The mushrooms are largely used as a foodstuff from historical details as a traditional food and it have greater importance in the diet of mankind most recently. The cultivation and production of edible mushrooms are on the increase, particularly in Europe, America and Asia due to its increased nutritional values. Their increased nutritional importance is almost equals to that of the milk [1]. The cultivated and wild mushrooms contain reasonable amounts of proteins, carbohydrates, minerals, fibers and vitamins [2-5]. The available literature review indicates that mushrooms have phytochemicals with the compounds of strong antioxidants [6]. The phenolic compounds, alkaloids, saponins, flavonoids, tannins, sterols, triterpenes, coumarins and cyanogenic glycosides have been detected and reported with wild mushrooms of Sudan’s and Nigerian regions of African continent [7]. Immune modulation is a bio-chemical phenomenon which is explained “as any change in the immune response and may involve induction, expression, amplification of any part or phase in the immune response of the organism”. The effect of modulation may be very specific and limited to a given antigen of an agent or a non-specific subtract, with a great effect on immune responsiveness. The stimulation of the immune response is preferred with certain type of people namely immune compromised patient. Whereas, it suppresses the immune responsiveness of transplant recipient or patient who are auto allergic or prone with inflammatory diseases [8]. Several in vivo and in vitro models are available for screening immuno modulation activity [9] in which Phagocytosis is one of the extensively adopted methods for screening the immune response.

Collection of sample

The mushrooms have been collected from the neighboring places of Coimbatore City. This shade dried mushrooms are used for this present investigation with standardard procedures and protocols.

Extract preparation by cold maturation method: The accurately weight 150 g of power sample has been collected and it is mixed with 100 ml of 50% ethanol in a round bottom flask and kept for incubation for 24 hrs. Then this mixture has been filtered and the filtered sample is kept in the evaporation stand at 70°C. The dried sample is collected and allowed for the phytochemical screening and Immunomodulatory activities.

Animals used: Swiss albino mice of either sex of Indian region, weighing 18-25 g each, have been selected for this study with proper permissions. They are housed under standard conditions at the temperature of 25 ± 10ºC and at a relative humidity of 60 ± 10%, for 12/12 h light/dark cycle, and they are fed with standard pellets and water. These Animal experiments were performed in accordance with the CPCSEA norms and are obtained from Nandha College of Pharmacy, Animals House.

The qualitative phytochemical analysis

The pytochemical analysis carried out with the standard procedure, adopted by Kokate, in which, 1 g the sample is transferred into a clean screw cap bottle of 10 ml capacity and a solvent ethanol, 10 ml is added into it separately and the extracts are separated and collected and stored at 4°C which are used for the remaining analysis. The tests for Sterols, Saponins, Flavonoids, Coumarin glycosides, Anthraquinone glycosides, Amino acid/Protein, Lactone, Resins, Alkaloids, Tri-terpenes, Carbohydrates, Cynophoric glycoside, Tannins are all performed and is tabulated.

Systematic and qualitative studies are carried out as per the prescribed by standard protocols with the crude drug samples on primary and secondary metabolites. The extracts of the algae species are subjected to standard chemical tests as a normal routine procedure in order to identify the phyto constituents followed by the protocol adopted by Harborne.

In-vivo immunomodulation study

Carbon clearance test: Swiss albino mice were divided into 4 groups, each containing 6 animals. Group I (control) was given 1.0% sodium carboxy methyl cellulose (CMC) in water (0.3 ml/mouse) for 5 days. Group II-IV were given different concentrations of ETF (50, 100 and 200 mg/kg, p.o.) for 7 days. At the end of 7th day, after 48 hrs, mice were injected via the tail vein with carbon ink suspension (1:50 dilution of Indian ink, Camel, and 10 μl/gm body wt.). Blood samples were withdrawn (in EDTA solution 5 μl) from the retro-orbital vein at 0 and 15 min, a 25 μl sample has been mixed with 0.1% sodium carbonate solution (2 ml) and its absorbance at 650 nm has been determined. The rate of carbon clearance (phagocytic index k) is calculated from the slope of each time-concentration curve drawn by plotting as ordinate on semi logarithmic paper against time as abscissa. Results are expressed and keeping with the arithmetic mean ± S.E.M. of six mice [8,9].

All experiments have been repeated thrice and results are expressed as mean ± SD.

The phytochemical screenings of the Agaricus bisporus fraction are performed. Sterols, Saponins, Coumarin glycosides, Amino acid/ protein, Resins showed the positive results for ethanol extract; while Alkaloids, Tri-terpenes, Carbohydrates, Flavonoids, Anthraquinone glycosides, Cynophoric glycoside, Lactone, Tannins showed the negative results (Table 1). It has been revealed that Agaricus bisporous has the stimulated chemotatic, phagocytic and intracellular killing of human neutrophils at a dose of 100 μg/ml and 200 μg/ml respectively. In vivo studies of Agaricus bisporous have shown moderate immunostimulator activity at a concentration of 200 mg/ml as compared with control, whereas fungal species have shown prominent immunostimulator activity at the same dose (Table 2).

Table 1: Qualitative Phytochemical screening of Agaricus bisporus.

Table 2: Effect on Agaricus bisporus on Carbon clearance test.

The use of immunostimulants, mostly as adjuvant to chemotherapy, to control and prevention of infection holds great promise [10-12]. Significant interest has now been generated in research on bioactive molecules from Agaricus bisporus designated as immunomodulatory agents in alternate systems of medicine. The ethanol fraction of Agaricus bisporus was tested in the present study to determine their possible effect on immune function at three dose levels. This investigation reveals the ethanol fractions have significantly increased the phagocytic function of human neutrophils, when compared with control and increase the movement of neutrophils towards the foreign body which is the most important step in the phagocytosis process or activity.

The ethanol extracts of Agaricus bisporus on in vivo carbon clearance test have shown moderate immunostimulatory effect at a dose of 100 mg/kg body weight po route. This may suggest that macrophage probably stimulate production of cytokines which in turn stimulates other immunocytes which may help in defence mechanisms of body to counter various infections. A preliminary observation of these Agaricus bisporus extracts also requires further investigations for exact mechanism of immunomodulation.

The authors are thankful to Dr. R. Duraisamy, Associate Professor, Department of Pharmacology, Nandha College of Pharmacy, Erode, Tamilnadu, India who provides the animal house facilities.