Registration of subject for dissertation a protocol for the project work entitled

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REGISTRATION OF SUBJECT FOR DISSERTATION

A PROTOCOL FOR THE PROJECT WORK ENTITLED

“PHYTOCHEMICAL INVESTIGATION AND BIOLOGICAL ACTIVITY OF STEM BARK OF Lawsonia inermis Linn.”

By

BHAVSAR KAJALBEN NAYANKUMAR

M.Pharm, Part-I

Department of Pharmaceutical Chemistry

NGSM Institute of Pharmaceutical Sciences

Paneer, Deralakatte

Manglore-574160

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALORE

ANNEXURE – II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

	NAME OF THE CANDIDATE AND ADDRESS	BHAVSAR KAJALBEN NAYANKUMAR NGSM INSTITUTE OF PHARMACEUTICAL SCIENCES, PANEER,DERALAKATTE MANGALORE-574160
	NAME OF THE INSTITUTION	NGSM INSTITUTE OF PHARMACEUTICAL SCIENCES,PANEER,DERALAKATTE MANGALORE-574160
	COURSE OF STUDY AND SUBJECT	MASTER OF PHARMACY (PHARMACEUTICAL CHEMISTRY)
	DATE OF ADMISSION TO THE COURSE	JUNE 2008
	Title of the Topic : “*PHYTOCHEMICAL INVESTIGATION AND BIOLOGICAL ACTIVITY OF STEM BARK OF Lawsonia inermis* Linn.”**

6.	Brief Resume of the Intended Work Introduction Lawsonia inermis Linn. (Fam. Lythraceae) is commonly known as “Mehndi”in hindi.¹ A shrub with opposite leaves and very fragrant flowers; up to about 6 m high; generally considered a native of Africa and Asia; widely cultivated in tropical regions of the world. Commercially cultivated in Punjab, Madhya Pradesh, Rajasthan, Gujarat, & Tamilnadu.^2-3 The leaves are emetic & expectorant. The seeds are astringent to the bowels & antipyretic; cure insaning (ayurveda). The seeds are tonic to the brain. The flowers are vulnerary; an infusion cures headache. The bark is given in jaundis and enlargement of the spleen, also in calculous affection; & as an alternative in leprosy & obstinate skin disease; in decoction it is applied to burns, scalds etc.¹
	6.1 Need for the Study The plant Lawsonia inermis Linn belongs to the family Lythraceae.This Lawsonia inermis Linn are claimed to possess antimicrobial activity from the stem bark. Lawsonia inermis are used by local people as an emetic, expectorant, astringent and antipyretic. As the chemical investigation of Lawsonia inermis are not dealt in deep, detailed phytochemical and biological investigation of stem bark of the plant can be done. Investigation of the constituents will give us an insight mechanism into the therapeutic action, which can be useful for research and therapeutic activities.
	6.2 Review of Literature: 1. Devendra K, et al. (1977)⁴ reported two new Xanthone from Lawsonia inermis & characterized as 1-3 dihydroxy-6, 7-dimethoxyxanthone & 1-hydroxy-3, 6-diacetoxy-7-methixyxanthone. 2. Singh VK, et al. (1989)⁵ carried out the fungitoxic studied on bark extract of Lawsonia inermis against ring worm fungi. The extract showed the broad fungitoxic spectum when tested against 13 ring worm fungi. 3. Sharma VK. (1990)⁶studied the tuberculostatic activity of Lawsonia inermis Linn in vitro & in vivo and reported that the growth of tuberculi bacilli from the sputum and of Mycobacterium tuberculosis H37RV was inhibited by Lawsonia inermis Linn. 4. Gupta S, et al. (1991)⁷ reported an inflammatory agent from the Lawsonia inermis stem bark. A new needle shaped oranged colored naphthalene derivative has been isolated from the stem bark of the plant. It has been identified as 2-hydroxy-6-methyl-1, 4-naphthaquinone. It showed anti-inflammatory activity against carrageenan induced acute paw odema in rats. 5. Anand KK, et al. (1992)⁸ carried out an evaluation of Lawsonia alba extract as hepatoprotective agent. This activity of an ethanol- water (1:1) extract of Lawsonia alba has been studied against CCl₄ induced liver toxicity The effects of the extract on hexobarbitol induced sleep, BSP clearance & on certain biochemical parameters indicated its protective role. The extract did not show any signs of toxicities. 6. Alia BH, et al. (1995)⁹ reported that crude ethanolic extract of Lawsonia inermis Linn. Produced significant and dose dependent anti-inflammatory, analgesic, & antipyretic effects in rats. 7. Endrini S, et al. (2002)¹⁰ reported anticarcinogenic and antioxidant activity of chloroform extract of Lawsonia inermis. 8. Yogisha R, et al. (2002)¹¹ carried out trypsin inhibitory activity of Lawsonia inermis.The ethanolic extract of Lawsonia inermis leaves & lawsone tested for trypsin inhibitory activity showed an IC₅₀ value. 9. Mandawgade SD, et al. (2003)¹² isolated naphthaquinone derivative, lawsone from the leaves of lawsonia alba Lam. and reported wound healing activity of Lawsonia alba Lam. leaves using rat excision and incision would models. 10. Bhandarkar M, et al. (2003)¹³ reported the protective effect of aqueous suspension of extract of Lawsonia alba bark against CCl₄ induced hepatic damaged in albino rats. 11. Mikhaeils BR, et al. (2004)¹⁴ determined the antioxidant & Immunomodulatory constituents of henna leaves resulted in the isolation of seven compounds. Their immunomodulatory profile was studied using an in vitro immunoassay, the lymphocyte transformation assay. 12 .Saussriasari R, et al. (2007)¹⁵ carried out cytotoxicity of lawsone & cytoprotective activity of antioxidant in catalase mutant Escherichia coli. They carried out CAT assays; Ames mutagenecity assay & H₂O₂ generation assay.Lawsone generated slightly H₂O₂ in phosphate buffer system & was not mutagenic in Ames assay using TA98, TA100 & TA102 in the absence & presence of metabolic activation. They suggest that lawsone cytotoxicity is probably mediated, at least in plant, by the release of O₂, H₂O₂& OH^-. 13. Sultana N, et al. (2007)¹⁶ evaluated the protein glycation inhibitory activity of ethanolic extract of Lawsonia inernis plant and reported that the alcoholic extract of Lawsonia inermis can effectively protect against protein damage. 14. Mohammad Hosein HK, et al. (2007)¹⁷ studied the effect of addition of Lawsonia inermis extract on the stability of soyabin oil and determined the content total phenolic compounds in the extract by spectrophotometric method and reported antioxidant activity of leaves of Lawsonia inermis.
	6.3 Objectives of the Study 1. Investigation of phytoconstituents of stem bark of Lawsonia inermis Linn. 2. The stem barks of the plant Lawsonia inermis Linn is selected for the study. The stem bark is exhaustively extracted in the soxhlet extractor using ethanol. This ethanolic extract will be subjected to successive extraction by employing different solvent like petroleum ether, diethyl ether, ethyl acetate and butanol. 3. The individual extracts will be subjected to column chromatography to isolate pure component. The pure sample is purified, recrystallised and characterized by spectral data. 4. Screening of Anti-microbial activity of ethanolic extract of stem bark of Lawsonia inermis and its various fractions.
7.	Material and Methods 7.1 Source of data Laboratory based studies. Journals and publications. CD-ROM and Internet. 7.2 Method of collection of data 7.2.1 Collection of plant material and extraction Stem bark will be collected from Karnataka. The air dried powdered stem bark will be subjected to soxhlet extraction with ethanol (95%). The ethanolic extract thus obtained will be concentrated and evaporated under reduced and controlled temperature. The ethanolic extract thus obtained will be subjected to the study of anti microbial activity.
	Method of extraction, isolation and characterization The powdered form of stem bark of Lawsonia inermis will be exhaustively extracted by soxhlet extractor, using ethanol as solvent. The alcohol will be distilled off and the concentrate is evaporate on a water bath into sticky mass. This sticky mass is further fractionated into petroleum ether soluble, ethyl acetate and ether solubles.The dry extracts from all the fractions are first subjected to column chromatography using silica as adsorbent and eluted first with petroleum ether(60-80^oC), petroleum ether(60-80^o C): benzene graded mixture (95:5, 90:10, 80:20 and 50:50) then with benzene followed by graded mixture of benzene: chloroform (95:5, 90:10, 80:20 and 50:50), chloroform and finally chloroform: methanol (95:5, 90:10, 80:20 and 50:50). The elute from different solvent are concentrated to give dry residue which is further recrystallised from the solvent. It is then subjected to characterization; the characterization will be carried out as follows. Elemental Analysis UV absorption studies. IR absorption studies. NMR spectra and Mass spectra.
	7.2.3 Antimicrobial activity: The residues obtained from the different elutes are checked for antimicrobial properties. 1. Evaluation of antimicrobial activity is done against gram +ve organism and gram –ve organism by: Cup plate method/ Disc plate technique. 2. Interpretation is based on the measurement of inhibition zone diameter produced around Cup/disc, Minimum Inhibitory Concentration (MIC)²⁰ is used for the comparison with standard drug.
	7.3 Does the study require any investigations or interventions to be conducted on patients or other humans or animals? No
	7.4 Has ethical clearance been obtained from your institution in case of 7.3? Not Applicable

List of References:

1. Kirtikar KR, Basu BD. Indian Medicinal Plant, vol-2. 2^nd ed. New Delhi: Periodical Expert Book Agency; 1991. p. 1076-8.

2. Nandkarni AK. Indian Materia Medica, vol-1. 3rd ed. Mumbai: Popular Prakashan, 1982. p. 730-2.

3. Khare CP. Encyclopaedia of Indian Medicinal Plant. New York: Springer -Verlag Berlin Heidelburg, 2004. p. 281-2.

4. Devendra K, Bhardwai, Tiravenkata R, Seshadri, Singh R. Xanthones from Lawsonia inermis. Phytochemistry. 1977; 16(10) : 1616-7.

5. Singh VK, Pandey DK. Fungitoxic studies on bark extract of Lawsonia inermis against ring worm fungi. Hindustan Antibiotics Bulletin. 1989; 31(1-2) : 32-5.

6. Sharma VK. Tuberculosis activity of henna (Lawsonia inermis Linn.). Tubercle. 1990 Dec; 71(4) : 293-5.

7. Gupta S, Ali M, Alam S. Anti-inflammatory agents from Lawsonia inermis stems bark. International Journal of Toxicology, Occupational and Environmental Health. 1991; 1(1) : 237.

8. Anand KK, Singh BN, chand D, Chandan BK. An evaluation of Lawsonia alba extract as hepatoprotective agent. Planta Medica. 1992; 58(1) : 22-5.

9. Alia BH, Bashir AK, Tanira M. Anti-inflammatory and analgesic effects of Lawsonia inermis Lann. (Heena) in rats. Pharmacology. 1995; 51 : 356-63.

10. Endrini S, Rahmat A, Ismail P, Taufiq-Yap Yum Hin. Anticarcinogenic properties and antioxidant activity of henna (Lawsonia inermis). Journal of Medical Science. 2002; 2(4) : 194-7.

11.Yogisha R , Samiulla DS , Prashant D ,Padamaja R , Ami A. Trypsin inhibitory activity of Lawsonia inermis. Fititerapia. 2002 Dec; 73(7-8) : 690-1.

12. Mandawgade SD, Ptil KS. Wound healing potential of some active principal of Lawsonia alba Lann. Leaves. Indian Journal of Pharmaceutical Science. 2003; 65(4) : 390-4.

13. Bhandarkar M, Khan A. Protective effect of Lawsonia alba Lann, against CCl4 induced hepatic damage in albino rats. Indian Journal of Experimental Biology. 2003; 41(1) : 85-7.

14. Mikhaeil BR, Badria FA, Matooq GT, Mohamed MA Amer. Antioxidant and immunomodulatory constituents of henna leaves. Z Naturforsch. 2004; 59 : 468-76.

15.Sauriasari R , Wang Da-Hong, Takemura Y , Tsutsui K , Masuoka N , Sano K et al. Cytotoxicity of lawsone and cytoprotective activity of antioxidant in catalase mutant Escherichia Coli. Toxicology. 2007; 235(1-2) : 103-11.

16.Sultana S ,Choudhary MI , Khan A. Protein glycation inhibitory activity of Lawsonia inermis and its active principals. Phytother Res. 2007 Sep; 21(9) : 827-31.

17. Mohammad Hosein HK, Dezashibi Zinab. Phenolic compounds and antioxidant activity of henna leaves extract (Lawsonia inermis). World Journal of Dairy & Food science. 2007; 2(1) : 38-41.

9.	Signature of candidate	(BHAVSAR KAJALBEN NAYANBHAI)
10.	Remarks of the guide	The candidate is working under my direct supervision in laboratories of N.G.S.M Institute of Pharmaceutical Sciences, Paneer, Deralakatte, Mangalore- 574160.
11.	Name & Designation of
	11.1 Guide 11.2 Signature	Dr. CHANDRASHEKAR K.S M.Pharm., Ph. Assistant Professor in Pharmacognosy
	11.3 Head of the Department 11.4 Signature	Prof. (Dr.) EVS Subrahmanyam M.Pharm., Ph.D
12.	12.1 Remarks of the chairman & principal Recommended and Forwarded 12.2 Signature PROF.(Dr.) EVS Subrahmanyam M.Pharm., Ph.D. (PRINCIPAL)

Registration of subject for dissertation a protocol for the project work entitled

Brief Resume of the Intended Work

Recommended and Forwarded