ANTI-ALLERGIC ACTIVITY OF MATRICURIA RECUTITALINN. IN MAST CELL MEDIATED ALLERGIC MODELS
Protocol of Dissertation Submitted
By
Mr. KALLAPPA.S.HALAGALI
To
RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES
BANGALORE, KARNATAKA.
Under the guidance of
Dr. I S MUCHANDI
Principal and Professor
Department of Pharmacology,
HANAGALSHRIKUMARESHWARCOLLEGE OF PHARMACY,
BAGALKOT- 587101, KARNATAKA
(2009-10)
RAJIVGANDHIUNIVERSITY OF HEALTH SCIENCES
KARNATAKA-BANGALOR
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR
1. /Name of the Candidate and Address
/ KALLAPPA. S. HALAGALIDEPARTMENT 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 / 15-06-2009
5. /
Title of topic:
Anti-allergic activity of Matricuria recutita Linn. in mast cell mediated allergic models
DISSERTATION
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 minutesafter the combination of an antigen with antibody to mast cell in individuals previously sensitized to the antigen. These reactions are called allergic reactionsandantigens eliciting them are called as allergens. Allergic reaction mayoccur 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, proteases,LTC4,LTB4,PGD2, platelet activating factor2. 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 includeSodium cromoglycatebut 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 Siddhafor the treatment of allergic conditions including asthma. With continuation of new drug discovery, plants or their preparation scientifically to prove for their clinicalapplicability
is required. In this view the present study has been selected for evaluation of Matricariarecutita for its anti-allergicproperty in compound 48/80 induced allergic models.
6.2 Review of literature:
Plant profile:
Title of plant:Matricaria recutita Linn.
Family :Asteraceae (Compositae)
Synonyms :Matricaria, German chamomile, Sweet false chamomile’s
Habitat :German chamomile is annual herb originally from Europe which has escapedto the wildand now naturalized in almost every continent. It can now be found growing along fence rows, roadsidesand in sunny open field from Southern Canada to Northern U.S. west to Minnesota.Chemical constituent :
The plant containsTerpenoids like α-bisabolol, α-bisabolol oxide A and B, Chamazulene, Sesquiterpenes. Flavonoids like apigenin, luteolin, quercetin. Coumarins,umbelliferone andothers areanthemic acid, choline, tannin and polysaccharides.
Medical uses:
Matricaria recutita Linn.(Chamomile) has been used medicinally for thousands of years and is widely used in Europe. It is a popular treatment for numerous ailments including sleep disorders, anxiety, digestion, intestinal conditions, skin infections, inflammation (including eczema), wound healing, infantile colic, teething pains and diaper rash. In the United States, Chamomile is best known as an ingredient in herbal tea preparations advertised for mild sedative effects. Chamomile is used externally for wounds, ulcers, eczema, gout, neuralgia, rheumatic pain, hemorrhoidsand leg ulcers4.
7. /Pharmacological actions:
Matricaria recutitais potentially used as an Antioxidant5, diabetic complication6, cardiac effects7, anti microbial activity8, anti proliferative and apoptic effects9.Cardiovascular effects as increase atrial rate, relaxed thoracic aorta, hemodynamic effectsand anti-ulcer activity. Chamomile’s main active constituents are apigenin and bisabolol. Apigenin having cardio protective activity10, 11, 12, 13.6.3 Objectives of the study :
The traditionally well known Matricaria recutita (M.R) will be extracted by using nonpolar and polar solvents and extract will be used for the present study with fallowing objectives.
1)Effect of Matricaria recutita extract on mast cells stabilization.
2)Effect of Matricaria recutitaextract on compound 48/80 induced anaphylactic shock.
3) Effect of Matricaria recutitaextract on compound 48/80 induced histamine release.
4) Effect of Matricaria recutitaextract on compound 48/80 induced nitric oxide level.
Materials and methods :
7.1 Source of data :
The data will be based on animal experiment as per the parameter studied for the model. Selection of dose will be based on earlier acute toxicity studyby dividing the animal in following groups.
Group I: Effect ofmethanolic extract of M.R ( 100, 200 and 300 mg/kg) on compound48/80
induced mast cell activation (n=6).
Group II:Effect ofmethanolic extract of M.R (100, 200 and 300 mg/kg)on compound48/80
induced anaphylactic shock (n=6).
Group III:Effect ofmethanolic extract of M.R ( 100, 200 and 300 mg/kg) on compound 48/80
induced blood histamine release (n=6).
Group IV:Effect ofmethanolic extract of M.R ( 100, 200 and 300 mg/kg) on compound48/80
induced nitric oxide (n=6).
7.2 Materials :
Plant : Matricaria recutita Linn.
Chemicals : Compound 48/80, Griess reagent, methanol.
Animals : Male Sprague Dawley rats, Swiss Albino Mice.
Instruments : Research microscope, Centrifuge, Laminar flow system, Fluorimeter,
UV-spectrophotometer.
7.3 Method:
7.3.1Preparation of plant extract:
The plant authenticated and collected in ideal conditions will be air dried under the shade and powdered to a fine texture of uniform size by passing through the sieve no.44. The powder collected will be extracted with polar and non-polar solvent and the extract obtained will be used for present study.
7.3.2 Mast cell stabilizing activity.The extracts of Matricaria recutita and disodium chromoglycate (10 mg/kg; ip) were given orally to rats daily 5 days prior to the collection of mast cells. The animal is anesthetized by diethyl ether and injected normal saline (10 ml) into peritoneal cavity.After gentle massage the peritoneal fluid was collected and transferred into siliconised test tube containing RPMI-1640 (pH 7.2-7.4). Mast cells are washed three times by centrifugation at low speed (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)14.
7.3.3 Blood histamine Determination.
The histamine in blood was estimated by o-phthalaldehyde method by usingfluorimetrically15. 2 ml of aqueous phase of aliquotwas taken, mixed 0.1 ml of OPT reagent and then added 0.2 ml of 3N HCL. The fluorescence of acidified solution was stable foratleast 90min. The fluorescence intensity was proportional to histamine concentration over the range 0.005 to 0.5µg/ml.
7.3.4 Antianaphylactic activity.
Compound 48/80 induced anaphylactic reaction is examined. Mice were given an injection (ip) of 8mg/kg of mast cell degranulatior compound 48/80. Matricariarecutita extract at 100, 200 and 300 mg/kg 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 protection16.
7.3.5Measurement of nitric oxide released by mast cell.
Nitric oxide will be assayed by measuring nitrite the primary, stable and non- volatile breakdownproductof NO. The Griess reagent uses N-1-napthylethylene diamine dihydrochloride (NED) andsulphanilamideunder acidic condition in the presence of nitrite to yield azo compound that can bemeasured 546 nm by Spectrophotometric method17.
7.3.6 Statistical Evaluation.
The collected data will be evaluated by ANOVAmethod followed by Dunnett’s multiple comparison and Chi-square test for anaphylactic reaction carried for conclusion of result.
7.4 Does the study requires any investigations or interventions to be calculated on patients or
otherHumans/animals? If so please describe briefly
Yes,For this study rats and mice will be used before withdrawing the peritoneal fluid from the ratswill 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.
8. / References:
- Kay A.B. Allergy and allergic diseases. N Eng J Med 2001;344:30,109.
- Shirwaikar A., Somashekar A.P.Anti-inflammatory activity and free radical scavenging studies ofAristolochia bracteata Lam. Pharmacol 2003;65(1):67-69.
- 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.
- NewelC.A., Anderson L.A., Phillipson J.D. Herbal medicines. A guide for health care Profession- als. London, Pharmaceutical press 1996; 9: 296.
6Kato A., Minoshima Y., Yamamoto J., Adachi I., Watson A.A., Nash R.J. Protectiveeffects of dietary Chamomile tea on diabetic complication. J Agric Food Chem 2008; 56(17):8206-11.
7Lawrence., Ramana Reddy C.V., Robert FG. CardialEffects of Chamomile tea. J Clin Pharmacol 1973;13:475-479.
8Noqueira J.C., Diniz Mde F., Lima E.O. Anti microbial activity of plants in acute otiti externa. Braz J Otorhinolarygol 2008; 74(1):118-24
9Srivastava J.K., Gupta S. Antiprliferative and apoptic effects of chamomile extract invarious human cancer cells. J Agric Food Chem 2007; 55 (23):9470-8.
10Lorenzo P.S., Rubio M.C., Medina J.H., Involvement of Monoamine oxidase and noradrenalin uptake in the positive chronotropic effects of apigenin in rat atria. Euro J Pharmacol 1996; 312:203-7.
11.Ko F.N., Huang T.F., Teng C.M. Vasodilatory action mechanisms of apigenin isolatedfrom Apium graveollens in rat thoracic aorta. Biochem Biophy Acta 1991;1115:69-74.
12.Gould L., Reddy C.V.R., Gomprecht R.F. Cardiac effects of chamomile tea. J ClinPharmacol New Drugs 1973;13:475-479.
- Tamasdon S., Cristea E., Mihele D. Action upon gastric secretion of Rbiniae Flores,chamomillaeflores and strobuli lupuli extracts. Farmacia 1981;29:71-75.
- Singh R., Nath A., Gupta P.P., Shukla M.,Khare S.K., Kundu B. Antiallergic/antiasthemetic activity of oligopeptide related to IgE. Pharmacol Res 1998;37(5):350.
- Shore P.A., Burkhalter a., Cohn V.H Jr. A method for the fluorimetric assay of histamine in tissues.J Pharmacol Exp Ther 1959;127;182-189.
- Jin-Mu Y., Hong S.H., Lee H.J., Woon J.H., Kim J.M., Jeong J.M. lxeris dentate green sap inhibits both compound 48/80 induced anaphylaxis-like response an IgE mediated anaphylactic response in murine model.Biol Phama Bulln 2001;25(1):5.
- Chitme H.R., Patel N.R.Anti-arthritic activity of Aristolochia bractiata. Pharmcol Res 2006. (In-press).
9. / SIGNATURE OF CANDIDATE / KALLAPPA.S.HALAGALI
10. / REMARKS OF THE GUIDE / Matricaria recutita Linnhas been mentioned as very good medicinal plant in various literatures, The present study was evaluated for its antallergic activity scientifically.
11. / NAME AND DESIGNATION OF THE GUIDE / Dr. I.S.MUCHANDI
Principal and Professor
12. / SIGNATURE
13. / CO-GUIDE
14. / SIGNATURE
15. / HEAD OF THE DEPARTMENT / Dr. I.S.MUCHANDI
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
PRINCIPAL / The above mentioned information is correct and I recommended the same for approval.
18. / NAME OF THE PRINCIPAL / Dr. I.S.MUCHANDI
H.O.D. , Department of Pharmacology
H.S.K.College of Pharmacy,
B.V.V.S. Campus, Bagalkot-587101.
19. / SIGNATURE
OFFICE OF THE INSTITUTIONAL ANIMAL ETHICS COMMITTEE (IAEC)
HANAGALSHRIKUMARESHWARCOLLEGE 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. KALLAPPA S HALAGALI. a student of first M.Pharm is
permitted to carry out experiments on animals for the dissertation / thesis work entitled
as “Antiallergic activity of Matricaria recutita Linn. 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 PHARMACYBAGALKOT, KARNATAKA.INDIA
(2) / Date and Registration Number of the Establishment: / 821/01/a CPCSEA
(3) / Name, address and Registration NO. of the
breeder from whom acquired and the date of
acquisition: / OFFICE OF CPCSEA,
MINISTRY OF ENVIROMENT AND FOREST, 3rd SEAWARD ROAD, VALMIKINAGAR,THRIRUVANMIYUR,
CHENNAI-600041.
(4) / Place where the animals are presently kept: / ANIMAL HOUSE
H.S.K.COLLEGE OF PHARMACY
BAGALKOT, KARNATAKA.
(5) / Place where the experiment is to be performed: / DEPARTMENT OF PHARMACOLOGY
H.S.K.COLLEGE OF PHARMACY
BAGALKOT, KARNATAKA.
(6) / The date on which the experiment is to commence and the duration of the experiment: / 15 MAY 2010
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.
Signature
Dated: (Name and Designation)
Place:
PART – BProtocol 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 Matricaria recutita Linn.in mast cell mediated allergic models.
2. / Investigation (s) :
Designation / Dr. I.S. Muchandi
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 : / 8 months
(2)Date of start of the Project :
(Experiment) / 15th May 2010
(3)Date of termination of the project : / 15th Dec 2010
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). / ----
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
Rat Mice / Sex / Weight
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
first 12 months. / 80
Justification of number (define treatment group and
number per group) / Fifteen groups, Each group containing six animals.
Number of animals housed per weeks / 20
8. / List all invasive Non Surgical Animal Procedures and Potentially Stressful Noninvasive procedures to be used (Example IM injection, foot pad injection, venapunctures). / Invasive surgical animal procedures.
Procedure and Approximate Frequency: / Enclosed
9.
10.
11. / Anesthetic and/or Analgesic and Dosage:
Not applicable.
Test substance injected and/or applied:
Test substance will administer post Orally.
Does the protocol prohibit the use of anesthetic and analgesic for the conduct of painful procedures?
No.
With surgical procedure/Experimental procedure be performed?
No.
(a) Will the animal be sacrificed after surgery?
No.
(b) Give anticipated post operative survival time:
----
Will hazardous agent such as radioisotopes, carcinogens, radiation exposure, microbial and parasitic agent be administered to animals?
No
INVESTIGATOR SIGNATURE
DATE______
1