Anti-Allergic Activity of Matricuria Recutit Linn

ANTI-ALLERGIC ACTIVITY OF BUCHNANIA LANZAN SPRENG. IN MAST CELL MEDIATED ALLERGIC MODELS

Protocol of Dissertation Submitted

Mr. VIJAYKUMAR SHEVALE

Rajiv Gandhi University of Health Sciences Bangalore, Karnataka.

Under the guidance of Dr. I. S. MUCHCHANDI Principal and professor

Department of Pharmacology, HANAGAL SHRI KUMARESHWAR COLLEGE OF PHARMACY, BAGALKOT- 587101, KARNATAKA. (2012-13)

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA-BANGALORE

ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR

DISSERTATION

1. Name of the Candidate and Address VIJAYKUMAR SHEVALE DEPARTMENT OF PHARMACOLOGY, H.S.K. COLLEGE OF PHARMACY, B.V.V.S CAMPUS, BAGALKOT-587101, KARNATAKA.

2. Name of the Institution H.S.K. COLLEGE OF PHARMACY, B.V.V.S CAMPUS, BAGALKOT-587101, KARNATAKA.

3. Course of Study and Subject MASTER OF PHARMACY IN PHARMACOLOGY

4. Date of Admission to Course 16 Augast 2012

5. Title of topic: Anti-allergic activity of Buchnania lanzan Spreng. in mast cell mediated allergic models.

2 6. Brief resume of the intended work

6.1 Need for the study:

Immediate or type I, hypersensitivity is a rapidly developing immunologic reaction occurring

within minutes after the combination of an antigen with antibody to mast cell in individuals

previously sensitized to the antigen. These reactions are called allergic reactions and antigens

eliciting them are called as allergens. Allergic reaction may occur as two types: Systemic reaction

and local reaction usually follows injection of antigen to which host has become sensitized often

with in a minutes, a state of shock is produced which is sometimes fatal. The nature of local

reaction varies depending on portal entry of allergen and may take the form of localized cutaneous

swelling (skin allergy), nasal and conjunctival discharge (allergic rhinitis & conjunctivitis), hay

fever, bronchial asthma in which mast cells are the principal target cells for the immediate

hypersensitivity1. As part of allergic response to an antigen, antibodies are generated and bind to

the surface of mast cell via high affinity Fc receptors that are specific for IgE. Mast cells release

histamine during these reactions. Mast cell mediators of inflammatory processes are histamine,

2 proteases, LTC4, LTB4, PGD2, platelet activating factor . Mast cell degranulation is believed to be

involved in the pathophysiology of CHF, asthma, allergic hypersensitivity reaction and

inflammation. The modern medicines available as anti-allergic drugs include disodium

cromoglycate but these drugs are associated with unwanted effects including local irritation,

transient bronchospasm. These drugs are also restricted to use in pregnancy and prolonged use3.

However, there are several plant-derived preparations in the ancient text of Ayurveda and

Siddha for the treatment of allergic conditions including asthma. With continuation of new drug

discovery, plants or their preparation scientifically to prove for their clinical applicability is

required. In this view the present study has been selected for evaluation of Buchnania lanzan for its

anti-allergic property in compound 48/80 induced allergic models.

6.2 Review of literature:

Plant profile-

Title of plant : Buchnania lanzan Spreng. 3 Family : Anacardiaceae

Synonyms : Char, chironji, Almondette Tree, Murkali, Nurkale.

Habitat : Hill Plant, Plain Land commonly found throughout the greater part of India (400-500 rpm) discarding the supernatant and taking the pellet of mast cells into the medium.

Mast cells from the control group and treated group were incubated with compound 48/80

(1µg/ml) at 37oc for 10 min. After incubation, mast cells were stained with toluidine blue (0.1%) and percent of protection against degranulation was counted under high-power microscope

(45x)15.

7.3.3 Blood histamine Determination:

The histamine in blood was estimated by o-phthalaldehyde method by using fluorimetrically16.

2 ml of aqueous phase of aliquot was taken, mixed 0.1 ml of OPT reagent and then added 0.2 ml of 3N HCL. The fluorescence of acidified solution was stable for atleast 90 min. The fluorescence intensity was proportional to histamine concentration over the range 0.005 to

0.5µg/ml.

7.3.4 Antianaphylactic activity:

The compound 48/80 induced anaphylactic reaction is examined. Mice were given an injection

(ip) of 8mg/kg of mast cell degranulation compound 48/80. Buchnania lanzan extract at 100, 200 and 300 mg/kg and disodium cromoglycate were given orally 1 hr prior to compound 48/80.

Mortality was monitored for 1 hr after induction of anaphylactic reaction in animals on those bases to check the percent of protection17.

7.3.5 Measurement of nitric oxide released by mast cell:

The nitric oxide will be assayed by measuring nitrite the primary, stable and non- volatile breakdown product of NO. The Griess reagent uses N-1-napthylethylene diamine dihydrochloride (NED) and sulphanilamide under acidic condition in the presence of nitrite to yield azo compound that can be measured 546 nm by Spectrophotometric method18.

7.3.6 Statistical Evaluation: 4 The collected data will be evaluated by ANOVA method followed by Dunnett’s multiple comparison and Chi-square test for anaphylactic reaction carried for conclusion of result.

7.4 Does the study require any investigations or interventions to be calculated on patients or other Humans/animals? If so please describe briefly

Yes, For this study rats and mice will be used before withdrawing the peritoneal fluid from the rats will be sacrificed by deep anesthesia using anesthetic ether.

7.5 Has ethical clearance been obtained from your institution in case of 7.2 and?

Yes, The study is cleared from Institutional Animal Ethics Committee (IAEC). The copy is enclosed with this protocol.

5 8. References:

1. Kay A.B. Allergy and allergic diseases. N Eng J Med 2001;344:30,109.

2. Shirwaikar A., Somashekar A.P. Anti-inflammatory activity and free radical scavenging

studies of Aristolochia bracteata Lam. Pharmacol 2003;65(1):67-69.

3. Bradley J.U., Lawrence M.L. Drugs used in the treatment of asthma, In: Goodman and

Gilman’s. The Pharmacological basis of therapeutics, Hardman JG., Limberd LE. 11th Ed. Mc

Graw Hill Publication, New Delhi 2001;748-749.

4. Arya R., Babu.V., Ilyas.M and Nasim.K.T. Myricetin-3-Rhamnoside-3-galactoside from

Buchanania lanzan Phytochemistry. MAPA 1992;31(7):2569-2570.

5. Anupam Sengupta and Sajal K. Roychoudhury. Department of pharmacy, Jadavpur University,

Culcutta-700032, India. J Sci Food Agri 1977;28(5):463-468.

6. Rajesh K., Mishra. Shambhu., P. Patel., Anupam Srivastava., Rajiv K. Vashistha., Ajay Singh

and Ashok K. piskar. Ethnomedicinally important plants of Pachmarhi region, Madhya

Pradesh, India. Nat Sci 2012;10(4):24.

7. Kirtikar KR and Basu BD. Indian Medicinal Plants. 2nd Edition. Dehradun: International Book

Distributors, Book Sellers and Publishers 1999;2:1020-1023.

8. S. K. Mali., R. Chaudhury., N. S. Panwar., O. P. Dhariwal., Ravish Choudhary and Susheel

Kumar Genetic resources of Chironji (Buchanania lanzan Spreng.) Genet Resour Crop Evol

2012;59:615-623.

9. Thatoi HN., Panda SK., Rath SK and Dutta SK. Antimicrobial activity of Ethnomedicinaluses

of some Medicinal plants from Similipal Biosphere Reserve, Orissa. Asi Jou plant Sci

2008;7(3):260-267.

10. Anju puri., Sahai R., Kiran L Singh., Saxena RP., Tandon JS and Saxena KC.

Immunostimulant activity of dry fruits and plant materials used in Indian traditional medical

system for mothers after child birth and invalids. J Ethnopharmacol 2000;71(200):89-92.

11. Archana Kumari and Poonam Kakkar. Screening of Antioxidant potential of selected Barks of 6 Indian Medicinal plants by Multiple invitro Assays. Biomed Environment Sci 2008;21:24-29.

12. Xue Y and Chen H. Study on the anti-carcinogenic effect of three compounds of Buchanania

lanzan L, Xei Shen Yan Jiu 2002;31(4):247-248,251. 9. SIGNATURE OF CANDIDATE VIJAYKUMAR SHEVALE

Buchnania lanzan Sperng. has been 10. REMARKS OF TH GUIDE mentioned as very good medicinal plant in various literatures, The present study was evaluated for its anti-allergic activity scientifically.

11. NAME AND DESIGNATION OF THE Dr. I. S. MUCHCHANDI GUIDE Principal and professor

12. SIGNATURE

13. CO-GUIDE

14. SIGNATURE

15. HEAD OF THE DEPARTMENT Dr. I. S. MUCHCHANDI H.O.D., Department of Pharmacology H.S.K. College of Pharmacy, B.V.V.S. Campus, Bagalkot-587101.

16. SIGNATURE

17. REMARKS OF THE The above mentioned information is correct PRINCIPAL and I recommended the same for approval.

Dr. I.S.MUCHCHANDI 18. NAME OF THE PRINCIPAL H.O.D., Department of Pharmacology H.S.K. College of Pharmacy, B.V.V.S. Campus, Bagalkot-587101.

19. SIGNATURE

7 OFFICE OF THE INSTITUTIONAL ANIMAL ETHICS COMMITTEE (IAEC) HANAGAL SHRI KUMARESHWAR COLLEGE OF PHARMACY, BAGALKOT-587101, KARNATAKA

REG NO.821/01/a/CPCSEA, Dated: 6th AUG 2004 UNDER THE RULES 5(a) OF THE “BREEDING OF AND EXPERIMENTS ON ANIMALS (Control and Supervision) RULES 1998”

Ref: HSKCP/IAEC, Clear / 2009-10/1-8

CERTIFICATE

This is to certify that Mr.VIJAYKUMAR SHEVALE. A student of first

M.Pharm is permitted to carry out experiments on animals for the dissertation / thesis work entitled as “Anti-allergic activity of Buchnania lanzan Spreng. In mast cell mediated allergic models” per details mentioned and after observing the usual formalities laid down by IAEC as per provision made by CPCSEA.

Animal house in charge CHAIRMAN

FORM B See rule [6 (a) and 8(a)] PART A

(1) Name and address of the Establishment: H.S.K. COLLEGE OF PHARMACY BAGALKOT, KARNATAKA. INDIA.

(2) Date and Registration Number of the 821/01/a CPCSEA. Establishment:

(3) Name, address and Registration NO. of the OFFICE OF CPCSEA, breeder from whom acquired and the date of MINISTRY OF ENVIROMENT AND FOREST, acquisition: 3rd SEAWARD ROAD, VALMIKINAGAR, THRIRUVANMIYUR, CHENNAI-600041.

ANIMAL HOUSE (4) Place where the animals are presently kept: H.S.K. COLLEGE OF PHARMACY BAGALKOT, KARNATAKA.

DEPARTMENT OF PHARMACOLOGY (5) Place where the experiment is to be H.S.K. COLLEGE OF PHARMACY performed: BAGALKOT, KARNATAKA.

(6) The date on which the experiment is to commence and the duration of the 15 MAY 2013, (6 months). experiment:

The protocol form for the research proposal - PART B in the case of experiments using other than non-human primate animals for new projects, PART C for use of non-human primates for new projects and PART D for use of non-human primate for extension of ongoing projects – should be duly filled, singed and annexed with this form.

Dated: Signature

Place: (Name and Designation)

PART – B

Protocol form for Research Proposal to be submitted to the Committee on use of small animals / animals other than non human primate in Biomedical Research for NEW PROJECTS

1. Project Title : Anti allergic activity of Buchnania lanzan Spreng. In mast cell mediated allergic models.

2. Investigation (s) : Dr. I.S. Muchchandi Designation Principal and professor

3. Department (s) : DEPARTMENT OF PHARMACOLOGY H.S.K. COLLAGE OF PHARMACY BAGALKOT, KARNATAKA.

4. (a) Funding Source (s): if any ----

(b) Are sufficient funds available for purchase and maintenance of the animals yes

(C) Duration of present project :

(1) Number of months : 6 months

(2) Date of start of the Project : (Experiment) 15th May 2013

(3) Date of termination of the project : 15th Dec 2013

5. Date by which approval is needed in case the project is to be funded by outside ---- agency (If less than six weeks from the date of admission, please justify below).

10 6. Summary of project briefly summarize in laymen’s term the background, the objective and the experiment approach.

(a) Background Enclosed

(b) Objectives Enclosed

(c) Experimental procedure: Enclosed

7. (a) Name of species Swiss Albino (Mice)

Spague-Dawley ( rats) Age Sex Weight Rat Mice Rat Mice

4-6 weeks 2-5 weeks Male 200-250 g 20-25 g

(b) Rationale for selection

Approximate number of animals required during the 80 first 12 months.

Justification of number (define treatment group and Fifteen groups, Each group containing six animals. number per group)

Number of animals housed per weeks 20

8. List all invasive Non Surgical Animal Procedures and Invasive surgical animal Potentially Stressful Noninvasive procedures to be used procedures. (Example IM injection, foot pad injection, venapunctures).

Procedure and Approximate Frequency: Enclosed

11 Anesthetic and/or Analgesic and Dosage:

Not applicable.

Test substance injected and/or applied:

Test substance will administer post Orally.

9. Does the protocol prohibit the use of anesthetic and analgesic for the conduct of painful procedures?

10. With surgical procedure/Experimental procedure is performed?

(a) Will the animal be sacrificed after surgery?

(b) Give anticipated post operative survival time:

---- 11. Will hazardous agent such as radioisotopes, carcinogens, radiation exposure, microbial and parasitic agent be administered to animals?

INVESTIGATOR SIGNATURE

DATE______