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RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE, KARNATAKA, ANNEXURE II PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION 1. Name of the Candidate and Address PANKTI KALARIA a. Permanent Address H-6, Janakpuri app. Zanzarada Road, Junagadh-362 001, Gujarat. b. Postal Address Shree Devi College of Pharmacy, Airport Road, Kenjar Village, Malavoor Panchayat, Mangalore-574 142 Karnataka. 2. Name of the Institute Shree Devi College of pharmacy, Airport Road, Kenjar Village, Malavoor Panchayat, Mangalore-574 142 Karnataka. 3. Course of Study and Subject Master of Pharmacy in Pharmacology 4. Date of Admission to Course July 2010 5. Title of the Topic: “EVALUATION OF REJUVANATIVE SCHOLARIS LINN LEAVES” ACTIVITY OF ALSTONIA 1 BRIEF RESUME OF THE INTENDED WORK: 6. 6.1 Need of study: Immunology is the study of immunity which is the ability of body to resist infections. It provides us with the specific mechanism to resist against a particular pathogen or foreign substance involving production of antibodies or activation of T-cells. Moreover, it can discriminate between self and foreign antigens and if this ability is lost, it leads to autoimmune diseases [1]. Immunomodulators are the diverse range of metabolites which keep the body in homeostasis condition. These stimulate, suppress or modulate the components of immune system [2] . In the recent past, there has been growing interest on exploiting the biological activities of different Ayurvedic medicinal herbs, owing to their natural origin, cost effectiveness and lesser side effects [3]. During the last couple of years the use of herbal products have shown promising responses to modulate immune system compared to modern medicines [4] . Some of the traditional herbs which magnify effect of immune system include Allium sativum, Ocimum sanctum, Aegle marmelos etc[5]. Alstonia scholaris Linn R.Br.commonly known as Devil’s tree belongs to the family Apocynaceae. It grows throughout India, in deciduous and evergreen forests, also in plains. Alstonia scholaris has been mentioned in Ayurveda and is known as saptaparna. Scientific studies have established the multifarious utility of this plant in wide array of pharmacological activities including antimicrobial, antipyretic, antidiarrhoeal, anticancer, anthelmentic and antifertility [6]. It is useful in malarial fevers, abdominal disorder, leprosy, asthma, skin diseases etc. It is also used as an analgesic in case of rheumatic pain and ear ache. The bark extract of Alstonia scholaris is reported to produce immunostimulating effect [7] . However there is no report available on immunomodulatory activity of plant leaf [6] . Therefore present study is designed to examine the rejuvenative activity of Alstonia scholaris Linn R.Br. leaves extract in different experimental models of cellular and humoral immunity in animals. 2 6.2 Review of literature: Alstonia scholaris Linn belongs to family Apocyanaceae commonly known as Devil’s tree. Alstonia scholaris is significantly known for its medicinal value which grows up to 3 meters in height, distributed throughout the sub-Himalayan belt, west-Bengal, Bihar, peninsular India and southwest Asia. The bark, stem, roots and leaves have been used traditionally as folk remedies for the treatment of many diseases [8]. The plant is known to contain alkaloids such as ditamine and echitamine, flavonoids and phenolic acids [6] . The alkaloid fraction of leaves contains three main alkaloids picrinine, vallesamine and scholaricine [9]. The plant is already reported for anthelmintic, digestive, febrifuge, antipyretic, laxative, galactogogue, stomatic, cardiotonic properties. It is useful in fevers, abdominal disorders, dyspepsia, pruritis, leprosy, asthma, bronchitis and debility. Milky juice is applied on wounds, ulcers and rheumatic pain; mixed with oil and dropped into ear it relieves ear ache. Drug is also used in case of snake bite[7]. Potent alpha glycosidase inhibitory activity was found in plant extract of Alstonia scholaris that contributes towards the treatment of diabetes [10]. It has been reported to be used in management of hypertension [11]. Methanolic extract of plant was found to exhibit pronounced antiplasmodial activity. The plant is reported to have antimutagenic effect. The bark extract of Alstonia scholaris has immunostimulating effect [7]. Alstonia scholaris was documented as an effective herb for treatment of chronic respiratory disease and its leaf crude extract is used for relieving tracheitis and cold symptoms [12]. 6.3 Objective of study: The objective of the proposed study is to investigate the effect of the leaf extract of Alstonia scholaris Linn (LEAS) on different experimental models of cellular and humoral immunity in animals. 3 SPECIFIC OBJECTIVES: To carryout extraction of leaf extract of Alstonia scholaris (LEAS) using suitable solvent and extraction procedure. To determine the phytochemical constituents of LEAS. To arrive at the therapeutic dose range after acute toxicity studies following WHO guidelines. To explore the influence of LEAS on the following models of immunity evaluation in animals: 1. Mice lethality test 2. Indirect haemagglutination test 3. Cyclophosphamide induced neutropenia 4. Serum immunoglobulins 7. MATERIALS AND METHODS: 7.1 Source of Data: Data will be obtained from laboratory based studies by using albino rats or albino mice of either sex weighing between 150-200 gms and 25-30 gms respectively, maintained at room temperature, having free access to food (std pellet diet), tap water ad libitum. These studies will be carried out using different models of immunity such as mice lethality test, indirect haemagglutination test, cyclophosphamide induced neutropenia, serum immunoglobulins. 7.2 Method of Collection of Data: Chemicals and reagents will be procured from standard companies. Extraction of Alstonia scholaris leaf extract (LEAS) using suitable solvent and extraction procedure will be carried out and extract will be subjected to different methods of evaluation of immunity as discussed in objectives. 4 EXPERIMENTAL MODELS: 1. Acute toxicity studies: . The dose will be selected by the acute toxicity studies following limit test procedure according to OPPTS guidelines12. Briefly, a test dose of 2 g/kg and 5 g/kg will be given to the mice. Around 1/10th and 1/20th of the maximum safe dose (either 2 g/kg or 5 g/kg) will be used as low and high dose respectively. 2.Mice lethality test1[3]: Albino mice of either sex will be treated with different doses of LEAS for 21 days. On 7th and 17th day, they will be vaccinated with formalin inactivated potash alum adsorbed pasteurella vaccine and at the end of treatment; all animals will be challenged with broth culture of 25LD50 of duck pasturella organism containing 335 CFU. Following animals groups will be used for the study (n=6): Group I: No vaccination, no treatment. Group II: Vaccination on 7th and 17th day with 0.2 ml of formalin inactivated potash alum adsorbed pasteurella vaccine (Vaccine). Group III: Standard (Ocimum sanctum 100 mg/kg, p.o for 21 days) + vaccination on 7th and 17th day. Group IV: Low dose of LEAS orally for 21 days + vaccination on 7th and 17th day. Group V: High dose of LEAS orally for 21 days + vaccination on 7th and 17th day. On 21st day, the animals will be challenged with broth culture of 25 LD50 of duck pasturella organism containing 335 CFU in 0.2ml of nutrient broth approximating to 1017 cells. The animals will be observed for a period of 72 hr and the mortality ratio will be determined using the formula. Mortality ratio: Number of animals dead Total number of animals 5 3. Indirect heamagglutination test[14]: Rats will be pretreated with the drugs for 14 days and each rat will be immunized with 0.5x109 sheep red blood cells (SRBCs) intraperitoneally, including control rats. The day of immunization will be referred to as day 0. The drug treatment will be continued for another 14 more days and blood samples will be collected from each rat at the end of the drug treatment and the titre value will be determined by titrating serum dilutions with SRBC (0.025x109 cells) in microtitre plates. The plates will be incubated at room temperature for 2 hr and examined visually for agglutination. The highest dilution of serum showing haemagglutination will be expressed as haemagglutinating titre. Animal groups will be made as follows (n=6): Group I: Control, no treatment. Group II: Standard (Ocimum sanctum 100 mg/kg, p.o for 28 days). Group III: Low dose of LEAS orally for 28 days Group IV: High dose of LEAS orally for 28 days. 4.Cyclophosphamide induced neutropenia [15]: On 10th day of LEAS/standard administration, single dose of cyclophosphamide 200 mg/kg s.c will be given. The TLC and DLC will be determined before and 3 days after cyclophosphamide administration. Albino mice of either sex will be divided into four groups as given in Indirect heamagglutination test. 5. Serum immunoglobulin(Ig) levels[16]: Albino rats of either sex weighing between 150-200 gm will be treated with LEAS/standard for 21 days. Six hours after the last dose of drug, blood will collected and the serum will be used for estimation of immunoglobulin levels spectrophotometrically. Animals are divided into four groups of six each as discussed in Indirect heamagglutination test. 6 7.3 Does the study require any investigation or interventions to be conducted on patients or the human or animals? If so please describe briefly: YES Study requires investigation on animals. The effects of the drug will be studied on various parameters using rats and mice as experimental animals. 7.4 Has ethical clearance been obtained from your institute Ethical Committee approval letter is enclosed 8. List of References: 1. Sharma HL, Sharma KK. Immunomodulation and immunotherapy, Principles of Pharmacology. Paras Publication, 2007;1:902-920. 2. Smith A, O’Byrne A, Brandt BV, Bianchi I. Introduction to Bioregulatory medicine, Thieme Publication 2009:22. 3. Chopra RN, Nayal SL, Chopra IC. Glossary of Indian Medicinal Plants, CSIR, New Delhi, 2009:185-210. 4. http:// www.banyanbotanicals.com, retrieved on 3/12/2010, 11:20 pm. 5. Dr.Ansari SH. Essential of Pharmacognosy. Birla publication 2004:705708. 6. Kulkarni MP, Juvekar AR. Effect of Alstonia scholaris Linn on stress and cognition in mice. Indian J Exp Biol, 2009;47:47-52. 7. Arulmozhi S, Mazumder PM, Narayan LS, Thakurdesai PA. Invitro antioxidant and free radical scavenging activity of fractions from Alstonia scholaris Linn R. Br. International Journal of Pharm Tech Research 2010;2:18-25. 8. Thomas PS, Kanarijia A, Ghosh D, Duggar R, Katiyar CK. Alstonoside, a secoiridoid glucoside from Alstonia scholaris. Indian J Chem 2008; 47:1298-1302. 9. Shanq JH, Caj XH, Feng T, Luo XD. Pharmacological evaluation of 7 Alstonia scholaris Linn R.Br.: antiinflammatory and analgesic activity.J Ethanopharmacol 2010;129(2):174-181. 10. Anurakkun NJ, Bhandari MR, Kawabata J. Alpha-glucosidase inhibitors from Devil’s tree (Alstonia scholaris). Food chem 2007;103:1319-1323. 11. Bhogayata K, Sharma PP, Patel BR. A clinical evaluation of saptaparna (Alstonia scholaris Linn R.Br.). Ayu 2009;30:318-322. 12. Shanq JH, Caj XH, Feng T, Luo XD. Pharmacological evaluation of Alstonia scholaris Linn R.Br.: antitussive, antiasthmatic and expectorant activities. J Ethnopharmacol 2010,129(3):293-298. 13. Ramanatha KR, Lakshmanarayana R, Gopal T. Potency test of Duck pasturella vaccine in mice. Mysore J Agri Scie 1995;29:155-157. 14. Takuo S, Richard BR, Keith RR. Indirect Hemagglutination test that uses glutardehyde-fixed Sheep Erythrocytes sensitized with extracts, antigens for detection of pasturella antibody. J Clin Microbial 1982;15(5):752756. 15. Heppner GH, Calabresi P. Selective suppression of humoral immunity by antineoplastic drugs. Annu Rev Pharmacol Toxicol 1976;16:367-379. 16. Mullen PA. Zinc sulphate turbidity test as an aid to diagnosis. Veter Ann 1975;15:451-455. 9. SIGNATURE OF THE CANDIDATE: 10. REMARKS OF THE GUIDE: “EVALUATION OF REJUVENATIVE ACTIVITY OF ALSTONIA SCHOLARIS LINN LEAVES EXTRACT IN ANIMALS” to be carried out by Miss Pankti Kalaria of M. Pharm has been discussed and worked out under my directions and supervision as an official guide. The project work envisaged is of great importance in the field of herbal research. The work can be carried out in 8 pharmacology laboratory of Shree Devi College of Pharmacy for which facilities are available. Hence the project is viable and is recommended for clearance and approval. 11. Name and Designation of 11.1 Guide Mr. Talha Jawad Asst. Professor, Dept. of Pharmacology Shree Devi College of Pharmacy, Mangalore-574 142 11.2 Signature 11.3 Head of the Department Dr. Jagadish V. Kamath Dept. of Pharmacology Shree Devi College of Pharmacy, Mangalore-574 142 11.4 Signature 12. 12.1 Remarks of the Principal The Programme and the Research work that is undertaking by Miss Pankti Kalaria have potential implication in the field of Pharmacology. The work can be carried in the Research Laboratories of Pharmacology Department at Shree Devi college of Pharmacy. Hence the Project is recommended and requested for clearance and approval. 12.2 Signature 9 10