STUDY ON PHYTOCHEMICAL AND NEUROPHAMACOLOGICAL EFFECTS OF CASSIA AURICULATA LEAVES

M.PHARM DISSERTATION PROTOCOL SUBMITTED TO THE

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCE, BANGLORE

BY MOHAMMAD HAROON SIDDEQUE UNDER THE GUIDANCE OF

MOHAMMED SHAFIUDDIN
M.Pharm (Ph.D) ASST. PROFESSOR

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA BANGALORE

DEPARTMENT OF PHARMACOLOGY ANNEXURE-II

LUQMAN COLLEGE OF PHARMACY ,GULBARGA-85102
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RAIIV GANDHI UNIVERSITY OF HEALTH SCIENCES,KARNATAKA BANGALORE ANNEXURE-II PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION 1. Name of the Candidate and MOHAMMAD HAROON SIDDEQUE Address (In block letters) S/O MOHAMMAD MAROOF SIDDEQUE VILLEGE NAWADA, DISTT: BUDAUN (243601) (UTTAR PRADESH) 2. 3. 4. 5. Name of the Institution Course of Study and Subject Date of Admission to Course Title of the Topic LUQMAN COLLEGE OF PHARMACY, GULBARGA 585 102 M.PHARMACY (PHARMACOLOGY) 05/07/2010 STUDY ON PHYTOCHEMICAL AND NEUROPHARMACOLOGICAL EFFECTS OF CASSIA AURICULATA LEAVES. 6 BRIEF RESUME OF THE INTENDED WORK 6.1 Need for the Study Neuropharmacology is a branch of neuroscience involving the study of drugs that alter the nervous system and its functioning, specifically within the brain. The goal of neuropharmacology in general is to understand the basic functioning of impulses and signals within the brain in order to discover ways in which drugs can be used to treat neurological disorders and drug dependence 1. 21st Centaury is called the centaury of neurological disorders. Neurological related problems are so common today that approximately 18% population suffer from disorders each year 2 Neurological disorders are disorders that can affect the central nervous system, peripheral system and autonomic nervous system. These disorders produces serious health problems like behavioral / cognitive syndrome, sleep disorders, peripheral disorders, epilepsy, neurodegenerative disorders, parkinsonism, neoplasm and many others3 In spite of the recent advances in understanding neurological disorders,
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there are still no effective therapies. Because in modern medicine treatment is empirical symptom oriented and not disease specific. And also the currently available drugs which are in clinical use have characteristic profile and side effects4 Hence management of neurological disorders with agents devoid of any side effect is still a challenge to the medical system. This has led to an increase in the demand of natural products with neurological activity having lesser side effects. Many natural products having neurological effect have been discovered and used by man since prehistoric times4 for instance, The methanolic leaf extract of Cissus cornifolia has got remedy against mental derangement 5. The aqueous extract of Ficus sycomorus stembark possesses a sedative and anticonvulsive properties6. Myristica fragrans seeds possesses anxiogenic, sedative and analgesic activity7 Like above one of the most commonly available plant is Cassia auriculata belongs to family leguminosae, is an erect or biennial shurb found through out India in open areas of forest. The leaves are bitter, astringent, acrid, thermogenic, haematinic,constipating and expectorant. Seeds are bitter, astringent, acrid, cooling, ophthalmic, diuretic,alexeteric and vulnerary. The leaves are used for ulcer, leprosy and skin diseases, the flowers are useful in diabetes and throat troubles and the fruit is useful in vomiting. The leaf of this plant has been used in the traditional system of Indian medicine for treatment of jaundice, liver diseases, leprosy and ulcers. The powdered dried flower bud is used as a substitute for tea in the case of diabetic patient and it is also supposed to improve the complexion in women and also considered to be one of the important dye yielding plants in India.8 The literature survey revealed the presence of alkaloid, flavonoids, saponins, tannins, phenols, cardiac glycosides and resins.9,10 The saponins and flavonoids have been reported to be responsible for neuropharmacological effects. These results are supported by several researchers11 To the best of our knowledge the neuropharmacological effects of Cassia auriculata have not been explored yet. In the light of literature survey which explored the presence of flavonoids and saponins in cassia auriculata and also based on the knowledge of traditional use of this plant as an analgesic and anticonvulsant in folk medicine practice, an attempt could be made to evaluate its neuropharmacological effects in experimental animals using various scientific animal models.

6.2 Review of the literature Literature survey reveals that, the human nervous system is an extremely complex structure, having more than 12 billion nerve cells. Together with the endocrine system it coordinates and regulates the functioning of all body organs12. Central nervous system or psychiatric disorders are the serious health problems. Despite introduction of neurological agents from natural and synthetic sources neurological disorders and its complication continued to be a major problem in the world population. How ever synthetic medicines are available in the treatment of neurological disorders but having large number of side effects13. Indian traditional medicine is one of the richest medicinal systems, among those available around the world. Literature survey indicates that the petroleum and chloroform extracts of Laportea crenulata Roots has got potent CNS Stimulant activity
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Similarly petroleum ether

extract of Peperomia pellucida leaves in the treatment of mental disorder15 Literature review also shows that good piece of work has been done on various pharmacological activities of Cassia auriculata. But there are no reports on its neuropharmacological effects. Some of the published reports on pharmacological and phytochemical aspects of Cassia auriculata are listed as below:
1. 2. 3. 4. 5. 6.

1. Antioxidant activity of cassia auriculata leaves by using in vitro model.16 2. Antidiabetic activity of the aqueous and ethanolic extract of cassia auriculata leaves was assessed in alloxan-induced diabetic rat.17 3. Nephroprotective activity of cassia auriculata.18 4. Tissue lipid peroxidation and Antioxidant activity of cassia auriculata.19 5. Antibacterial and antioxidant activity of cassia auriculata.20

6.3 Objectives of the Study The literature review reveals that neuropharmacological activity of Cassia auriculata leaves has not been reported. In view of this, the present study is aimed to investigate the Phytochemical and Neuropharmocological activity of various extracts of Cassia auriculata leaves in different experimental animal models.

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MATERIALS AND METHODS

7.1 Source of Data The whole work is aimed to generate data from the laboratory that is experiments on animals. Albino rats and mice will be used for this purpose. The scheme of proposed work is as follows. 1. Collection of plant material. 2. Identification and authentication of the plant material 3. Shade drying and powdering of the plant material. 4. Soxhlet extraction of powdered leaves from suitable solvents systems. 5. Preliminary phytochemical investigation of various extracts.
6. 7.

Study of the acute toxicity for determination of LD50 of the extract in mice. To evaluate the Neuropharmocological effect of Cassia auriculata leaf extracts in rodents.

7.2 Methods of Collection of Data 1. Phytochemical studies: The leaves of Cassia auriculata will be dried in shade and processed to a coarse powder. The coarse powder is percolated with successive extraction in increasing polarity as explained by Kokate21. The solvents used are petroleum ether, chloroform, alcohol and distilled water. The crude extracts obtained by removing the solvent will be subjected to preliminary phytochemical screening, some extracts are used for the pharmacological investigations. The detailed methodology is as under. Acute Toxicity test: It is planned to study the acute toxicity of solvent extracts of Cassia auriculata in albino mice of either sex (25-30 gms). Fixed dose methods OECD guidelines 420 is adopted for toxicity study. Exploratory behavior: Albino mice were devided into six groups containing ten animals each. The test was carried out 30 minutes after pretreatment of the various extracts of cassia auriculata leaves and vehicle. Chlorpromazine hydrochloride (1mg/kg i.p.) was used as standard control drug. In this method a white printed wooden board (40cm40cm) with four equidistant holes (1cm diameter2cm depth) is used. The mice are placed at the center of the board and allowed to move freely in the box. A head dip into holes was used to indicate exploratory behavior. The number of dip is observed for ten minutes22.
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Analgesic activity: Hot plate method: The albino mice (n=5) devided into 6 groups were placed individually on a hot plate maintained at 551 C, after 0, 30, 60, 90, 120 and 150 mins of administration of vehicle, various extracts of Cassia auriculata leaves and Tramadol (10mg/kg) I.P. and the time required to lick the paws was measured for each mouse. The percentage of analgesia was calculated. Acetic acid induced writhing: Albino mice devided into groups of six (n=10) received various extracts of Cassia auriculata leaves, Ibuprofen(40mg/kg orally) and vehicle 30mins before intraperitonial injection 0.1ml of 0.6% solution of acetic acid. The number of abdominal constrictions (writhing) is measured for 20mins and percentage analgesia was calculated23. Diazepam induced sleeping time: The sedative- hypnotic effect of the extracts will be assessed using diazepam (85mg/kg i.p.) induced sleeping time in mice. Albino mice are devided into five groups with 10 animals per groups and later treated with different extracts of Cassia auriculata leaves and the control , 30 mins later animals are given Diazepam. Sleeping time is measured as a time interval between loss and regain of righting reflex 24. Maximal electroshock seizure (MES) test: A total 36 mice were divided into 6 groups and alternating current of 50Hz and 150mA is delivered to experimental animals through bicorneal electrodes for 0.2 seconds. A drop of 0.9% saline solution was poured into both eyes prior to placing the electrodes. The different extracts of Cassia auriculata leaves as well as valproic acid are injected intraperitonially 45 mins before induction of MES and control group will receive only vehicle. Duration of tonic convulsion and the percentage of seizure protection and mortality is recorded25.

INCLUSION CRITERIA Normal and healthy animals weighing between 150-180 gms for rats and 2530gms for mice will be included in the study.

EXCLUSION CRITERIA
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The albino rats and mice which do not fall the above mentioned weights are excluded from study. STATISTICAL ANALYSIS All values will be expressed as meanSEM from any group. Results will be subjected to statistical analysis using one- way ANOVA and allowed by post hoc test (Dunnet T test). p<0.05 will be considered as statistical significant. 7.3 Does the study require any investigation or intervention to be conducted on patients or other humans or animals? If so, please describe briefly. Yes, the above study requires investigation on animals like albino rats and mice.

Has ethical clearance been obtained from your Institution in case of 7.3? Yes, the study is cleared from institutional animal ethics committee (IAEC copy enclosed).

8. LIST OF REFERENCES 1. Everitt B. J and Robbins T. W. Neural systems of reinforcement for drug addiction from actions to habits to compulsion. Nature Neuroscience 2005; 8(11):1481-1489.
2. 3. 4.

www.phobias.about.com/od/prevalence/a/phobprevus http://en.wikipedia . org/wiki/The__Neurologist Tripathi KD. Essential medical pharmacology. 2nd Edition. New Delhi. Jaypee brothers publication 1988; 388-389p. Musa A. M, Yaro A. H, Usman H, Magaji M.G and Habu M. Phytochemical and some Neuropharmacological studies on the Methanolic Leaf Extracts of Cissus cornifolia in Mice. International Journal of Pharmacology 2008;4(2): 145-148p. Umar Kyari Sandabe, Patrik Azubuike Oneyili and Gregory Anene Chibuzo. Sedative and anticonvulsant effects of aqueous extract of Ficus sycomorus L. steambark in rats. Veterinarski archive 2003; 73(2):103-110 Ganeshchandra Sonavane, Vikram Sarveiya, Veena Kasture, Sanjay B. Kasture. Behavioral actions of Myristica fragrans seeds. Indian Journal of Pharmacology 2001; 33: 417-424.

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M. Ayyanar and S. Ignacimuthu Entomology Research , Pharmacological Actions of Cassia auriculata L. and Cissus quadrangularis, Jounral of Pharmacology and Toxicology 2008 3 (3): 213-221,

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Botanical Classification of cassia auriculata/ http://en. Wikipedia . org/wiki/The chemical constituents, Shipra Gupta , Suman bala Sharma, Krishana madhava prabhu and Surender kumar bansal, Protective role of cassia auriculata leaf extract on hyperglycemiainduced oxidative stress and safety evaluation, Indian journal of biochemistry and biophysics 200(10):371-377.

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Viswanatha Swamy A.H.M, Thoppeswamy A.H.M, Manula D.V. and Mahendra kumar C.B. Some Neuropharmacological studies on the Methanolic Leaf Extracts of Cissus Quadranfularis in mice. African Journal of Biomedical Research 2006;9:69-75.

12.

Barar FSK. Essentials of Pharmacotherapeutics, publishers.New Delhi.1999.62p.

3rd edition.

S.Chand

13. Tripathi KD.Essential medical pharmacology. 2nd Edition. New Delhi. Jaypee brothers publication 1988; 388-389p.

14. Alam khan, Ashik Mosaddik, Mukhlesur Rahman, Motiur Rahman and Eqramul Haque S.S.Jahan, M.S.Islam, Sohel Hassan. Neuropharmacological effect of Laportea crenulata Root es in mice. Journal of applied sciences Research 2007.3(7):601-606. 15. Khan A, Rahman M.Islam M.S. Neuropharmacological effects of Peperomia pellucida leaves in Mice.DARU 2008 ,16(1):35-40 16 .Pankaj GJ,Dinesh KJ,Pankaj S.Patil,PMC, Dheeraj TB. Antioxidant activity of ethyle acetate extract of cassia auriculata leaves Journal of pharmacy Research 2009,2(9):1412-1413. 17 .Lukmanul FH, Giriga S, Senthil KS, MD Jalaludeen MD. Effect of aqueous and ethanol extract of cassia auriculata L. flower on diabetes using alloxan induced diabetic rats Int J Diabetes and metabolism 2007, 15: 100-106 18 . Senthil kumar,Rajagopal Ponmozhi Manickam, Viswanathan periyasamy, research article on activity of cassia auriculata leaf The journal of Nutritional Biochemistry,2003:14(8):452-458. 19..Annie S, Rajagopal Malini S, Research article on cassia auriculata leaves phytomedicines 2005,12 : 555-560. 20. C.Anushia, P. Sampathkumar and LRramkumar,Antibacterial and antioxident activity in cassia auriculata, Global journal of pharmacology,2009, 3(3):127130. 21. Kokate CK,Pratical pharmacognosy. Pune , Nirali Prakashan 2006,107-111p. . 22. Wakeel OK, PI.Aziba, Ashorobi R.B, Umuloro S, Dderibigbe A.O and Awe E.O. Neuropharmacological activities of Ficus platyphylla stem bark in Mice. African Journal of Biomedical Research 2004, 7 : 75-78 23. Ganeshchandra Sonavane, Vikram Sarveiya, Veena Kasture, Sanjay B. Kasture Behavioral actions of Myristica fragrans seeds. Indian Journal of Pharmacology 2001; 33: 417-424 24. Wakeel OK, PI.Aziba, Ashorobi R.B, Umuloro S, Dderibigbe A.O and Awe E.O. Neuropharmacological activities of Ficus platyphylla stem bark in Mice. African Journal of Biomedical Research 2004, 7 : 75-78. 25. Hossein Hosseizadeh and Vahid Khosravan. Anticonvulsant effects of Aqueous and Ethamolic extracts of Crocus sativus L. stigmas in mice . Arch Irn Med 2002, 5(1):44-47

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9.

Signature of Candidate

MOHAMMAD HAROON SIDDEQUE

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Remarks of the guide Name & Designation of (in block Letters )

The work is highly justifiable and is feasible to work in the institution.

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MR. MOHAMMED SHAFIUDDIN M Pharm (Ph.D) ASST.PROFESSOR 11.1 Guide DEPT. OF PHARMACOLOGY, LUQMAN COLLEGE OF PHARMACY, GULBARGA. 11.2 Signature 11.3 Co-guide 11.4 Signature

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12.1 Remarks of the Chairman & Principal

We will provide all the necessary facilities required for the proposed research work. So, recommended for registration

12.2 Signature

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