Comparative Evaluation of Leaves and Roots Of

“COMPARATIVE EVALUATION OF LEAVES AND ROOTS OF

WITHANIA SOMNIFERA COLLECTED FROM DIFFERENT GEOGRAPHICAL LOCATIONS AND ISOLATION OF PHYTOCHEMICAL CONSTITUENTS/MARKERS FROM WITHANIA SOMNIFERA LEAVES”

SYNOPSIS FOR

M.PHARM DISSERTATION

SUBMITTED TO

RAJIV GANDHI UNIVERSITY OF

HEALTH SCIENCES

KARNATAKA

BY

ARSHEE MEHBOOB

I M.PHARM

DEPARTMENT OF PHARMACOGNOSY

AL AMEEN COLLEGE OF PHARMACY

BANGALORE - 560027

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALORE

ANNEXURE – II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. / NAME OF THE CANDIDATE AND ADDRESS / ARSHEE MEHBOOB
D/O MOHD MEHBOOB KHAN GAURI.
B-1 SUSWEE APARTMENT, 17-CROSS, 15- MAIN , 5TH PHASE J. P NAGAR.
BANGALORE
KARNATAKA.
2. / NAME OF THE INSTITUTION / AL- AMEEN COLLEGE OF PHARMACY
HOSUR ROAD, BANGALORE – 560027
3. / COURSE OF STUDY AND SUBJECT / M. PHARM - PHARMACOGNOSY
4. / DATE OF ADMISSION / JULY – 2008
5. / TITLE OF TOPIC: -
“COMPARATIVE EVALUATION OF LEAVES AND ROOTS OF WITHANIA
SOMNIFERA COLLECTED FROM DIFFERENT GEOGRAPHICAL LOCATIONS AND ISOLATION OF PHYTOCHEMICAL CONSTITUENTS/MARKERS FROM WITHANIA SOMNIFERA LEAVES”
6.0 / BRIEF RESUME OF THE INTENDED WORK :
6.1 / NEED OF STUDY
Withania somnifera, also known as ashwagandha, Indian ginseng, and winter cherry, has been an important herb in the Ayurvedic and indigenous medical systems16 for over 3000 years. Historically, the plant has been used as an aphrodisiac, liver tonic, anti-inflammatory agent, astringent, and more recently to treat bronchitis, asthma, ulcers, emaciation, insomnia, and senile dementia. Clinical trials and animal research support the use of ashwagandha for anxiety, cognitive and neurological disorders, inflammation, and Parkinson’s disease. Ashwagandha’s chemopreventive properties make it a potentially useful adjunct for patients undergoing radiation and chemotherapy. Ashwagandha is also used therapeutically as an adaptogen for patients with nervous exhaustion, insomnia, and debility due to stress, and as an immune stimulant in patients with low white blood cell counts. The major biochemical constituents of ashwaganda root are steroidal alkaloids and steroidal lactones in a class of constituents called withanolides. At present, 12 alkaloids, 35 withanolides, and several sitoindosides from this plant have been isolated and studied. A sitoindoside is a withanolide containing a glucose molecule at carbon 27. Much of ashwagandha’s pharmacological activity has been attributed to two main withanolides, withaferin A and withanolide D. For the preparation of methanolic and aqueous extracts from roots and leaves conventional methods like sonication4, refluxation8,9,10, Percolation11, and countercurrent extractions12 are used .Various withanolides isolated from different parts of Withania somnifera have been analysed by U V15 and by HPLC method.4
Withanolides are present in both leaves and roots, which are medicinally used. But from the leaves the withanolides are not much used. In the present investigation an attempt will be made to develop protocol for isolation of major withanolides from the leaves of Withania somnifera and chemical comparison of them with the roots by HPTLC fingerprinting. To improve the commercial outlook for the production of withanolides, advanced techniques will be used. The present study will help in the isolation of withanolides from leaves, and comparing the yield chemical entities with that present in the root of Withania somnifera.
6.2 /

LITERATURE REVIEW

6.3 / ·  Aqueous suspension of Withania somnifera root powder was investigated for there in vitro and in vivo immunomodulatory properties. It showed potent inhibitory activity towards the compliment system, mitogen induced lymphocyte proliferation and delayed type hypersensivity reaction.1
·  The effects of ethanolic extracts of Withania somnifera Dunal (REWS) against Dalton’s Ascitic lymphoma has been evaluated in Swiss albino mice by Christina .et al. A significant increase in the life span and a decrease in the cancer cell number and tumour weight were noted in tumour-induced mice after treatment with REWS.2
·  Growth inhibition of Human tumour cell lines by Withanolides from Withania somnifera leaves by Muraleedharan .Nair et al.3
·  HPLC method for determination of withaferinA and withanolide D was developed and this system was successfully used to investigate the presence of the markers in different Withania somnifera plant parts as well as to analyze their content in market products, which was reported by .Ganzera. 4
·  Invitro flowering, in vitro fruiting and effective micro propagation protocol were studied in Withania somnifera, an antitumor medicinal plant using axillary bud plants by Naidu et al..5
·  A significant dose dependent diuretic effect produced in the lab rats by hot water infusions and methanolic extracts of Withania aristata Ait., has been reported by J. Gutierrez-Luis et al. 6
·  Muraleedheran .Nair et al. studied the potent lipid peroxidation inhibitors from Withania somnifera fruits.7
·  M.Owais reported antibacterial efficacy of Withania somnifera and showed that aqueous as well as alcoholic extracts of the plant found to possess strong antibacterial activity against a spectrum of bacteria including Salmonella typhimurium.8
·  Many withanolides have been reported from Withania species like Hisashi Matsuda et al. reported isolation of withanosides I, II, III, IV, V, VI and VII from roots of Withania somnifera and their structures were determined based on chemical and physicochemical evidence and studied the inhibitory activity of withaferin VI and withaferin A for tachyphylaxis to clonidine in isolated guinea pig ileum. 9
·  Effect of Withania somnifera root extract on cell cycle and angiogenesis was evaluated and finding suggest that roots of Withania somnifera possess cell cycle disruption and anti-angiogenic activity, which may be a critical mediator for its anti-cancer action.10
·  There is report of isolation of withanolides from Datura species like Withafastuosin F from flowers of Datura fastuosa .11
· Withania somnifera have been investigated for its antistress property in the study done by Namasivayam and Archana using adult Wistar strain albino rats and cold-water swimming stress test. The treated animals showed better tolerance limits.12
·  Namasivayam et al., studied the Antistessor effect on withania somnifera.13
· Muruganandam et al., studied adaptogenic activity of Withania somnifera in an experiment using a rat model of chronic stress.14
·  Garg K.N et al. worked on spectroscopic on a withanolide from Withania coagulans.15
·  Swinbourne et al. Withasomnine studied the pyrazole alkaloid from Withania somnifera Dun. Tetrahedron.16
·  Lavie et al. studied the Chemotaxonomic study of Withania somnifera. 17
·  Jarnal et al. studied the structure and bioactivities of withanolides from Withania somnifera.18
·  Kaur et al. Evaluated the anti-proliferative and anti-oxidative activities of leaf extract from in vivo and in vitro raised Ashwagandha.19
·  Abraham et al.studied the withanolides of withania somnifera chemotypes 1&2.20
AIMS AND OBJECTIVES OF THE STUDY
AIM:
AIM AIM : - To carry out comparative evaluation of leaves and roots of Withania somnifera collected from different geographical locations and isolation of phytochemical constituents/markers from Withania somnifera leaves.
OBJECTIVES: -
1. To carry out extraction of Withania somnifera Roots & Leaves.
2. TLC Method development for the Extracts prepared.
3. Isolation of the Phytochemical reference substance /Markers from the
leaves extract by various chromatographic techniques like Column
Chromatography/MPLC/HPLC.
4. Identification & Characterization of the isolated Phytochemical
constituent by various spectroscopic techniques like 1H NMR, 13C NMR,
FTIR & MASS.
5. Comparison of Roots & Leaves extract by HPTLC/HPLC.
7.0
7.1 / MATERIALS AND METHODS
SOURCES OF DATA
vAl-Ameen college of Pharmacy Information Center,
Bangalore.
vLiterature survey.
vJournal and Publications.
vSearch on - Science direct, Pub med, Medline, Google.
vJ-Gate@HELINET search.
PLACE OF WORK:-
·  Naturals remedies Pvt. Ltd., Bangalore.
·  Al-Ameen college of Pharmacy, Bangalore.
7.2 /

METHOD OF COLLECTION OF DATA

1. Raw material will be Collected from natural remedies Pvt Lmt bangalore.
2.Preparation of chloroform and water extracts of Withania somnifera from Roots& Leaves.
3.TLC Method development for the Extracts prepared.
4. Isolation of the Phytochemical reference substance /Markers from the leaves extract by various chromatographic techniques like Column Chromatography/MPLC/HPLC.
5. Identification & Characterization of the isolated Phytochemical constituent by various spectroscopic techniques like 1H NMR, 13C NMR, FTIR & MASS.
6.Comparison of Roots & Leaves extract by HPTLC/HPLC.
7.3 / Does the study require any investigations or interventions to be conducted on patient or other humans or animals? If so please describe briefly.
-NA-
7.4 / Has ethical clearance been obtained from your institution in case of the above query?
-N A-
8.0 / REFERENCES:
1. Varalakshmi P, Rasool M: Immunomodulatory role of Withania somnifera root powder on experimental induced inflammation:An in vivo and in vitro study. Vascular Pharmacology 2006; 44:406-410.
2. Christina AJM, Joseph GD, Packialakshmi M, Kothaai S, Robert SJH, Chidambaranathan N, Ramasamy M: Anticarcinogenic activity of Withania somnifera Dunal aganint Dalton’s Asicitic Lymphoma. Journol of Ethnopharmacology 2004; 93:356-361.
3. Nair GM, Seeram NP, Zhang Y, Jayaprakasam B.L. Growth inhibition of human
tumor cell lines by Withanolides from Withania somnifera leaves. Life Sciences
2003; 74:125-132.
4. Ganzera M, Choudhary MI, Khan IA. Quantitative HPLC analysis of
withanolides in Withania somnifera. Fitoterapia 2003; 74:68-76.
5. Naidu CV, Saritha KV.In vitro flowering of Withania somnifera Dunal- An
important antitumor medicinal plant. Plant sciences 2007; 172:847-851.
6. Herrera MD, Abdala S, Benjumea, Luis- GJ. Diuretic activity of Withania
aristata Ait. fractions. Journal of Ethanopharmacology 2008;496-499.
7. Jayaprakasam B, Strasburg AG, Nair. GM. Tetrahedron 2004; 60:3109-3121.
8. Owais.M, Sharad.KS, Shehbaz A, Saleemuddin M. Antibacterial efficacy of
Withania somnifera an indigenous medicinal plant against experimental
murine salmonellosis.Phytomedicine2005;12:229-235.
9. Mastuda H, Murakami M, Kishi A, Yoshikawa M. Structures of withanosides I,II,III,IV,V,VI AND VII, New withanolide glycosides from the roots of Indian Withania somnifera DUNAL.and inhibitory activity for tachyphylaxis to clonidine in isolated guinea-pig ileum.Bioorganic and medicinal chemistry 2001; 9:1499-1507.
10. Rajani M. Evaluation of the effect of Withania somnifera root extracts on cell cycle and angiogenesis.Journal of Ethnopharmacology 2006;105:336-341.
11. Manickam.M, Srivastava.A, Ray AB.Withanolides from the flowers of Datura fastuosa.Phytochemistry 1998; 47:1427-1429.
12. Rajpal.V.Standardisation of botanicals.Eastern publishers,2002; Vol.1.
13.Narasivayam A, Archana R. Antistressor effects of Withania somnifera. Journal of Ethanopharmacology 1999;64:91-93
14. Muruganandam A.V, Bhattacharya. S.K. Adaptogenic activity of Withania somnifera: an experimental study using a rat model of chronic stress. Pharmcology, Biochemistry and Behaviour 2003; 75:547-555.
15. Ramaiah AP, Lavie D, Budhiraja D, Ramji, SS, Garg NK.Spectroscopic studies on a withanolide from Withania coagulans. Phytochemistry 1984; 23:143-149.
16. Swinbourne FJ, Katritzky AR. Withasomnine: A pyrazole alkaloid from Withania somnifera Dun. Tetrahedron 1996; 22:2895-2897.
17.Lavie D, Glotter E, Kirson I. A Chemotaxonomic study of Withania somnifera. Phytochemistry 1968; 7; 957-962.
18. Jamal SA, Qureshi S, Ali SN, Choudhary MI, Atta -ur -Rahman. Bioactivities and structural studies of withanolides from Withania somnifera.
19. Kaur K, Rani. G, Widodo N, Nagpal A, Taira K, Kaul SC, Wadhwa. R. Evaluation of anti-proliferative and anti-oxidative activities of leaf extract from in vivo and in vitro raised Ashwagandha. Food and Chemical Toxicology 2004; 42:2015-2020.
20. Abraham A, Kirson I ,Glotter E. The withanolides of Withania somnifera
chemotypes 1 & 2. Phytochemistry 1975, 14: 189-194.
9. / SIGNATURE OF THE CANDIDATE:
10. / REMARKS OF THE GUIDE: / RECOMMENDED FOR RESEARCH AND SUBMISSION OF DISSERTATION
11. / NAME AND DESIGNATION OF THE GUIDE: / Dr. RAMAN DANG
PROFESSOR
Department of Pharmacognosy,
Al-Ameen College of Pharmacy,
Bangalore.
11.1. / SIGNATURE:
11.2 / CO-GUIDE / Dr. DEEPAK M
SENIOR MANAGER
Dept. of Phytochemistry,
Natural Remedies pvt. Ltd.,
Bangalore.
11.3 / SIGNATURE:
11.4 / HEAD OF THE DEPARTMENT: / Dr. SALMA KHANAM
PROF. AND H.O.D.
Department of Pharmacognosy,
Al-Ameen College of Pharmacy,
Bangalore.
11.5 / SIGNATURE:
11.6 / PRINCIPAL: / Prof. B.G. SHIVANANDA
Al-Ameen College of Pharmacy,
Hosur road, Bangalore.
11.7 / REMARKS OF THE PRINCIPAL: / RECOMMENDED FOR RESEARCH AND SUBMISSION OF DISSERTATION
11.8 / SIGNATURE: