Introduction

Immune system dysfunction is responsible for various diseases like arthritis, ulcerative colitis, asthma, allergy, parasitic diseases, cancer and infectious diseases (Patwardhan B et al, 1990) ¹. The degree to which the patient becomes abnormally susceptible to infections by this microbial environment depends on the extent of immunosuppression. The suppression of the immune system is characterized by reduction in number and phagocytic function of the neutrophils and macrophages, as well as an impairment of the intracellular bactericidal capacity of these cells. This immunosuppression allows opportunistic pathogens to overwhelm the host to cause secondary infection (Rao CS et al, 1994) ².

This problem can be overcome by boosting the immune system by the use of immunomodulatory drugs (Fulzele SV et al, 2003) ³. ^{Many medicinal plants are known to have Immunomodulatory properties and maintain organic resistance against infection by reestablishing the body's immune system such as Azadirachta indica (Nat VD et al, 1987) 4, Terminalia chebula (Sohni YR et al, 1996) 5, Lawsonia alba (Kulkarni SR et al, 1998) 6 etc. The phytochemical constituents like terpenoids, steroids, proteins and tannins (Biswas K et al, 2002) 7, are considered to exhibit this immunomodulatory property.}

A number of in-vitro and in-vivo test system are available for assessing immunomodulatory activity. Phagocytosis is one such widely available for assessing immunomodulatory activity. Phagocytosis is one such widely used method for screening the immune response (Ponkshe CA et al, 2002) ⁸. Phagocytosis is the primary defence mechanism against any foreign bodies entering the body, which is offered by neutrophils and macrophages. The process of phagocytosis consists of sequential stage as motility, adhesion to microorganisms, ingestion of microorganisms, degranulation and intracellular killing of microorganisms (Daniel PS et al 1994) ⁹.

Gymnema sylvestre R.Br. (Asclepiadaceae) leaves, commonly known as Gudmar is a large woody, much branched climber with pubescent young parts in dry forest up to 600 meter height (Arya Vaidya Shala et al, 1997) ¹⁰. Gymnema sylvestre leaf has been widely used in Ayurvedic traditional medicine; Leaves of the plant as anti- diabetes (11), ^{astringent, bitter, acrid, thermogenic, anti-inflammatory, anodyne, digestive and liver toinc (Kokate C.K. 1999) 12. Tannins are the main chemical constituents present in Gymnema sylvestre and are known to possess anti-inflammatory and immunomodulatory properties (Trease EG, 1993) 13.}

In our present study, we have attempted to evaluate immunomodulatory potency of the extract using different in-vitro methods for locomotion, phagocytic and intracellular killing potency of neutrophils, which are subsequent events involved in the process of phagocytosis by neutrophils.

Materials And Method

Plant material

Preparation of extracts

Preparation of the test sample

Study of immunomodulatory activity

Neutrophil locomotion and chemotaxis test

Phagocytosis of killed Candida albicans

Qualitative Nitroblue Terazolium (NBT) test

Results

Preliminary phytochemical investigation reveals the presence of tannins (Fayez E.K et al, 1998) ¹⁵. Aqueous extract have showed significant activity in the some of the parameters at higher concentrations. However highly significant were obtained in all the parameter in all the concentrations of aqueous extract.

The neutrophil locomotion and chemotaxis showed significant activity at all concentrations; extract have showed significant activity at 20µg/ml concentrations (table1). In case of phagocytosis of killed Candida albicans, extract showed significant activity even at low concentration of 10µg/ml concentrations (table2). In Nitro Tetrazolium qualitative tests, extract showed significant activity. The extract showed predominantly very good at 10µg/ml concentrations (table3)

Figure 1

Table No.1 Neutrophil Locomotion and Chemo tactic Activity

Figure 2

Table No -2 hagocytosis of Killed Candida albicans

Figure 3

Table No.3 Qualitative Nitroblue Tetrazolium Test (NBT)

Discussion

Immunomodulatory agents of plant and animal origin increase the immune responsiveness of the body against pathogens by activating the non-specific immune system. However there is a need to subject such medicinal plants to systemic studies to substantiate the therapeutic claims made with regard to their clinical utility (Fulzele SV et al, 2003) ³.

In the present study, the extract of Gymnema sylvestre leaves significantly increased the phagocytic function of human neutrophils, when compared to control indicating the possible immunostimulating effect.

The engulfment of microorganism by leukocytes called phagocytosis and which is one of the main defence mechanisms of an organism (Daniel P.S, 1987) ¹⁶. The Gymnema sylvestre leaves extract have significantly increased the neutrophil chemo tactic movement as indicate by the increase in number of cells reached the lower surface of filter, there by extracts acts as chemo attractant.

The final step of phagocytosis is the intracellular killing of microorganisms by the neutrophils, which is dependent on metabolic thrust generated through the hexose monophosphate shunt activation, and activation which also necessary for the normal microbicidal activity (Daniel PS et al 1994) ⁹.

The extracts have significantly increased the intracellular reduction of NBT dye to formazan (deep blue compound) by the neutrophils confirming the intracellular killing property and overall metabolic integrity of phagocytosing neutrophils.

Tannins obtained from the leaves are found to possess anti-inflammatory and immunomodulatory properties. Thus it can be also be concluded that immunomodulatory activity may be due to the presence of tannins in the Gymnema sylvestre