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 AND PHARMACOLOGICAL INVESTIGATION OF Anacardium occidentale Linn.



By

DAHAKE AKASH P.

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

DAHAKE AKASH P.

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 2007



Title of the Topic :

PHYTOCHEMICAL AND PHARMACOLOGICAL INVESTIGATION OF Anacardium occidentale Linn.









Brief Resume of the Intended Work

introduction

Anacardium occidentale Linn. (Fam: Anacardiaceae) is commonly known as kaju in hindi. It is wide spread tree, grown in all tropical countries. Generally it has a crooked trunk.The leaves are oval in shape with rounded tips. The yellowish flowers are small, crowned at the tips of the branches and the petals are sometimes with pink stripes. The fruit is pear-shaped, producing a kidney-shaped nut outside and under the fruit. The cashew nut is well-known as delicacy.1

The bark of the tree is used in decoction for diarrhoea, diabetes, swellings and mouth ulcers.Infusion of the leaves and the bark relieves toothache, sore gums, and dysentry.The juice from the pericarp promotes flow of urine and the alcoholic solution of the pericarp expels worms. The juice of the ripe fruit is good for dysentry.The gum extracted from the bark is an effective insecticide.1






6.1 Need for the Study

The plant Anacardium occidentale Linn. belongs to family Anacardiaceae .This Anacardium occidentale Linn.. are claimed to posses hypoglycaemic activity.

As the chemical investigation of the leaf Anacardium occidentale not dealt in deep, detailed phytochemical and pharmacological investigation of the leaves of the plant can be done. Investigation of the constituent will give us an insight mechanism into the therapeutics action, which can be useful for research and therapeutic activities





6.2 Review of Literature

1. Mota ML et al., (1985)2 A mixture of tannins obtained from the bark of Anacardium occidentale Linn. demonstrated the apparent anti-inflammatory activity in carrageenan and dextran induced rat paw oedemas, cotton pellet granuloma test and adjuvant induced polyarthritis in rats.



2. Marques MR et al., (1990)3 The gum exudates from the Anacardium occidentale Linn.tree has been analyzed for carbohydrate, kjeldhal nitrogen, protein and phenols and for the activities found to antagonize the permeability increasing effects of certain mediators of inflammation in rats and inhibits the migration of leucocytes to the inflammatory site.

3. Patil MB et al., (2003)4 Ethanolic crude extract of Anacardium occidentale leaves and it’s five different crude fractions, petroleum ether, solvent ether, ethyl acetate, butanol and butanone were subjected to preliminary qualitative chemical investigation. The ethanolic extract and all other fractions were screened for anti-inflammatory activity in albino rats (300 mg/kg).Ethanolic extract and Butanone fraction exhibited significant anti-inflammatory activity when compared with control and standard drug diclofenac sodium (100 mg/kg).

4. Torquato DS et al., (2004)5 Crude and purified cashew tree gum extract was evaluated as carbon source for microbial growth. Cashew gum presented a weak activity against saccharomyces cerevisiae and no activity was observed against all other micro organisms tested.

5. Tedong L et al., (2006)6 The study was undertaken to investigate antihyperglycemic and renal protective activity of hexane extract of Anacardium occidentale (Anacrdiaceae) leaves in streptozotocin induced diabetic rats.The results indicate that hexane extract of the leaves of Anacardium occidentale possesses anti-diabetic activity and hexane extract was found to effectively improve the renal function and reduce lesions associated with diabetic state in sreptozotocin-diabetic rats.

6. Kubo I et al., (2006)7 Ancardic acids, 6-pentadec(en)ylsalicylic acids isolated from the cashew Anacardium occidentale Linn. (Anacardiaceae) nut and apple, Anacardic acids were found to possess preventive anti-oxidant activity while salicylic acid did not show this activity..

7. Nair GM et al., (2007)8The study was undertaken to investigate the possible effects of the hexane extract of the bark of Anacardium occidentale and the fractions collected at different stages of the purification process on glucose tolerance in normoglycaemic dogs.The results indicate that stigmast-4-en-3-one isolated from hexane extract of the bark of Anacardium occidentale possesses hypoglycaemic activity

8.Barcelos GR et al., (2007)9 The study was undertaken to assess the mutagenicity and anti-mutagenciity of cashew stem bark methanolic extract (CSBME) on cell cultures of chinese hamster lung fibroblasts (V79).The anti-mutagenic effect observed in this work indicates the safe use of this extract.

9.Konan NA,Bachhi EM et al., (2007)10 The anti-ulcerogenic effect of a hydroethanolic extract of Anacardium occidentale Linn. was studied.The extract inhibited gastric lesions induced by HCl/ethanol in female rats.






6.3 Objectives of the Study

1. Investigation of phytochemical constituents of leaves of Anacardium occidentale Linn.



2. The leaves of the plant Anacardium occidentale Linn. is selected for the study. The leaves will be exhaustively extracted in the soxhlet extractor using ethanol as solvent. This ethanolic extract will be further subjected to successive extraction by employing different solvent like petroleum ether, diethyl ether, ethyl acetate and butanol.

3. The extracted samples from various solvents will be subjected to column chromatography to isolate pure component. The pure sample will be purified, recrystallised and characterised further by spectral data.

4. Screening of Anti-diabetic activity of ethanolic extract of leaf of Anacardium occidentale Linn.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 Operational Definitions

7.2.1 Collection of plant material and extraction

Leaves will be collected from Mangalore, Karnataka. The air dried powdered leaf will be subjected to soxhlet extraction with ethanol (95%). The ethanolic extract thus obtained will be concentrated and evaporated under reduced pressure and controlled temperature. The ethanolic extract thus obtained will be subjected to the study of acute toxic effect and anti-diabetic activity.







      1. Method of extraction, Isolation and Characterization

The powered form of leaves of Anacardium occidentale Linn. will be exhaustively extracted by soxhlet extractor, using ethanol as solvent. The ethanol will be distilled off and the concentrate is evaporated in a flash evaporator in reduced temperature and pressure.This residue 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 gel as adsorbent and eluted first with petroleum ether(60-80°C), petroleum ether(60-80°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 Assessment of acute toxic effect

  • Acute toxicity study of ethanolic leaf extract will be carried out in adult albino rats (150-250 gm. 5-6 weeks old) by “Up and Down method” as per OECD 425 guidelines.

  • Different dose levels (up to 2000 mg/ kg body wt.) of the extract will be administered orally to different groups of rats consisting of three animals in each group. Following the administration of the extract animals will be observerd continuously for 2-3 hours for general behavioural, neurological, autonomic profiles and death for a period of 24 hours.



7.2.4 In-vivo method:




Alloxan induced diabetes:

Alloxan Monohydrate (150 mg/kg, body weight) will be dissolved in normal saline and injected intraperitonealy to rats of Wistar albino or Sprague-Dawley strain weighing 150-200 g, after 18 hr fasting to induce hypoglycaemia. After 1 hr of alloxan administration, the animal will be fed on standard pellets and water ad libetum. The blood glucose level will be monitored after alloxabization collected by tail prick12 method after 72hr of alloxanization, the rats having blood glucose level above 150 mg/dl of blood will be selected for the study and will be divided in to five groups of six rats each.

Group1-Control (2ml distilled water,orally)

Group2-Diabetic control (Alloxan, 150mg/kg i.p)

Group3-Diabetic + Anacardium occidentale leaf extract

Group4-Diabetic + Glibenclamide (600 mg/kg orally)

Group5- Anacardium occidentale leaf extract +distilled water(300 mg/kg orally).

The extract will be carried out in each group of animals for 7 consecutive days. The blood glucose will be monitored at the end of 1,2,3 and 7 days.

The reaction time will be noted and compared with control group and a suitable standard drug group and analysed by one way ANOVA12 followed by Dunnet”s test.





7.3 Does the study require any investigations or interventions to be conducted on patients or other humans or animals?

Yes, Male/Female rats, and mice will be used for pharmacological investigation.






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

The application is forwarded to the ethical clearance committee of the institution and the studies on animals will be undertaken after obtaining the ethical committee clearance.



8

List of References:

1.Kurian JC. Plant that Heal. 7th ed. Pune.Oriental Watchman Publishing House.2004; vol.1:18.

2.Mota ML,Filho JM.Anti inflammatory action of tannins isolated from the bark of Anacardium occidentale Linn.J Ethnopharmacol.1985;13(3):289-300.

3.Marques MR,Filho JX.Enzymatic and inhibitory activities of cashew tree gum exudates.J Ethnopharmacol.1985;13(3):289-300.

4.Patil MB,Jalalpure SS,Pramod HJ,Manvi FV.Anti-inflammatory activity of the leaves of Anacardium occidentale Linn.Indian J Pharmaceutical science.2003;65(1):70-72.



5.Torquato FS,Ferreira ML,Brito ES,Pinto GA,Azevedo EH.Evaluatiom of antimicrobial activity of cashew tree gum.World J Micro Biotech.2004;20(5):505-507.

6.Tedong L, Dimo T, Dzeufiet PD,Asongalem AE,Sokeng DS,Callard P, et al.Antihyperglycemic and renal protective activities of Anacardium occidentale (Anacardiaceae) leaves in Streptozotocin induced diabetic rats.Afr J Trad CAM.2006;3(1):23-35.

7.Nair GM,Alexander RL,Errol Y,Mc Growder DA.Effect of the fractions of the hexane bark extract and stigmast-4-en-3-one isolated from Anacardium occidentale on blood glucose tolerance test in an animal model.International J Pharmacology.2007;3(1):41-47.

8.Kubo I,Masuoka N,joung TH,Tsujimoto K.Anti-oxidant activity of anacardic acid.Food Chemistry.2006;99(3):555-562.

9.Barcelos GR,Shikamburo F,Mori MP,Maciel MA,Colus IM.Evaluation of mutagenicity and antimutagenicity of cashew stem bark methanolic extract invitro.Phytochemistry.1991;30(5):1431-1433.

10.Konan AM,Bachhi EM.Anti-ulcerogenic effect and acute toxicity of a hydroethanolic extract from the cashew (Anacardium occidentale Linn.).J Ethnopharmacol.2007;112(2):237-242.

11.New OECD 425 guidelines,OECD guidelines for testing of animal;2001 Dec;1/26,p:1-26

12.Kulkarni SK.Hand book of experimental pharmacology 1st ed.Delhi Vallabha Prakashan;1987:950,959-966.





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