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Immunomodulatory and antimicrobial activity of mangifera indica


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IMMUNOMODULATORY AND ANTIMICROBIAL ACTIVITY OF MANGIFERA INDICA L. BARK OIL

M. Pharm Dissertation Protocol Submitted to



Rajiv Gandhi University of Health Sciences, Karnataka

Bangalore– 560041

By

Mr. CHETAN SAVANT B.Pharm

Under the Guidance of

Dr.A.R. KULKARNI M.Pharm, Ph D.



Department of Pharmacology

S.E.T’s COLLEGE OF PHARMACY

S. R. Nagar, Dharwad–580002

2010-2011

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

BANGALORE, KARNATAKA

ANNEXURE –II

PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERATION

1.

NAME OF THE CANDIDATE AND ADDRESS

CHETAN SAVANT

DEPT. OF PHARMACOLOGY,

SET’s COLLEGE OF PHARMACY,

S.R.NAGAR,

DHARWAD-580002


2.

NAME OF THE INSTITUTION


SET’s COLLEGE OF PHARMACY

S. R. NAGAR,

DHARWAD-580002


3.

COURSE OF STUDY AND SUBJECT

MASTER OF PHARMACY IN PHARMACOLOGY



4.

DATE OF ADMISSION TO THE COURSE


JUNE-2010

5.

6.

7.

8

TITLE OF THE TOPIC
IMMUNOMODULATORY AND ANTIMICROBIAL ACTIVITY OF MANGIFERA INDICA L. BARK OIL”

BRIEF RESUME OF THE INTENDED WORK:

6.1 Need for the Study:

Immunomodulation is a procedure in which the immune system of an organism is altered results in an enhancement of immune reactions it is named as an immunostimulative drug which primarily implies stimulation of non specific system, i.e. granulocytes, macrophages, complement certain T-lymphocytes and different effector substances. Immunosuppression implies mainly to reduce resistance against infections, stress and may occur on account of environmental or chemotherapeutic factors.1

Immunological diseases are growing at epidemic proportions that require aggressive and innovative approaches to develop new treatments. These diseases include a broad spectrum of autoimmune diseases such as rheumatoid arthritis, type 1 diabetes mellitus, systemic lupus erythematosus, and multiple sclerosis: solid tumours and hematologic malignancies; infectious diseases; asthma; and various allergic conditions. 2 In addition, infectious diseases are now primarily considered immunological disorders, while neoplastic diseases and organ transplantation and several autoimmune diseases may involve in an immunosuppressive state. The immune system is known to be involved in the etiology as well as the path physiologic mechanism of many diseases.3

The various immunosuppresants available are glucocorticoids, cyclosporine4 and antibodies like antilymphocyte antibodies, antithymocyte antibodies, immunoglobulins like Rh0(D) immunoglobulin, monoclonal antibodies like trastuzumab, rituximab, dacliaumab, basiliximab, absiximab, palivizumab, entanercept.5 The important immunostimulants available are interferon, thalidomide, levamisole6, inosiplex, murabutide7 and pentoxyfylline8 . There are many vaccines which are prepared to increase the immune resistance such as diphtheria antitoxoid, Bacille Calmette Guerin (BCG), rabies vaccines, polio vaccines etc.

An antimicrobial is a substance that kills or inhibits the growth of microorganisms such as bacteria, fungi, or protozoans. Antimicrobial drugs either kill microbes (microbicidal) or prevent the growth of microbes (microbistatic). The history of antimicrobials begins with the observations of Pasteur and Joubert, who discovered that one type of bacteria could prevent the growth of another. Considering that the first truly effective antimicrobial agents date from the mid 1930s (the sulfonamides) and the first antibiotics came into use in the 1940s (the penicillins), it is amazing that we have already grown complacent9.

Many infectious diseases are known to be treated with herbal remedies throughout the history of mankind. In India, herbal medicines have been the basis of treatment and cure for various diseases in traditional methods practiced such as Ayurveda, Unani and Siddha . Most of the medicinal plants contain tannins, gallic acid, quinine flavonoids and alkaloids. Antimicrobials of plant origin are not associated with many side effects and have an enormous therapeutic potential to heal many infectious diseases10.



Mangifera indica Linn belongs to family Anacardiaceae a plant widely used in the traditional medicinal systems of India, has been reported to possess antiviral, antibacterial and anti-inflammatory activities11. The alcoholic extract of stem bark of Mangifera indica has been also reported to possess immunomodulatory activity with immunostimulant properties. Hence, the present study is designed to evaluate the immunomodulatory and antimicrobial activity of Mangifera indica L. bark oil.
6.2 Review of Literature :

The immune system evolved to discriminate self from nonself. Multicellular organisms were faced with the problem of destroying infectious invaders (microbes) or dysregulated self (tumors) while leaving normal cells intact1. The immune system are antigen-specific (they recognize and act against particular antigens), systemic (not confined to the initial infection site, but work throughout the body), and have memory (recognize and mount an even stronger attack to the same antigen the next time). Self/non-self recognition is achieved by having every cell display a marker based on the major histocompatibility complex (MHC). Any cell not displaying this marker is treated as non-self and attacked. The process is so effective that undigested proteins are treated as antigens. Sometimes the process breaks down and the immune system attacks self-cells.12



Mangifera indica, commonly used herb in ayurvedic medicine. It is native tropical Asia and has been cultivated in the Indian subcontinent for over 4000 years and is now found naturalized in most tropical countries. Various parts of Mangifera indica are used as a dentifrice, antiseptic, astringent, diaphoretic, stomachic, vermifuge, tonic, laxative and diuretic and to treat diarrhea, dysentery, anaemia, asthma, bronchitis, cough, hypertension, insomnia, rheumatism, toothache, leucorrhoea, haemorrhage and piles. All parts are used to treat abscesses, broken horn, rabid dog or jackal bite, tumour, snakebite, stings, datura poisoning, heat stroke, miscarriage, anthrax, blisters, wounds in the mouth, tympanitis, colic, diarrhea, glossitis, indigestion, bacillosis, bloody dysentery, liver disorders, excessive urination, tetanus and asthma.

Ripe mango fruit is considered to be invigorating and freshening. The juice is restorative tonic and used in heat stroke. The seeds are used in asthma and as an astringent. Fumes from the burning leaves are inhaled for relief from hiccups and affections of the throat. The bark is astringent, it is used in diphtheria and rheumatism, and it is believed to possess a tonic action on mucus membrane. The gum is used in dressings for cracked feet and for scabies. It is also as considered anti-syphilitic. The kernels are converted into flour after soaking in water and eliminating the astringent principles. Most parts of the tree are used medicinally and the bark also contains tannins, which are used for the purpose of dyeing.11 To our best knowledge no scientific data regarding the immunomodulatory and antimicrobial activity of Mangifera indica L. bark oil is available in literature. Therefore, we undertake this project with following objectives.



6.3 Objective of study:

The objective of the present study is to investigate the immunomodulatory and antimicrobial activity of Mangifera indica L bark oil on different experimental models.



  • To evaluate immunomodulatory activity by,

  • Neutrophil Adhesion test: In this method the percent neutrophil adhesion is calculated.

  • Carbon Clearance test: To evaluate the effect of drug on the reticuloendothelial system.

  • Indirect Haemagglutination test: To confirm the effect of test drug on humoral immune system.

  • To evaluate in vitro antimicrobial activity of Mangifera indica Linn. bark oil by agar well diffusion technique and minimum inhibitory concentration assay.

MATERIAL AND METHODS

7.1 Source of Data:

The source of data are



  • Laboratory experiments on animals

  • Literature survey

  • Books and journals

  • Internet

7.2 Method of collection of Data:

Materials:

Animals:

Albino Wistar male rats weighing 200-250g and albino mice weighing 20-30g will be used. The animals will be maintained under controlled conditions of temperature (23 ± 2C) and 12-h light-dark cycles. The animals are randomized into experimental and control groups and housed separately in sanitized polypropylene cages containing sterile paddy husk as bedding. They will have free access to standard pellets as basal diet and water ad libitum.



Acute toxicity studies :

The guidelines described by OECD will be adopted for the determination of LD50 on Swiss albino mice and 1/10th of LD50 will be taken as dose for the study13.



Methods:

1) Neutrophil adhesion test14

In this method rats will be divided into different groups. And animals are treated with the test drug for 14 days, then blood samples will be analysed for total leukocyte count (TLC) and differential leukocyte count(DLC). Blood will be incubated with 80 mg/ml of nylon fibres for 15 mins at 370C and will be again analyzed for TLC and DLC. The product of TLC and % neutrophil gives Neutrophil Index (NI) of blood sample. Percent Neutrophil adhesion will be calculated as shown below.

Neutrophil adhesion(%)= (NIu-NIt/NIu)×100

Where NIu= Neutrophil index of untreated blood sample and NIt=Neutrophil index of treated blood samples.


2) Carbon clearance test15

In this method mice will be divided into different groups. At the end of treatment with the test drug for 10 days, i.v injection of 0.3 ml per 30 g body weight of dilute Indian ink will be given to albino mice, Blood samples will be collected at 0 and 15 min, 50 µl of blood will be diluted with 4 ml of 0.1% sodium carbonate and absorbance of the diluted blood will be measured spectrophotometrically at 660 nm.

The phagocytic index (K) will be calculated using the following equation:

K=(Loge OD1 – Loge OD2)/15

Where OD1 and OD2 are the optical densities at 0 min and 15 min, respectively.
3) Indirect heamagglutination test16

In this method rats will be divided into different groups. And animals are pretreated with the test drug for 14 days and each rat will be immunized with 0.5×109 sheep red blood cell (SRBCs) intraperitoneally, including control rats. The day of immunization will be reffered 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 treament and the titre value will be determined by titrating serum dilution with SRBC (0.025×109 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 heamagglutination will be expressed as heamagglutination titre.


Antimicrobial activity :

The antimicrobial activity of the extracts was determined by using the agar well diffusion technique17. The agar plates will be seeded with 0.1ml of overnight culture (the test micro organisms i.e., Gram positive:- Streptococcus aureus, Bacillus subtillis; Gram negative:- Escherichia coli, Pseudomonas aeruginosa), allowed to set and wells made by sterile cork borer (0.85cm). Extract (200µl) will be added and the bacterial plates will be incubated (370C) for 24 hours.

Microbial growth was determined by measuring the diameter of zone of inhibition. Chloramphenicol discs were used as control. The experiment was done three times and the mean values are presented18.

Minimum inhibitory concentration (MIC) Assay :

The MIC values will be studied for the bacterial strains, being sensitive to the extract in agar well diffusion technique.19


7.3 DOES THE STUDY REQUIRE ANY INVESTIGATION OR INTERVENTIONS TO BE CONDUCTED ON PATIENTS OR OTHER HUMANS/ANIMALS? IF SO PLEASE DESCRIBE BRIEFLY.

Yes. The investigation involves adult Wistar rats and Albino mice as an experimental animal.



7.4 HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTION IN CASE OF 7.3 ?

The copy of the ethical clearance certificate obtained from Institutional Animal Ethical Committee (IAEC) is attached.



REFERENCES :

  1. Makare N, Bodhankar S, Rangari V. Immunomodulatory activity of alcoholic extract of Mangifera indica L. in mice. J Ethnopharmacology 2001;78:133–7.

  2. Krensky AM, Vincenti F, Bennett WM. Immunosuppressants, tolerogens, and immunostimulants In: Brunton LL, Lazo JS, Parker KL, editors. Goodman and Gilman’s The Pharmacological Basis of Therapeutics. 11th ed. New York (NY): McGraw Hill; 2006. p. 1405-7.

  3. Rinku M, Prasanth VV, Parthasarathy G. Immunomodulatory activity of the methanolic extract of Urena lobata Linn. J Pharmacol 2009;7:1-4.

  4. Katzung BG. Basic and Clinical Pharmacology. 9th ed. New York(NY): McGraw- Hill Companies 2000. p. 940-3.

  5. Katzung BG. Basic and Clinical Pharmacology. 9th ed. New York(NY): McGraw-Hill Companies 2001. p. 945-8.

  6. Roy R, Kalla G, Singh MK. Levamisole in vitiligo of eyelids. Indian J Dermatol Venereal Leprol 1996;62:199-200.

  7. Darcissac CA, Marie-Jose Truong, Joelle Dewulf, George M. The Synthetic Immunomodulator Murabutide Controls Human Immunodeficiency Virus Type 1. Bahr J Viirol 2000;74(17):7794-802.

  8. Gandhi V, Singal A, Sachdeva B, Bhattacharya SN. Treatment of schamberg’s Disease with pentoxifylline. Indian J Dermatol Venereol Leprol 2003;69(1):25-6.

  9. Williams DA, Lemke TL, Foye’s Principles of Medicinal Chemistry. 5th ed. International Student Edition by Lippincott Williams and Wilkins. A Wolters Kluwer Company; 2002. p. 819-20.

  10. Bhagyalakshmi B, Shridhar D, Ponmurugan P. Antimicrobial activity of important Indian medicinal plants against pyogenic infection. Journal of phytology 2009;1(6): 391-6.

  11. Shah KA, Patel MB, Patel RJ, Parmar PK. Mangifera Indica (Mango). Phcog Rev 2010 Nov 25;4:42-8.

  12. Immune system [online]. march 2001 Available from: URL: http://uhaweb.hartford.edu/BUGL/immune.htm.




  1. Ghosh MN. Fundamentals of Experimental Pharmacology. 2nd ed. Calcutta: Scientific Book Agency; 1984. p. 154-7.

  2. Fulzele SV, Satturwar PM, Joshi SB, Dorle AK. Study of immunomodulatory activity of Haridradi Ghrita in rats. Ind J Pharmacol 2003;35:51-4.

  3. Rishi P, Batra N, Sood S, Tiwari, RP. Modullatory effects of Salmonella lap-las on murine macrophages. Ind J Med Microbiol 2002;20 (4):187-3.

  4. Takuo S, Richard BR, Keith RR. Indirect Hemagglutination test that uses ghtardehyde-fixed sheep erythroeytes Sensitised with Extracts, antigens for detection of pasteurella anitbody. J Clin Microblol 1982;15 (5):752-6.

  5. Bauer AW, Kirby WMM, Sherris JC, and Truck M. Antibiotic susceptibility testing by a standard single disc method. Am. J. Clin. Pathol 1996;45:493-6.

  6. Bhagyalakshmi B, Sridhar D, Ponmurugan P. Antimicrobial activity of important Indian medicinal plants against pyogenic infection. J Phytol 2009;1(6):391-6.

  7. Guangrong H, Jiaxin J, Dehui D. Antioxidative and antibacterial activity of methanol extract of Artemisia anomala S. Moore. African J Biotech 2008;7(9):1335-8.




9

SIGNATURE OF THE CANDIDATE



10

REMARK OF THE GUIDE:


The above information and literature has been extensively investigated, verified and was found to be correct. The present study will be carried out under my supervision and guidance.

11

11.1 NAME AND DESIGNATION OF GUIDE

11.2 SIGNATURE

Dr. A.R. KULKARNI. M.Pharm., Ph.D.

PROFESSOR,

DEPT.OF PHARMACOLOGY,

S E T’s COLLEGE OF PHARMACY,

S. R. NAGAR, DHARWAD-580002


12

12.1 HEAD OF THE DEPARTMENT
12.2 SIGNATURE

Dr. A.R. KULKARNI. M.Pharm., Ph.D.

PROFESSOR,

DEPT.OF PHARMACOLOGY,

S E T’s COLLEGE OF PHARMACY,

S. R. NAGAR, DHARWAD-580002


13

13.1 REMARKS OF THE PRINCIPAL

The above mentioned information is correct and I recommend the same for approval.





13.2 SIGNATURE




Dr. V.H. KULKARNI. M.Pharm., Ph.D.

PROFESSOR AND PRINCIPAL

S E T’s COLLEGE OF PHARMACY,

S. R. NAGAR, DHARWAD-580002




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