MATERIALS AND METHODS

The study was carried out at Pharmacognosy and Pharmacology Departments of R.C. Patel Institute of Pharmaceutical Education and Research, Shirpur Dist-Dhule, North Maharashtra University (MS), India between December 2007 and August 2009.

Reagents
Fresh blood was collected from sheep’s sacrificed in the local slaughter house. Sheep Red Blood Cells (SRBC) were washed three times in normal saline and adjusted to a concentration of 0.1 mL containing 1x10⁸ cells for immunization and challenge.

Plant Material and Extraction
The aerial parts of Tridax procumbens were collected in the month of December and January from locality of Gondia, Maharashtra State, India and authenticated by the Dr. D.A. Patil, HOD Botany Dept, SSVPS College, Dhule (MS), India. A voucher specimen (SA-01) was submitted at institute’s herbarium department for future reference. Dried material was coarsely pulverized to powdered form. One kilogram of powdered plant material was extracted by maceration with 500 mL alcohol for 3 complete cycles. The alcoholic extract was dried at 30-40°C using a vacuum evaporator. The resulting dried extract was solubilized in purified water and fractionated in to chloroform fraction (CFTP), ethyl acetate fraction (EFTP), n-Butanol fraction (NFTP) and remnant water soluble fraction (RWSFTP), by liquid-liquid partitioning. These fractions were investigated for immunomodulatory activity, by dissolving in 1% sodium carboxy methyl cellulose and filtered through 0.22 μm membrane filter.

Experimental Animals
Swiss Albino Mice, strain C57BL6 weighing between 20-40 g were used in the study with prior approval and scrutinization from the Institutional Animal Ethical Committee (RCPIPER/IAEC/2008-09/30). The animals were housed in clean and spacious cages provided with net and feeding bottle, at ambient temperature of 25±2°C with 12 h light and 12 h dark cycles and provided free access to standard laboratory chow mixture provided water ad libitum for fixed period so as to acclimatize all animals and to achieve normal constant basal food intake in all.

Effect on in vitro Phagocytosis of Candida albicans by Human PMN Cells
The extract and all the fractions obtained there of were evaluated for immunomodulatory activity, using the PMN function test. Peripheral venous blood, 10 mL, was collected from volunteers in a sterile heparinised tube. Neutrophils were isolated by Ficoll Hypaque density gradient sedimentation (Boyum, 1968, 1976). The RBC-PMN pellet was then subjected to dextran sedimentation. The supernatants, containing more than 90% of PMN cells, were collected and the cell density adjusted to 1x10⁶ cells mL^-1 using MEM.

Candida albicans (cell density adjusted to 1x10⁶ cells mL^-1 using MEM) was used as the test microorganism. The PMN cells (cell density adjusted to 1x10⁶ cells mL^-1 using MEM) were mixed with 1x10⁶ cells mL^-1 of Candida albicans and incubated at 37°C for 1 h in 5% CO₂ atmosphere, in the presence of the test fractions. The control was the identical solution minus the test fraction. Cytosmears were prepared after incubation. The smear was fixed with methanol, stained with Giemsa and studied under 100X oil immersion objective to determine the phagocytic activity of PMN cells. Neutrophils (100 numbers) were scanned and the cells with ingested microorganisms were counted (Lehrer and Cline, 1969; Gabhe et al., 2006).

Treatment of Mice
Twelve groups of mice each consisting of six animals received all extract (0.1-1.0 g kg^-1) and fractions (0.02 and 0.04 g kg^-1) Body Weight (BW) for 7 consecutive days intraperitoneally. The control group was treated with 1% sodium carboxy methyl cellulose solution, whereas native group was devoid of any treatment. No mortality or any toxic effects were observed in the above mentioned doses of these all fractions, administered intraperitoneally.

Heamagglutination Antibody Titre
Mice were intraperitoneally immunized with 1x10⁸ SRBC on day 0. Blood samples were collected from individual animals from the orbital plexus on day 7. Antibody levels were determined by the haemagglutination technique (Ray et al., 1991). Two-fold diluted sera in saline (0.025 mL) were mixed with 0.025 mL of 0.1% v/v SRBC suspension in microtitre plates. The plates were incubated at 37°C for 1 h and then inspected for haemagglutination. The antibody titer was determined by a two-fold serial dilution of one volume (100 μL) of serum and one volume (100 μL) of 0.1% Bovine Serum Albumin (BSA) in saline. One volume (100 μL) of 0.1% SRBCs in BSA in saline was added and the tubes were mixed thoroughly. They were allowed to settle at room temperature for about 60-90 min until the control tube showed, a negative pattern (a small button formation). The value of the highest serum dilution showing visible haemagglutination was taken as the antibody titer (Puri et al., 1993).

Delayed Type Hypersensitivity
Mice were primed with 0.1 mL of SRBC suspension containing 1x10⁸ cells intraperitoneally on day 0 and challenged on day 7 with 1x10⁸ SRBC in right-hind foot pad. The contra lateral paw received saline alone. The thickness of foot pad was measured at 24 h after challenge using a Vernier Caliper. The difference in the thickness of right hind paw and left hind paw was used as a measure of DTH reaction (Ghazanfari et al., 2002; Fulzele et al., 2003).

Statistical Analysis
The data was analysed using One-Way Analysis of Variance (ANOVA), followed by Dunnett’s test. The p<0.05 was considered significant.

RESULTS

Effects of Extract and all Fractions Obtained thereof on PMN Cell in Phagocytic Activity
Methanol extract and fractions obtained from the extract of Tridax procumbens were evaluated for their phagocytic activity. In this assay, ingested and associated C. albicans per PMN cell were measured. The results have been expressed as Candida per cell, the average number of Candida cells associated with PMN cells. Methanol extract exhibited significant phagocytic activity in dose of 200 μg mL^-1 (Table 1). Among all the groups of fractions studied, EFTP and NBTP groups shows the significant engulfment of the C. albicans per PMN cell in a dose of 25 μg mL^-1 (Table 2), as compared to the negative control.

Heamagglutination Antibody Titre
A dose-related increase in humoral antibody titer was observed in mice treated with the methanol extract in a dose of 200 mg kg^-1 and EFTP and NFTP of T. procumbens in a dose of 20-40 mg kg^-1, per oral (Table 3, 4).

Table 1:	Effect of alcoholic extract on in vitro phagocytosis of C. albicans by PMN cells

Table 2:	Effect of fractions on in vitro phagocytosis of C. albicans by PMN cells

Table 3:	Effect of alcoholic extract on SRBC induced delayed type hypersensitivity and haemagglutination titer in mice
Control: 1% Sodium carboxy methyl cellulose; n = 6 per group. p-values are *p<0.05, **p<0.01, ***p<0.001 as compared with control were considered

Table 4:	Effect of fractions on SRBC induced delayed type hypersensitivity and haemagglutination titer in mice
Control: 1% Sodium carboxy methyl cellulose; n = 6 per group. p-values are *p<0.05, **p<0.01, ***p<0.001 as compared with control were considered

Table 5:	Effect of fractions on thymus and spleen weight using SRBC as an antigen in mice
Control: 1% Sodium carboxy methyl cellulose; n = 6 per group. p-values are *p<0.05, **p< 0.01, ***p<0.001 as compared with control were considered

Delayed Type Hypersensitivity Reaction
In the present study, SRBC-induced delayed-type hypersensitivity was used to assess the effect of the extract and fractions on cell-mediated immunity. In the control animals, the +48 h and +72 h response was either equal or slightly more than the 0 h response, therefore, the peak edema at +24 h was taken as a parameter for evaluating the reaction. The methanol extract (200 mg kg^-1) and fraction EFTP and NFTP of Tridax procumbens (20-40 mg kg^-1, p.o.) produced a significant, dose-related decrease from DTH reactivity in mice (Table 3, 4).

Effect of Plant Extract and Fractions on Body Weight, Lymphoid Organ Weight and Cellularity
No effect was observed in the spleen weight at any dose when compared with control (normal saline treated) animals (group I). At doses of 20 and 40 mg kg^-1 a significant decrease (p<0.05) in relative organ weight of thymus was observed with EFTP and NFTP but, there was no effect at a dose of 20 and 40 mg kg^-1 in its weight for CFTP and RWSTP. Lymphoid organ cellularity (Table 5) data indicate no significant decrease in the spleen cellularity at any of the doses. A significant increase was also recorded in the cellularity of thymus at doses of 50 and 100 mg kg^-1 as compared with control animals (group I).

DISCUSSION

The present study not only proved the immunomodulatory activity of the alcoholic extract of Tridax procumbens, but also showed that EFTP and BFTP were more active than other fractions. Alcoholic extract and EFTP and NFTP treatment improved the heamagglutination antibody titer reflecting an overall elevation of humoral immune response supporting the role of flavonoids and saponins in the immunostimulation. Haemagglutination antibody titer was determined to establish the humoral response against SRBC as antigen. At neutral pH, red blood cells possess negative ions cloud that makes the cells repel from one another, this repulsive force is referred to as zeta potential. Because of its size and pentameric nature, IgM can overcome the electric barrier and get cross-link to red blood cells, leading subsequent agglutination. The smaller size and bivalency of IgG, however, makes them less capable to overcome the electric barrier. This characteristic may accounted for, IgM being more effective than IgG in agglutinating red blood cells (Kuby, 1997; Manosroi et al., 2003).

Delayed type hypersensitivity reaction is characterized by large influxes of non specific inflammatory cells, in which the macrophage is a major participant. In DTH, circulating T cells sensitized to the antigen from prior contact reacts with the antigen and induces specific immune response, which includes mitosis (blastogenesis) and the release of soluble mediators. This promotes phagocytosis activity and increases the concentration of lytic enzymes for more effective killing. The overall effects of these cytokines are to recruit macrophages into the area and activate them, promoting increased phagocytic activity vis-a-vis increased concentration of lytic enzymes for more effective killing. Several lines of evidence suggest that DTH reaction is important in host defense against parasites and bacteria that can live and proliferate intracellularly (Dash et al., 2006; Smith et al., 2000). Treatment of extract and EFTP and NFTP enhanced DTH reaction, which is reflected from the increased footpad thickness compared to control group suggesting heightened infiltration of macrophages to the inflammatory site. This study may be supporting a possible role of EFTP and NFTP in assisting cell-mediated immune response.

The increase in thymus weight was accompanied by increase in its cell counts. This may be partly due to stimulatory effect of plant extract and fractions on the lymphocytes and bone marrow haematopoietic cells, which ultimately home in the thymus. However, this homing may be temporary and in due course of time normalcy may ensue (Bin-Hafeez et al., 2003; Gayatri et al., 2005).

Through chemical analysis, EFTP and BFTP mainly consist of flavonoids and triterpenoidal saponins which are probably responsible for its immunomodulatory activities. Therefore, EFTP and BFTP could be an effective and useful candidate in the development of immunomodulatory drug.

CONCLUSIONS

The present study has shown the immunostimulatory activity of Tridax procumbens and suggests its therapeutic usefulness. The EFTP and NFTP have stimulated both humoral as well as cellular arms of immune system. Further detailed studies are required which might establish a possible use of hydro-alcoholic extract of Tridax procumbens in immunocompromised patients and as an adjuvant during vaccination programs in order to reduce number of non-responders to vaccines. However, detailed studies of phytoconstituents responsible and mechanisms of immunomodulation as well as probable use in immunocompromised individuals are still to be investigated.

ACKNOWLEDGMENT

The authors are thankful to the Principal, R.C. Patel Institute of Pharmaceutical Education and Research, Shirpur for availing all the facilities.