The fresh leaves of H. indicum were collected from the Udupi district, Karnataka between September-October. The leaves were authenticated by Prof Gopal Krishna Bhat, Department of Botany, Poorna Prajna College Udupi, India. A voucher specimen was preserved in our laboratory for future reference. The fresh leaves were shade-dried and reduced to a fine powder with a mechanical grinder. The powdered leaves are stored in airtight, hard polyethylene container with silica pouch up to 10-12 days. About 700 g of dry powder was extracted with chloroform using soxhlet apparatus. Freshly prepared H. indicum extract was subjected to phytochemical screening tests for the detection of various constituents using conventional protocol [₆]. The extract was concentrated to dryness using a rotary evaporator attached to a vacuum pump and stored at a temperature of -4oC until use. The yield was 7 g, in a dark brownish green residue form.

Swiss albino mice (17-25 g) of either sex are procured from Indian Institute of Sciences. They are maintained under standard conditions (temperature 22 ± 2oC, relative humidity 60±5% and 12 h light/dark cycle).The animals were housed in sanitized polypropylene cages containing sterile paddy husk as bedding. They had free access to standard pellet diet and water ad libitum. The Institutional Animal Ethics Committee approved the experimental protocol. All the animals received humane care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the “National Academy of Sciences” and published by the “National Institute of Health”.

EDTA, RPMI-1640, Hank's balanced salt solution (HBSS), dextran, phosphate buffered saline, fetal calf serum, streptomycin, penicillin, amphotericin, and Trypan blue were purchased from Himedia. Phytohaemagglutinin, ficoll hypaque and L-glutamine were purchased from Sigma Diagnostic, USA. Interferon α-2b and levimasole were obtained as gift samples from Fulford (I) Ltd. and Khandelwal Laboratories Ltd., Mumbai, respectively.

Fresh blood was collected from a healthy sheep from the local slaughter house. Sheep red blood cells (SRBCs) were washed thrice with normal saline and adjusted to a concentration of 0.1 ml containing 1X10 8 cells for immunization and challenge.

The mice were divided into six groups, each consisting of 10 animals. Group I (Control) was given 2% gum acacia in water (0.3 ml/mouse, p.o) for 7 days, Group II-VI were given different concentrations of chloroform extract (50-200mg/kg, p.o.) and standard drug (Levimasole 50 mg/kg, p.o.) respectively for 7 days. At the end of 7 days, the mice were injected carbon ink suspension (10 µl/g body weight) through tail vein. Blood samples were drawn (in EDTA solution 5 µl), from the retro orbital vein, at intervals of 0 and 15 min, a 25 µl sample was mixed with 0.1% sodium carbonate solution (2 ml) and the absorbance measured at 660 nm. The carbon clearance was calculated using the following equation: (Loge OD ₁ - Loge OD ₂) /15, where OD₁ and OD₂ are the optical densities at 0 and 15 min, respectively [₇].

The mice were divided into six groups, each consisting of 6 mice. Group I (Control) was given 2% gum acacia (0.3 ml/mouse) for 7 days, Group II-VI were given drug treatment which was exactly the same as with the carbon clearance test. The animals were immunized by injecting 0.1 ml of SRBCs suspension, containing 1x10 8 cells, intraperitoneally on day zero. Blood samples were collected in micro centrifuge tubes from individual animals of all the groups by retro orbital vein puncture on day 8. The blood samples were centrifuged and the serum separated. Antibody levels were determined by the haemagglutination technique [₇]. Briefly, equal volumes of 50µl individual serum samples of each group were pooled. Serial two-fold dilutions of pooled serum samples were made in 50µl volumes of RPMI-1640 in micro titration plates. To this 50 µl of 1% suspension of SRBC in RPMI-1640 was added. After mixing, the plates were incubated at 37ºC for 1 h and examined for haemagglutination under the microscope (button formation). The reciprocal of highest dilution, just before the button formation, was observed and titre values were calculated.

On Day 8, the thickness of the right hind footpad was measured using Vernier calipers. The mice were then challenged by injection of 1x108 sub SRBCs in the right hind footpad. The footpad thickness was measured again after 24 h of challenge. The difference between the pre- and post challenge footpad thickness, expressed in mm, was taken as a measure of the DTH response [₈].

Preparation of microorganism

Human blood was collected from a local blood bank and the RBCs removed by sedimentation in 5% (w/v) solution of dextran in buffered saline (m.w. 200,000; 3 ml of solution to 10 ml of blood) for 30 min at 37ºC. The PMNC-rich supernatant layer was washed twice with heparin-saline, concentrated by centrifugation (10 min at 110 g), counted with a hemocytometer, and suspended in gelatin-HBSS to make up a concentration of 1x107 cells/ml.

To assess phagocytosis, different concentrations of chloroform extract (50-200µg/ml) and the standard drug, Interferon α-2b (0.5 million IU), in the final volume of 0.1ml, were incubated respectively with 2 ml of the PMNCs suspension (1x107 sub cells/ml), 2 ml of the suspended microorganisms (1x107 cells/ml) and 0.4 ml of fetal calf serum at 37°C for 1h in 5% CO₂ atmosphere in a slanting position. At 30 min intervals up to 120 min, 0.5 ml aliquot of the suspension was removed and added to 1.5 ml of the ice-cooled gelatin-HBSS to stop phagocytosis. The control was run using gelatin-HBSS in place of the test compounds. These samples were centrifuged at 110 g for 4 min. Under this condition, the non-ingested microorganisms remained in the supernatant fluid. The viable count of the microorganisms was undertaken using the colony counter [₉]. Phagocytosis was expressed as the percentage decrease in the initial number of viable extra cellular bacteria according to the formula: P (t) = (1 - N_t /N ₀) × 100, where P(t) is the phagocytic index at time t = t, N₀ and N_t are the number of viable extra cellular bacteria at time t = 0 and t = 30, 60, 90 and 120 min, respectively [₁₀].

This test was performed with peripheral mononuclear blood cells, following their separation from the blood by using ficoll-hypaque gradient centrifugation, according to manufacturer's instructions (Sigma Diagnostic, USA). The rate of proliferation of mononuclear cells, under the influence of mitogens, was measured [₁₁]. Under sterile conditions, the cells were diluted to 1x10 7sub cells/ml with RPMI-1640 (supplemented with 20% fetal calf serum). The cell suspension (2 ml) was transferred into a sterile culture tube and to each sample. Different concentrations of the plant extract (50-200µg/ml, filtered through 0.22 µ pore size filter) and standard drug Interferon α-2b (0.5 million IU), in the final volume of 0.1 ml, were added, respectively. The proliferation of cells was induced by 50 µl phythaemagglutinin (PHA, 0.1 mg/ml). The prepared samples were incubated for 72 h at 37oC in a CO₂ atmosphere, supplemented with 2 mM L-glutamine, 100 µg/ml streptomycin, 100 units/ml penicillin and 0.25 µg/ml amphotericin. The control incubated with cells minus the plant extract. The viability of the cells was assessed after incubation with test compounds, using the Trypan blue dye exclusion method [₁₂]. Briefly, 20 µl of the incubation mixture was mixed with 20 µl of Trypan blue dye. The total number of mononuclear cells and mononuclear stained blue (dead cells) were counted under an inverted microscope (Olympus, Japan), using the hemocytometer. The percentage of cell viability was taken as a measure of cell proliferation and calculated as per the following formula.

Figure 1

Similarly, the percentage of cell stimulation was calculated as per the following formula [₁₅]

Figure 2

Statistical analysis was performed using one-way ANOVA, followed by Dunnett's test. The significance in difference was accepted at P <0.05.

The authors are thankful to A.Shama Rao Foundation, for providing the facility and financial assistance to carry out this research work.