420 Immunostimulant activity of Safed Musli soluble portion was then reduced to half under reduced pressure and finally saponins were precipitated by addition of diethyl ether. The collected precipitate was hydrolyzed using hydrochloric acid (2 N HCl) and the precipitated sapogenins were collected (15). Alternatively, 500 g defatted drug powder was hydrolyzed by using 200 ml of 4 N H2SO4 in a round bottom flask which was kept overnight at room temperature and filtered to remove acidic solution, marc was washed three to four times with cold distilled water. After washing and drying the marc was refluxed for 30 min using fresh 200 ml of 4 N H2SO4 to ensure complete hydrolysis of drug. The mixture was filtered and filtrate was discarded. Hydrolyzed drug powder was further washed with distilled water three times to ensure complete removal of acid. The hydrolyzed drug powder was dried in an air oven to ensure completely dried drug powder. The hydrolyzed drug powder was fed in a Soxhlet extrac- tor and subjected to extraction with ethanol (95%). Ethanol was removed from the extract under reduced pressure and sapogenins were collected (16). The sapogenins isolated by this method were used for pharmacological studies. Sapogenins isolated by either method exhibited identical TLC profile suggesting suitability of both the process for isolation of sapogenins from the drug. HPTLC analysis of the ethanolic extract as well as isolated sapogenin fraction was performed. In brief, the chromatographic analysis of ethanolic extract was performed on silica gel-GF254 precoated plates (E Merck, Germany) using chloroform:glacial acetic acid: methanol:water (16:8:3:2, v/v) as mobile phase, 10 spots were visualized upon derivatization with anisaldehyde sulfuric acid reagent. Sapogenin fraction was characterized on chloroform: diethylether (1:1, v/v) mobile phase against b-sitosterol as standard marker. The pattern and standardization of the sapogenins against standard marker (b-sitosterol) has been reported by us previously (17). Materials Sheep red blood cells (SRBC’s) were obtained from Haffkine Biopharmaceuticals Ltd, Mumbai, India, and were washed thrice with large volumes of pyrogen-free sterile saline and adjusted to a concentration of 5 · 109 cells per ml for immunization and challenge. Azathioprine was obtained as gift sample from Troika Pharmaceuticals, Ahmedabad, India. Candida albicans was purchased from IMTECH (Chandigarh, India). Rats Wistar strain albino rats weighing between 140–150 gm of either sex were used. They were housed in departmental animal room under standard condition of temperature (24 ± 1 C), 12/12 h light/dark cycle and fed on standard pellet diet. Ethanolic extract and sapogenin were administered orally as suspension in 2% polyvinyl pyrolidine solution using metal canula. Toxicity studies were performed and dose of 1 gm kg 1 body weight (bw) of ethanolic extract or sapogenins did not cause any toxic effect. Statistical Analysis Data are expressed as mean ± SEM and analyzed for significance by Dunnet’s test (comparing all versus control) using Instat v.2.02 software (Graphpad software Inc.) residing in Pentium IV processor run on Windows Xp. Experimental Treatment Albino rats were divided into groups comprising of six animals each. Group I served as control and was administered vehicle only. Group II was administered 200 mg kg 1 bw ethanolic extract. Group III received 100 mg kg 1 bw sapogenin extract. Each experiment was performed on fresh group of animals unless specified. Non-Specific Immunity Determined by Survival Rate Against Fungal Infection Treatments of all the three groups began 14 days before challenge. On the day of challenge all groups were injected with 5 · 107 viable C. albicans cells and observed daily for mortality for a period of 10 days. In Vivo Phagocytosis Using Carbon Clearance Method The method of Biozzi et al. (1953) (18,19) was used. Treatments of all groups started 14 days before experimenta- tion. On 15th day of treatment, mice were injected with 0.1 ml of carbon suspension (Pellikan Tuschea Ink, Germany) intravenously through tail vein. Blood samples (25 ml) were collected from retro-orbital plexus just before and at 4, 8, 12 and 16 min after injection. Blood samples were lyzed with 2 ml of 0.1% acetic acid and absorbance of samples recorded at 675 nm (20). The graph for absorbance versus time was plotted for each animal in respective test group and phagocytic index was calculated using the formula: Ksample Phagocytic IndexðPIÞ ¼ Kstandard where Ksample represents the slope of absorbance versus time curve for extract-treated samples and Kstandard represents the slope of absorbance versus time curve for blood sample collected before treatment. SRBC-Induced Delayed-Type Hypersensitivity Reaction (DTH Response) The method of Lagrange et al. (1974) (21) was used. Treatments with extracts began 14 days before challenge. All the groups were immunized by injecting 20 ml of 5 · 109 SRBC per ml subcutaneously into the right foot pad. After