Pharmacognostical, Phytochemical and



Pharmacognostical, Phytochemical, Antipyretic

and Analgesic activity studies on the

stem bark of Millingtonia hortensis Linn.f

SYNOPSIS FOR

M. PHARM DISSERTATION

SUBMITTED TO

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

BANGALORE, KARNATAKA

BY

BHARATH.K.K

I- M. PHARM (2009-2010) PHARMACOGNOSY

DEPARTMENT OF PHARMACOGNOSY

M.S. RAMAIAH COLLEGE OF PHARMACY

BANGALORE- 560054

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

BANGALORE, KARNATAKA

ANNEXURE – II

PROFORMA FOR REGISTRATION OF SUBJECT FOR

DISSERTATION

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|1. |NAME OF THE CANDIDATE AND ADDRESS |BHARATH.K.K |

| | |# 74, IInd CROSS, LAKE SHORE GARDEN, THINDLU, VIDYARANYAPURA |

| | |POST, BANGALORE-560027. |

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| |NAME OF THE INSTITUTION |M.S. RAMAIAH COLLEGE OF PHARMACY, BANGALORE. |

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|3. |COURSE OF STUDY AND SUBJECT |M.PHARM |

| | |PHARMACOGNOSY |

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| |DATE OF ADMISSION |20.06.2009 |

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|5. | |Pharmacognostical, Phytochemical, Antipyretic and Analgesic |

| |TITLE OF THE TOPIC |activity studies on the stem bark of Millingtonia hortensis |

| | |Linn. f |

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|6. |BRIEF RESUME OF THE INTENDED WORK |

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|6.1 |NEED FOR STUDY |

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| |Aromatic plants synthesise and preserve a variety of biochemical products, many of which are extractable and useful as |

| |chemical feed stocks or as raw materials for various scientific investigations. Many secondary metabolites of plants are |

| |commercially important and find use in number of perfumery flavoring and pharmaceutical compounds. The characteristic |

| |property of the plant is due to a variety of complex chemical compounds and hence aromatic are generally referred to as |

| |natural ‘bio-chemical factories’ or ‘chemical goldmines’. Essential oils have a variety of therapeutic activities |

| |including antiseptic, antiviral, stimulant, relaxant, diuretic, invigorating, euphoric and digestive1. |

| |Fever if often the result of infection and is caused by release of chemicals (pyrogens) from damaged tissue and cells |

| |involved in inflammation. Pyrogens act on the hypothalamus, which release prostaglandins that reset the hypothalamic |

| |thermostat to the higher temperature. The body responds by activating heat promoting mechanisms, e.g. shivering and |

| |vasoconstriction until the new higher temperature is reached. When thermostat is reset to the normal level, heat loss |

| |mechanisms are activated. There is profuse sweating and vasodilation accompanied by warm, pink (flushed) skin until body |

| |temperature falls to the normal range again. Pain occurs when a local swelling compresses sensory nerve endings. It |

| |exacerbated by chemical mediators of the inflammatory processes, e.g. bradykinin, prostaglandins that potentiate the |

| |sensitivity of the sensory nerve ending to painful stimuli. Although pain is unpleasant experience, it may indirectly |

| |promote healing, because it encourages protection of the damaged site2. |

| |The genus Millingtonia belongs to the family Bignoniaceace. The vernacular names of plant are Indian cork tree, Tree |

| |jasmine(Eng), Biratu mara(Kan), Aakaasha mallige(San), Kaarkku maram(Tam), Kaarku mara(Tel), Kaatesam(Mal)3. |

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| |A monotypic genus of trees of native of Burma and Malaya has become naturalized in India. The tree is hardy. It grows best |

| |in moist climate but does fairly well in dry situations also.4 It is extensively planted in avenues and gardens |

| |throughout India. It flowers during Oct-Dec., but does not ripen seed in W. India, and is propagated by suckers from the |

| |roots5. |

| |Previous investigation revealed the presence of n-Hentriacontanol and (-Sitosterol in bark6,7. (-Carotene and Hispidulin |

| |in leaves8,9. Lapachol in root10 and Hortensin, Scutellarein and essential oil in flower11,12,13 of the plant Millingtonia |

| |hortensis. |

| |Millingtonia hortensis L. have been claimed it be useful as traditional medicine for the treatment of asthma and sinusitis |

| |(flower part), tonic (root part), antipyretic (bark part) and also antimicrobial activity (leaf part)14,15. |

| |The present investigation is aimed to carry out Pharmacognostical, phytochemical, antipyretic and analgesic activity |

| |studies on the stem bark of Millingtonia hortensis Linn. f., since no such comprehensive work is reported. |

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| |REVIEW OF LITERATURE |

| |Botanical name, Vernacular names and Morphological description of Millingtonia hortensis L.f. are given 3, 4, . |

| |Description, distribution and traditional uses of plant are mentioned4,5,. |

| |Vernacular names, distribution and chemical constituents such as n-Hentriacontanol and (-sitosterol isolated from bark are |

| |given6. |

| |Isolation of (-sitosterol and lapachol from the bark of Millingtonia hortensis was reported7. |

| |Flavonoids like 6-methoxy-5,7,4(-trihydroxyflavone(hispidulin=dinatin) from leaves, hispidulin from fruits was reported8. |

| |Chemical constituents isolated from leaves and flowers are given9. |

| |Chemical constituents such as Hentriacontane, Lapachol, Hentriacontanol-1, (-Sitosterol and Paulownin in the roots of |

| |Millingtonia hortensis Linn was reported10. |

| |Scutellarein-5-galactoside and Scutellarein from flowers of Millingtonia hortensis was reported 11. |

| |Hortensin, A flavonoid from Millingtonia hortensis was reported12. |

| |Anti-microbial activities of essential oils present in the flowers of Millingtonia hortensis Linn. was reported13. |

| |Botanical name and traditional use of the bark of Millingtonia hortensis are given14. |

| |Antiproliferation and apoptosis on RKO colon cancer by Millingtonia hortensis was reported15. |

| |Lapachol and other constituents from the Bignoniaceae was reported16. |

| |A phytochemical and pharmacological study of the flowers of the Millingtonia hortensis Linn.F was reported17. |

| |A diuretic flavone glycoside from Millingtonia hortensis Linn was reported18. |

| |Flavonoids of eight bignoniaceous plants was reported19 |

| |Phytochemical and pharmacological studies of the flowers of Millingtonia hortensis Linn.F. was reported20. |

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| |The inhibitory activity in 5-Lipoxgenase pathway of hispidulin from Millingtonia hortensis Linn. F. was reported21. |

| |Larvicidal activity of leaf extract of Millingtonia hortensis (Family: Bignoniaceae) against Anopheles stephensi, Culex |

|6.2 |quinquefasciatus and Aedes aegypti was reported22. |

| |Antiproliferative effect on colon cancer cell lines by aqueous extract from the bark of Millingtonia hortensis was |

| |reported23. |

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| |AIMS AND OBJECTIVES OF THE STUDY |

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| |AIMS |

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| |1. To undertake Pharmacognostical investigation on roots of Millingtonia hortensis Linn.f. |

| |2. To investigate and evolve Phytochemical parameters for the drug. |

| |3. To investigate and evaluate acute toxicity, antipyretic and analgesic activity of the drug. |

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| |OBJECTIVES |

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| |To study taxonomical characters of the plant which help in the investigation. |

| |To understand the macro and microscopical characters of the drug besides phytochemical analysis. This helps in |

| |evolving diagnostic characters for the identification of the drug and also contributes towards Pharmacopoeial |

| |standards. |

| |To carry out acute toxicity studies. |

| |To evaluate the antipyretic and analgesic activity of the drug. |

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| |MATERIALS AND METHODS |

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| |SOURCES OF DATA |

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| |Literature survey, Websites. |

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| |Journals and Publications. |

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| |Lab based studies. |

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| |METHOD OF COLLECTION OF DATA |

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| |Field and laboratory based studies |

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| |Field Work: |

| |The selected plant stem bark will be collected from forests. The voucher specimen will be collected and deposited at the |

| |herbarium/museum of the department. |

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| |Laboratory work: |

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| |Taxonomical studies: |

| |The plant material will be identified by using various Floras like the “Flora of Presidency of Bombay”5. |

|6.3 | |

| |Pharmacognostical studies: |

| |Free hand sections of the drug will be taken following Wallis24. Microscopical investigation and histochemical tests will |

|6.3.1 |be carried out following Evans25. |

| |Quantitative microscopy for carrying out the measurement of tissues will |

| |be done by using stage and ocular micrometers. Photomicrographs will be taken and images captured on computer. |

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| |Phytochemical studies: |

| |Physical constants of drug will be determined as per Indian Pharmacopoeia26, and Phytochemical tests for detection of |

| |organic constituents will be done as per Harborne27. The chromatographic, fluorescence and HPTLC |

| |studies will be carried out following Krebs et al28 and Kokoski CJ et al29. |

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| |Pharmacological studies: |

|6.3.2 |Acute toxicity studies: |

| |The acute toxicity study will be carried out as per OECD guidelines30. The procedure will be divided into two phases, Phase|

| |1 (observation made on day 1), and Phase 2 (observed the animals since next 14 days). Animals will be fasted |

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| |for 18 hours prior to the administration of extract orally and individually animals will be observed for 4 hours to find |

| |any clinical symptoms, any change in |

| |behaviour or mortality and 6 hours post dosing again body weights recorded. From the next day onwards, each day one hour |

| |the behavioural change, clinical symptoms or mortality will be observed in the same animals for next 14 days and animal |

| |body weights will be recorded on 8th and 14th day. The same procedure will be repeated with another set of animals to |

| |nullify the errors. |

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| |Antipyretic activity :31, 32, 33. |

| |Antipyretic activity of the drug extracts will be studied on albino rats |

| |(Wistar strain) of either sex in the weight range of 170-190 g. Animals outside this weight range will be excluded from the|

|7. |study. |

| |No. Of groups = 6 |

| |(Each containing 6 rats) |

|7.1 |Group 1: vehicle control group |

| |Group 2: Standard group, 45mg /kg of Paracetamol p.o. |

| |Group 3: 1st dose each of aqueous extract of drug p.o. |

| |Group 4: 2nd dose of aqueous extract of drug p.o. |

| |Group 5: 1st dose each of alcohol extract of drug p.o. |

| |Group 6: 2nd dose of alcohol extract of drug p.o. |

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| |Basal body temperature of all animals will be noted first. Fever will be induced in rats by subcutaneous injection of |

| |Brewer’s yeast in distilled water (2mg/kg). After 18 hours rats will show increased body temperature. Drugs will be |

| |administered to febrile rats once and body temperature will be noted at specific intervals of (4 hrs.) after drug |

| |treatment. |

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| |The results will be subjected to statistical analysis by one way ANOVA followed by Tukey-Kramer multiple comparison test. |

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|7.2 | |

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|7.2.1 | |

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| |Analgesic activity31, 32, 33,34: |

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| |Acetic acid induced abdominal writhing test: |

| |Acetic acid is used to induce the pain, which is indicated by writhings. |

| |Procedure: Pain will be induced by intra-peritoneal injection of acetic acid 0.6%v/v, 10ml/kg body weight. The animals will|

| |be pretreated respectively with vehicle, Diclofenac sodium (6.5mg/kg, i.p) and test drugs. Acetic acid will be injected |

| |intra-peritoneally 30 min later for standard and one hr. later for test drugs. The number of stretching or writhing will |

| |be recorded for 10 min. |

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| |Animals will be divided into 6 groups of 6 animals each. |

| |Group I (control group) : Untreated 6. |

| |Group II (standard) : Diclofenac sodium(6.5mg/kg, i.p) 6. |

| |Group III : 1st dose of Aqueous extract. 6. |

| |Group IV : 2nd dose of Aqueous extract 6. |

| |Group V : 1st dose of Alcohol extract. 6. |

| |Group VI : 2nd dose of Alcohol extract 6. |

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| |Eddy’s hot plate method: |

| |Here the pain threshold is considered to be reached when the animals lift/lick their paws, or jump. |

| |The hot plate will be used to measure the response latencies. |

| |Procedure: The animals will be placed on Eddy’s Hot Plate maintained at a temperature of 55°C±1°C. A cut off period of 15 |

| |sec, will be observed to avoid damage to the paws. Reaction time and the type of response viz. jumping/withdrawal of |

| |paws/licking of paws whichever comes first will be noted using a stopwatch. The latency will be recorded before and after |

| |15, 30, 60 and 120 min of treatment. |

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| |Animals will be divided into 6 groups of 6 animals each. |

| |Group I (control group) : Untreated – 6. |

| |Group II (Standard) : Tramadol (30mg/kg) – 6. |

| |Group III (Extract) : 1st dose of Aqueous extract - 6. |

| |Group IV (Extract) : 2nd dose of Aqueous extract- 6. |

| |Group V (Extract) : 1st dose of Alcohol extract-6. |

| |Group VI (Extract) : 2nd dose of Alcohol extract-6. |

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| |Does the study require any investigation to be conducted on patients or other humans or animals? If so, please describe |

| |briefly. |

| |Yes, Animal experiments on rats will be required for studying antipyretic activity and acute toxicity studies and mice will|

| |be required for analgesic activity studies. |

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| |Has ethical clearance been obtained from your institution in case of 7.3? |

| |Yes, ethical committee clearance has been obtained. Certificate copy enclosed |

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| |LIST OF REFERENCES |

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| |Baby PS, Joy PP, Mathews S, Mathews G, Joseph A, Joseph R. Aromatic plants: Horticulture science series1. New Delhi: NIPA |

| |2007; 1: 1-12. |

| |Waugh A, Grant A. Ross and Wilson Anatomy of physiology in health and illness. 10th ed. Elsevier: Churchill livingstone |

| |2006; 362,373. |

| |Gurudeva R Magadi. Botanical and Vernacular Names of South Indian plants. Bangalore: Divyachandra prakashana 2001: 277. |

| |Anonymous. The wealth of India, Raw material L-M. New Delhi: CSIR 1998; 6: 380. |

| |Cooke Theodore. The flora of the presidency of Bombay. Dehra Dun: Bishan singh Mahendra pal singh.2006; 334. |

| |Rastogi RP, Mehrotra BN. Compendium of Indian medicinal plants. Lucknow: CDRI and New Delhi: NISC 1970-1979; 2: 464. |

| |Joshi CK, Prakash Lalit and Singh Pahup. Chemical investigation of the Barks of Tecomella undulata, Heterophragma |

| |adenophylum and Millingtonia hortensis (Bignoniaceae). J Indian Chem Soc 1973 August; 50:561-562. |

| |Subramaniam Sankara S, Nagrajan S, Sulochana N. Flavonoids of Millingtonia hortensis. Current science (India) 1971 Jan |

| |15;40:194. |

| |Rastogi RP, Mehrotra BN. Compendium of Indian medicinal plants. Lucknow:CDRI 1985-1989; 4: 478 |

| |Prakash L and Mrs Garg gita. Chemical constituents of the roots of Millingtonia hortensis Linn. and Acacia nilotica (Linn.)|

| |Del. J Indian Chem Soc 1981 January;58:96-97. |

| |Sharma RC, Zaman A and Kidwai AR. Chemical examination of Millingtonia hortensis. Phytochemistry 1968;7:1891-1892. |

| |Bunyapraphatsara N, Bkasko G, Cordell AG. Hortensin, an unusual flavone from Millingtonia hortensis. Phytochemistry |

| |1989;28:1555-1556. |

| |. Sittiwet Chaiyasit. Anti-Microbial activities of Millingtonia hortensis Linn. Flowers essential oil. Journal of |

| |Pharmacology and Toxicology 2009; 4(1):41-44. |

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| |Singh VK, Govil JN, Hashmi S, Singh G. Recent progress in medicinal plants, Ethno medicine and pharmacognosy II. USA: |

| |studium press LIC;7: 243. |

| |Tansuwanwong Siwapong, Yamamoto H, Imai K, Vinitketkumnuen U. Antiproliferation and apoptosis on RKO colon cancer by |

| |Millingtonia hortensis. Plants foods Hum Nutr 2009;64:11-17. |

| |Singh pahup, Prakash Lalit and Joshi CK. Lapachol and other constituents from the Bignoniaceae. Phytochemistry 1972;2:1498.|

| |Anonymous. Phytochemical and pharmacological studies of the flowers of Millingtonia hortensis Linn. F. Chemical abstracts |

| |1988; 108: 45. |

| |Kar.K, Singh S and Khanna NM. A diuretic flavone glycoside from Millingtonia hortensis. The Indian journal of pharmacy |

| |Jan-Feb 1976; 38(1): 26-27. |

| |Subramanian Sankara.S, Nagarajan S and Sulochana N. Flavonoids of eight Bignoniaceous plants. Phytochemistry 1972;2:1499. |

| |Anulakanapakorn K, Bunyapraphatsara N, Satayavivad J. Phytochemical and pharmacological studies of the flowers of |

| |Millingtonia hortensis linn.f. J Sci Soc Thailand 1987;13:71-83. |

| |Moongkarndi P, Bunyapraphatsara N, Srisukh V, Wagner H. The inhibitory activity in 5-Lipoxygenase pathway of hispidulin |

| |from Millingtonia hortensis Linn. f. J Sci Soc Thailand 1991;17:51-56. |

| |Kaushik R, Saini P. Larvicidal activity of leaf extract of Millingtonia hortensis (Family: Bignoniaceae) against Anopheles |

| |stephensi, Culex quinquefasciatus and Aedes aegypti. J Vector Borne Dis 2008 March; 45:66-69. |

| |Tansuwanwong S, Yamamoto H, Imai K, Vinitketkumnuen U. Antiproliferative effect on colon cancer cell lines by aqueous |

| |extract from the bark of Millingtonia hortensis. Chiang Mai Med J 2007;46(2):61-66. |

| |Wallis TE. Text book of pharmacognosy. New Delhi: CBS publishers and distributors1985; 355-58. |

| |Evans WC. Trease and Evans Pharmacognosy. 15th ed. London Saunders 2002; 53-54. |

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| |Anonymous. Indian Pharmacopoeia. New Delhi: Controller of Publications. 1996; 2(A-89):53-54. |

| |Harborne JB. Phytochemical methods, A guide to modern techniques of plant analysis. 3rd ed. London Chapman and Hall 1998; |

| |74-83. |

| |Krebs KG, Heunsen D, Wimmer H. Thin layer chromatography, A laboratory hand books, 2nd ed. London ELBS. 1969; 204-55, |

| |855-09. |

| |Kokoski CJ, Kokoski RJ, Slama FJ. Florescence of powdered vegetable drugs under ultraviolet radiation. Journal of the |

| |American Pharmaceutical Association 1958 Oct; 47(10):715-717. |

| |OECD/OCDE, OECD guidelines for testing chemical-420: 2001 Dec; 1-14. |

| |Nandi D, Besra SE, Dey S, Babu S, Elango A, Roy S et al. Anti-inflammatory and analgesic activities of leaf extract of |

| |Wattakaka volubilis (Dreagea volubilis). International journal of Green Pharmacy 2009 July-Sep;195-200. |

| |Sabina EP, Chandel S, Rasool MK. Evaluation of analgesic, antipyretic and ulcerogenic effect of Withaferin A. International|

| |Journal of Integrative Biology 2009;6(2):52-56. |

| |Sawadogo WR, Boly R, Lompo M, Some N, Lamien CE, Guissou IP et al. Anti-inflammatory, Analgesic and Antipyretic activities |

| |of Dicliptera verticillata. International Journal of Pharmacology 2006;2(4):435-438. |

| |Kaushik D, Khora SL, Kaushik P, Saneja A, Chaudhary B, Koshy S et al. A study of Analgesic and Antimicrobial Potential of |

| |Mitragyna Parvifolia. International Journal of Pharmaceutical Science and Drug Research. 2009;1(1):6-8. |

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|7.3 | |

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| |Signature of the Candidate: |

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| |Remarks of the Guide: |

|7.4 | |

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| |Name and Designation: |

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| |11.1) Guide: Mr.Ashoka Babu V L |

| |Asst. Professor |

| |Department of Pharmacognosy |

| |M.S. Ramaiah College of Pharmacy |

| |Bangalore. |

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| |11.2) Signature: |

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| |11.3) Co-Guide: Not Applicable |

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| |11.4) Signature: Not Applicable |

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| |11.5) Head of the Department: Dr. V. Madhavan. M. Pharm, Ph.D. |

| |Principal, Professor & Head |

| |Department of Pharmacognosy |

| |M. S. Ramaiah College of Pharmacy |

| |Bangalore. |

| |11.6) Signature: |

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| |12.1) Remarks of the Principal: |

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| |12.2) Signature: |

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