Annexure- II proforma for registration of subject for




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RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

BANGALORE, KARNATAKA.


ANNEXURE- II



PROFORMA FOR REGISTRATION OF SUBJECT FOR

M. PHARMA DISSERTATION





1.



Name of the Candidate and Address:



SHILPA.S

D/o M SIDDAPPA, # 685, 3rd main, Maheshwaramma temple road,T.Dasarahalli



BENGALURU -560 057.


2.


Name of the Institute:


Government College of Pharmacy,

No.2, P. KalingaRao Road ,

Subbaiah Circle, Bengaluru-560 027.


3.


Course of Study and Subject:


Master of Pharmacy in Pharmacognosy



4.


Date of admission to course


14th June 2010


5.


Title of the Topic:
BIOACTIVITY GUIDED STUDIES OF ALBIZIA JULIBRISSIN FOR ANTI-ANXIETY ACTIVITY”.




6.

7.

8.



Brief Resume of Intended Work:

6.1: Need for the Study:

  • Anxiety disorder is characterized by excessive, uncontrollable and often irrational worry about everyday things that is disproportionate to the actual source of worry. They often exhibit a variety of physical symptoms as they include fatigue, headache, and numbness in hands, irritability, agitation, sweating, restlessness and insomnia.

  • About one-eighth of the total population world-wide is suffering from anxiety related diseases hence it has become an important area of research interest in psychopharmacology during this decade. Though there are many class of compound useful in anxiety but compounds with less side effects is an important regent. This has promoted many researchers to evaluate new compound in the hope that newer Anxiolytic drugs will have less undesirable effects.

  • The pharmacologist have not given importance to look at the chemical aspects and it was only crude drug as such or at the best their extracts have been pharmacologically evaluated. Very little efforts have been put in trying to attribute the activity to some particular constituents of the plants. Researches from a variety of scientific discipline are confronted with the challenge of extracting plant material with solvent often as a first step towards isolation and identifying the specific compound responsible for biological activities associated with a plant or plant part.1

  • Bioactivity guided isolation is a multidisciplinary approach involving pharmacological evaluation of extracts followed by fractionation of the most active extract. Active fractions again separated till the pure active compounds are obtained.

  • The objective is the targeted isolation of new bioactive plant products, Several “drug discovery hurdles” have to be taken. When an active extract has been identified, firstly it has to be isolated and the second hurdle is purification of the new compound by chromatographic method, the third hurdle is structure determination by spectroscopic method.1

  • Albizia julibrissin belonging to the family Fabaceace. It comprises of number of chemical constituents such as triterpenoidgenins, saponins, flavonoids and tannins. It is used as sedative, tumor, anti-angiogenic, and anti-inflammatory, in skin ulcer, wounds and others.

6.2: Review of literature

  • Albizia julibrissin belongs to the Family Fabaceace. It is a medium sized tree 6-9m tall with a spreading crown. The bark is light brown, nearly smooth and generally thin with lens shaped areas along the stem. Leaves are bipinnate, oblong and alternately arranged on the stem. Flower shows fragrant pink that resembles pompoms. Fruits are straw coloured pods containing light brown oval shaped seed.2

  • The study shows isolation of 2 flavanol glycosides from the flowers of Albizia julibrissin for the sedative action.Fresh flower was extracted with methanol and dissolved in water and partitioned with n-butanol. The n-butanol fraction was dissolved in water and partitioned with n-hexane to give n-hexane and methanolic fractions, then it was subjected to column chromatography using SephadexLH-20 column and methanol as eluent.The two compound isolated were identified using UV, H1and C13 NMR and others. Flavanol glycosides increased pentobarbital-induced sleeping time in dose dependent manner when administered intraperitonially to mice.3

  • Studies were carried out on cytotoxic saponins for structural elucidation and activity for stem bark isolated from Albizia julibrissin. The study involves isolation and structural elucidation of the new saponins. Powdered stem bark was extracted with 95%ethanol. The ethanolic extract was suspended in water, and was extracted with chloroform and n-butanol successively. Then butanolic fraction was subjected to column chromatography and HPLC studies. The product isolated was tested for the presence of triterpenoid saponin and on acid hydrolysis it gave sapogenin. The compound confirmed as Julibroside J27, II and J2. Julibroside exhibited good inhibitory action against KB and Bel cell line with ED500.6µM. No marked inhibitory action was shown against HL-60 cell line with the ED50 not more than 20µM invitro.4

  • The study was on Anxiolytic-like effects of the aqueous extract from Albizia julibrissin stem bark. The water extract was evaluated for its anxiolytic property using elevated plus maze model in rats. The extract of Albizia julibrissin was orally administered at 10, 50, 100 or 200 mg/kg to adult male rats, 1 h before behavioral evaluation respectively. Control rats were treated with an equal volume of saline, and positive control rat’s with buspirone (1mg/kg). After 30 minutes of drug administration the time spent by the rat in the closed arm and number of entries into the open arm was measured.5

  • The plant Albizia julibrissin was studied for isolation and anti-angiogenic activity of natural product from the stem bark of the plant. The powdered stem bark of Albizia julibrissin was extracted with 70% ethanol and sequentially extracted with chloroform, n-butanol. The n-butanol layer was fractionated on column chromatography (Diaion HP-20 resin column) with gradient elution (100%water-100%ethanol). TLC of the fractions was taken and determination by HPLC was carried out using water: methanol (65:35) as mobile phase. The cytotoxic activity was determined by MTT assay method against HMEC-1. In vivo anti-angiogenic effect was evaluated on a chorioallantoic membrane and in transplanted colon carcinoma cells in a nude mice neovascularisation model.6

  • Anti-Oxidant activity of Albizia julibrissin showed Oxygen radical absorbance capacity (ORAC) values of Methanolic extracts of Albizia julibrissin foliage displayed antioxidant activity. High performance liquid chromatography (HPLC) and mass spectrometry (MS) techniques were utilized in the identification of the compounds. The analysis confirmed the presence of three compounds in Albizia. Julibrissin foliage methanolic extracts of unknown quercetin derivative with mass of 610 Da, hyperoside (quercetin-3-O-galactoside), and quercetin (quercetin-3-O-rhamnoside). Fast performance liquid chromatography (FPLC) was employed to fractionate the crude Albizia julibrissin foliage Methanolic extract into its individual flavonoids components. The flavonoids were quantified in terms of mass and their respective contribution to the overall ORAC value. Quercetin glycosides accounted for 2.0% of total foliage.7

  • The mechanism of cytotoxic effects of Albizia julibrissin was investigated on Human acute leukemia Jurkat J cells. Stem bark sample was extracted with methanol and dissolved in water, water extract was fractionated with repeated extraction with (chloroform: butanol: water). Butanolic extract was further fractionated with preparative column chromatography. Evaluation of the fractions was helpful in separation of compound designated as HaBC18. Treatment of HaBC18 at concentration (0.5-2µ|ml) at Jurkat T cells. It induces apoptosis along with biochemical reaction such as mitochondrial cytochrome c release, activation of caspase-9 and -3, degradation of DNA and others. HaBC18-induced apoptosis was abrogated by an ectopic over expression of Bcl-xl, which is known to block mitochondrial cytochrome c release and it has not shown significant activity in primary human cultures of PBMC. This indicates the cytotoxicity is of HaBC18 apoptosis mediated by mitochondrial dependent death-signaling pathway.8

  • The study was conducted to investigate the effects of Albizia julibrissin on growth and brain monoamine neurotransmitters in chronic-stressed rats. The rats in both stressed and treated groups were stressed for seven days, and the rats in the treated group were fed with Albizia julibrissin by gavage administration for 10 days after stress.The results showed that the daily growth in mass of rats in the stressed group significantly decreased compared with the control group (P=0.011), while the Albizia julibrissin treated group had a higher growth than that of the stressed group (P=0.002). The results suggest that Albizia julibrissin alleviates the growth inhibition caused by stress, and regulates the levels of monoamine neurotransmitters of the brain in stressed rats.9

  • Isolation of oleananetriterpenoid saponin from stem bark of Albizia julibrissin was been carried out. The stem bark of Albizia julibrissin was extracted with boiling water, the residual extract was partitioned between n-butanol and water. The butanolic extract was separated by repeated chromatography on normal silica gel, reversed phase silica gel, Sephadex LH-20 and preparative HPLC to obtain a compound (1) Julibroside J32, Julibroside J35(2), Julibroside J36(3) and prosapogenin 9(4).10

  • Two new tri-O-Flavonoids were identified from the leaves of Albizia lebbeck. Dried powdered leaves of Albizia lebbeck were exhaustively extracted was using ethanol: water of (3:1) ratio. The concentrated extract was applied to a polyamide 6SCC and eluted with (water: ethanol) mixtures of decreasing polarities. The successive eluates were individually collected, dried in vacuum and subjected to 2D-PC. Two compounds were isolated from the 20% fraction through Sephadex LH-20 column fractionation using water as an eluent. Kaempferol and quercetin 3-O-alpha–rhamnopyranosyl(1-6)-beta-glucopyranosyl(1-6)-beta galactopyranosides were the structure established by conventional method of analysis and confirmed by ESI-MS.


6.3 Main objective of the study :

  • Collection, proper botanical identification and drying of plant material.

  • Preparation of appropriate extracts and preliminary chromatographic analysis.

  • Biological and pharmacological screening of crude extracts by on swiss albino mice using 2 models

  • Several consecutive steps of chromatographic separation of most active extract..

  • Evaluation of isolated constituent.

The in vivo models which are used are

1) Elevated plus maze model.

2) Rotarod apparatus.

Materials & methods

7.1 Source of the data:

The required data will be obtained from:



  • Electronic data (Internet).

  • Published research papers.

  • Review articles from journals.

  • Library of IISC, Govt.College of pharmacy.

  • Library of Natural remedies.

7.2 Methods of collection of data:

  • Authentification of the plant will be done from recognized institute.

  • Known quantity of drug wiil be taken and extracted with different solvent. Finding the weight of extract will be helpful in calculating percentage of extract.

  • Anti-anxiety studies will be carried out by two method, eight different group of animals will be taken each containing 6 animals .standard, control, samples will be fed in to the animals and change in motor activity and behaviour will be studied for anti-anxiety effects.

  • Extracts with maximum activity will be subjected to chromatographic separation by column chromatogrphy using different solvent system.

  • TLC and HPTLC studies are done depending on the elution Rf value is calculated and compared with standard.

  • Evaluation of isolated constituent.

In-vivo models used are:

Elevated plus maze test:12

Principle: The principle involved in elevated plus maze model is to determine motor activity and increase in the open arm exploratory time after treatment with Anxiolytics.

Procedure:

Mice in groups of 4 each containing six animals were treated with vehicle, diazepam (1mg\kg, i p) 2 solvent extract at various dose orally and the three fractions one hour before mice were placed individually in the EPM. The time spent in open arms and entries into open and closed arms were recorded for a period of 5min.



  1. Group I: Control receives water administered orally.

(b) Group II: Standard receives Diazepam.

(c) Group III: receives 1st solvent extract.

(d) Group IV: receives 2nd solvent extract.

Rotarod method:13

Principle: The test is used to evaluate the activity of drugs interfering with motor function. The dose which impairs the ability of 50%of the mice to remain on the revolving rod is considered the end point.

Procedure: Mice in groups of 4 each containing six animals were treated with vehicle, diazepam (1mg\kg, i p) 2 solvent extract at various dose, thirty minutes after oral administration the mice are placed for 1min on the rotarod. The number of animals falling from the roller during the time is counted

(a) Group I: Control receives water administered orally.

(b) Group II: Standard receives Diazepam.

(c) Group III: receives 1st solvent extract.

(d) Group IV: receives 2nd solvent extract.



7.3: Does the study require any investigations or intervention to be co

Patients other human or animals? If so, please describe briefly:

Yes

7.4: Has ethical clearance been obtained from your institute in case of 7.3.

Institutional Registration number: 185/CPCSEA. Applied for CPCSEA

List of References.

  1. Fabricant DS, Farnsworth NR. The value of plants used in traditional medicine for drug discovery. Environ Health Perspect.2001;109:69-75.

  2. Juli FM, Mortan C. Albizia julibrissin. Proc.fla.state Hort.soc.1983;96:173-8.

  3. Kang TH, Jeong SJ, Kim NY, Higuchi R, Kim YC. Sedative activity of flavanol glycosides isolated from the flowers of Albizia jubrissin Durazz. J Ethnopharmacol.2000;71:321-3.

  4. Kun Z, Jing R. A Cytotoxic Saponins with two Monoterpenoids from Albizia julibrissin, Chin Chem. Lett.2000;11(1):39-42.

  5. Won KI, Ji Hook J, and Nam YA et al. Anxiolytic like effects of extract from Albizia julibrissin bark in the Elevated plus-maze in rats. Life Sci.2004; 75(22):2787-

95.

  1. Hui H, Lei F, Xiao-ping Z, Lian-fen Z, Jian J. Anti-angiogenic activity of JulibrosideJ8, a natural product isolated from Albizia julibrissin.Phytomedicine.2009;16:703-11.

  2. Ching SL, Danielle JC, Robert RB. Identification and quantification of glycoside flavanoid in the energy crop Albizia julibrissin. Bioresource tech.2007;98(2):429-39.

  3. Hae JW, Chang HH, Young HK, Hyun JK,Byung WK, Jae SC et.al. Induction of apoptosis in human acute leukemia Jurkat T cells by Albizia julibrissin extract is mediated via mitochondria-dependent caspase-3 activation. J Ethnopharmacol. 2006;106:383–9.

  4. Zhand F, LI Fa-zeng.Effect of Albizia julibrissin on Growth and Brain Monoamine Neurotransmitters in Chronic-Stressed Rats. Zoolog Res.2006;27(6):621-5.

  5. Amani MD. Leaf flavonoids of Albizia lebbeck. Phytochemistry.1998;48(4):759-61.

  6. Lu Zheng, Jian Zheng, Qingying Zhang, Bin Wang, Yuying Zhao, Lijun Wu. Three new oleanane triterpenoid saponins acetylated with Monoterpenoids Acid from Albizia julibrissin. Fitoterapia.2010;81:859–63.

  7. Pellow S, Chopin PH, File SE, Beiley M. Validation of open: closed arm entries in an elevated plus-maze as a measure of anxiety in the mice. J Neurosci Meth.1985; 14:149-167.

  8. Dinham NW, Miya TS. A note on a simple apparatus for detecting neurological deficit in rats and mice. J Am Pharmaceut Association.1983;46:208-10.



9.

Signature of the candidate





(SHILPA S)

10.

Remarks of the Guide:





11.

Name and Designation of Guide

11.2 Signature


Dr. S SHASHIDHARA

Principal And Head

Dept. of Pharmacognosy

Govt. college of Pharmacy

Bengaluru-560027





11.5 Head of the Department

11.6 Signature

Dr. S SHASHIDHARA

Principal And Head

Dept. of Pharmacognosy

Govt. college of Pharmacy

Bengaluru-560027



12.


12.1 Remarks of the Chairman and Principal

12.2 Signature



DR. S. SHASHIDHARA

PRINCIPAL,



GOVT. COLLEGE OF PHARMCY, BANGALORE- 560027




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