ABSTRACT
Terminalia chebula. (Gaertn.) Retz. (Combretaceae), a plant widely used in the traditional medicinal systems of India, has been reported to possess antibacterial, laxative, antioxidant, and diuretic activities. In the current study, the aqueous fruit extract of Terminalia chebula. has been investigated for its effect on cell-mediated and humoral components of the immune system in mice. Administration of Terminalia chebula. extract produced an increase in humoral antibody (HA) titer and delayed-type hypersensitivity (DTH) in mice. It was concluded that the Terminalia chebula. extract is a promising drug with immunostimulant properties.
Introduction
Immunomodulation is a process that can alter the immune system of an organism by interfering with its functions; if an agent results in an enhancement of immune reactions, it is termed an immunostimulative drug, which primarily implies stimulation of nonspecific system (i.e., granulocytes, macrophages, certain T-lymphocytes, and different effector substances). Immunosuppression implies mainly to reduce resistance against infections or stress and may be due to environmental or chemotherapeutic factors.
Immunostimulation and immunosuppression both need to be investigated in order to regulate normal immunological functioning. Hence, both immunostimulating agents and immunosuppressing agents have their own importance, and the search for better agents exerting these activities is becoming a field of major interest all over the world (Patwardhan et al., Citation1990). Natural adjuvants, synthetic agents, and antibody reagents are used as immunosuppressive and immunostimulative agents. But there are major limitations to the general use of these agents, such as increased risk of infection and generalized effect throughout the immune system (Diasio & LoBuglio, Citation1996).
The use of plant products as immunomodulators is still in a developing stage. There are several herbs used in the indigenous systems of medicine that can modulate the body's immune system. A variety of plant-derived materials, such as polysaccharides, lectins, peptides, flavonoids, and tannins, have been reported to modulate the immune system.
Traditional Indian systems of medicines, such as Siddha and Ayurveda, have suggested a means to increase the body's natural resistance to disease. A number of Indian medicinal plants and various rasayanas. have been claimed to possess immunimodulatory activity. Medicines of the rasayana. group are believed to promote health, immunity, and longevity. According to Ayurveda, they strengthen all tissues of the body, prevent ageing, promote intellect, and prevent disease (Atal et al., Citation1986; Patwardhan et al., Citation1990; Puri et al., Citation1994; Ziauddin et al., Citation1996; Balachandran & Panchanathan, Citation1998).
Terminalia chebula. (Gaertn.) Retz. (Combretaceae) is found throughout India, especially in deciduous forests and areas of light rainfall. It possesses laxative, diuretic, cardiotonic, and hypoglycemic properties. A combination drug, Triphala, a composite mixture of Terminalia chebula, Terminalia bellerica. (Gaertn.) Roxb. (Combretaceae), and Emblica officinalis. (Gaertn.) (Euphorbiaceae), is a very popular traditional medicine used for the treatment of many chronic diseases such as ageing, heart ailments, hepatic diseases, and so forth (Jagetia et al., Citation2002; Kaur et al., Citation2002). However, there is paucity of scientific data on the in vivo. immunomodulatory activity of fruits of Terminalia chebula.. The objective of the current investigation was to study the immunomodulatory activity of the aqueous extract of the fruits of Terminalia chebula. in animal models.
Materials and Methods
Plant material
The fruits of Terminalia chebula. were collected in December 2004 from Sagar District, Madhya Pradesh, India Taxonomic identification was conducted by Dr. T.N. Shivananda, Senior Scientist, Indian Institute of Horticulture Research, Bangalore, India. A voucher was deposited in the herbarium of our laboratory under number PESCP/53.
Preparation of aqueous extract from the dry fruit of Terminalia chebula.
The air-dried fruits of Terminalia chebula. were finely powdered (mesh size #20) and stirred with eight parts of distilled water at about 70–80°C for 2 h. The liquid extract was filtered through sieve (mesh size #200). The filtrate was concentrated up to two parts on a rotary vacuum evaporator. The concentrated liquid was spray-dried to get the dry powder of the extract. The concentration is expressed as mg/ml.
Animals
Swiss albino mice of either sex weighing between 20 and 25 g were used for the study. Animals were housed under standard conditions of temperature (25°C), 12 h light/dark cycles, and fed with standard pellet diet and tap water.
Toxicity study
Terminalia chebula. dried extract was dissolved in water and administered orally to different groups of mice in dose ranging from 100 to 1000 mg/kg for the LD50 study using the method of Miller and Tainter (Ghosh, Citation1971). There was no lethality in any of the groups after 7 days of treatment.
Antigen
Fresh blood was collected from sheep sacrificed in the local slaughterhouse. Sheep red blood cells (SRBCs) were washed three-times in large volumes of pyrogen-free 0.9% normal saline and adjusted to a concentration of 0.5 × 109 cells/ml for immunization and challenge.
Effect of Terminalia chebula. extract on humoral antibody titer and delayed-type hypersensitivity response using SRBCs as an antigen in mice
The method described by Puri et al. (Citation1994) was adapted. Mice were divided into six groups, each group containing six mice. Drugs were administered in various groups: group I, control (normal saline); groups II–VI, Terminalia chebula. extract (5 dose levels 100–500 mg/kg p.o.).
The animals were immunized by injecting 0.1 ml of SRBC suspension containing 0.5 × 109 cells intraperitoneally on day 0. Blood samples were collected in microcentrifuge tubes from individual animals by retro-orbital puncture on day 7. The blood samples were centrifuged and serum was obtained. Antibody levels were determined by the hemagglutination technique. Briefly, equal volumes of individual serum samples of each group were pooled. To serial twofold dilutions of pooled serum samples made in 25-µl volumes of normal saline in microtitration plates was added 25-µl of 1% suspension of SRBCs in saline. After mixing, the plates were incubated at 37°C for 1 h and examined for hemagglutination under a microscopy. The reciprocal of the highest dilution of the test serum giving agglutination was taken as the antibody titer. On day 7, the thickness of the right hind foot pad was measured using a vernier caliper.
The mice were than challenged by injection of 0.5 × 109 SRBCs in right hind foot pad. Foot thickness was measured again 24 h after his challenge. The difference between the pre- and post-challenge foot thickness (expressed in mm) was taken as a measure of delayed-type hypersensitivity (DTH). The extract was administered orally on day 0 and continued until day 7 of challenge.
Statistical analysis
Data are expressed as the mean standard deviation of the means (SD), and statistical analysis was carried out employing Student's t.-test.
Results and Discussion
All the animals of groups I–IV were sensitized on day 0. The control group received only vehicle (saline) from days −7 to +7. The humoral antibody (HA) titer value was found to be 24.7 ± 9.9. Administration of Terminalia chebula. extract produced a dose-dependent increase in the HA titer after incubation with SRBCs (). Administration of higher doses (i.e., 300, 400 and 500 mg/kg) produced significant increase in HA titer as evident from hemagglutination after incubation of serum with SRBCs. DTH was determined 24 h after the challenge. Higher doses of Terminalia chebula. extract (400 and 500 mg/kg) showed statistically significant increase in mean paw edema.
Table 1 Effect of Terminalia chebula. extract on HA titer and DTH response using SRBCs as an antigen in mice.
The results obtained in the current study show that Terminalia chebula. extract displays a dose-dependent immunostimulatory effect in relation to antigenic stimulation. Injecting mice i.p. with 109 SRBCs suspended in saline sensitizes them for elicitation of DTH and also induces antibody formation, therefore this system has major advantage; that is, it enables two components of immune response to be measured in the same species under ideal conditions and is relatively simple and inexpensive to perform (Doherty, Citation1981). Terminalia chebula. extract produced a dose-dependent increase in both the parameters (i.e., antibody production and delayed-type hypersensitivity). Therefore, is concluded that the aqueous extract of fruit of Terminalia chebula. has promising immunostimulant properties.
Acknowledgments
The authors are grateful to Prof. M.R. Doreswamy, Founder, and Prof. D. Jawahar, Director, PES Group of Institutions, Bangalore, for their constant encouragement.
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