Agrobios; 2003:429 |

| |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 |

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| |10.1 |

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| |10.2 |

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| |11.1 |

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| |11.2 |

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| |11.3 |

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| |11.4 |

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| |11.5 |

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| |11.6 |

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| |11.7 |

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| |11.8 |

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| |12.1 |

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| |12.2 |

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| |NAME OF THE CANDIDATE |

| | |

| |SIGNATURE OF THE CANDIDATE |

| | |

| |REMARKS OF THE GUIDE |

| | |

| |NAME AND DESIGNATION OF THE |

| |GUIDE |

| | |

| |SIGNATURE |

| | |

| | |

| |HEAD OF THE DEPARTMENT |

| | |

| | |

| |SIGNATURE |

| | |

| |REMARKS OF CO-GUIDE |

| | |

| |NAME AND DESIGNATION OF CO-GUIDE |

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| | |

| |SIGNATURE |

| | |

| |REMARKS OF THE PRINCIPAL |

| | |

| | |

| |SIGNATURE |

| | |

| |SANJATA MAHAPATRA |

| | |

| | |

| |(SANJATA MAHAPATRA) |

| | |

| | |

| |Mrs. K.Girija. |

| |ASSISTANT PROFESSOR |

| |DEPT. OF PHARMACOGNOSY |

| |P.E.S. COLLEGE OF PHARMACY |

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| | |

| |Dr.K.Lakshman |

| |DEPT. OF PHARMACOGNOSY |

| |P.E.S. COLLEGE OF PHARMACY |

| | |

| | |

| | |

| | |

| |R.Srinath |

| |HOD & ASSISTANT PROFESSOR |

| |DEPT. OF PHARMACOLOGY |

| |P.E.S. COLLEGE OF PHARMACY |

| | |

| | |

| | |

| | |

| | |

| | |

| |Prof. Dr. S.Mohan PRINCIPAL AND DIRECTOR |

| | |

| |P.E.S. COLLEGE OF PHARMACY BENGALOORU -560 050 |

| | |

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