IMMUNO MODULATORY ACTIVITY OF THE ALCOHOLIC EXTRACTS OF MIMOSA PUDICA (LINN) IN MALE WISTAR RATS

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1 IMMUNO MODULATORY ACTIVITY OF THE ALCOHOLIC EXTRACTS OF MIMOSA PUDICA (LINN) IN MALE WISTAR RATS John Wesley J*, C. Suresh Raman Nadar, N. Chidambaranathan Department of Pharmacology, S. A. Raja College of Pharmacy, Tamil Nadu Abstract: The Immuno modulatory effect of alcoholic extract of the various aerial parts of mimosa pudica (Linn) Mimosaceae was investigated in male wistar rats. The assessement of immuno modulatory activity was carried out by various hematological and serological test. Futher immuno modulatory activity was studied by Cell Mediated Immune Response (CMIR) measured by delayed type of hypersensitivity reaction to SRBC and humoral immune response (HIR) measured by heamagglutination antibody titer. The alcoholic extract significantly enhanced humoral as well as cell mediated response in normal and treated rats. The results indicate the potential of mimosa pudica (Linn) as an immuno modulatory agent. Keywords: Mimosa Pudica, cell mediated, humoral. INTRODUCTION: Immuno modulation is a process that can alter the immune system of an Organism by interfering with its function. If enhancement of immune reactions named as immune modulatory, then immuno suppresant implies mainly to reduce resistance against infection on account of hemotherapeutic factors [1, 2]. Immuno modulatory agents may selectively activate either cell mediated or humoral immunity. The primary target of the immuno modulatory compounds is belived to be the macrophages, which play a key role in the generation of immune response. Number of Indian medicinal plants and various ( Rasayanas ) (charak samhita, 1000 B.C) have been claimed to possess immunomodulator activity. Rasayanas are group of non toxic herbal drug preparations that stimulate the immune response of the human. M. Pudica (Linn) is a sensitive plant, creeping annual or perennial herb belongs mostly to the subtropical areas of southern parts of India. The Juice of Fresh leaves are mixed with Ghee is used in the treatment of inflammation of the skin [3]. The leaves are also used for external applications for wounds and ulcers [4]. The effect of M. Pudica on the immune system at various levels. i.e hematopoiesis, unspecific mechanisms and cellular responses has not been extensively studied. Therefore the object of present *Corresponding Author John Wesley J 41 P a g e

2 study was to investigate the immuno modulatory effect of the alcoholic extract of mimosa pudica. MATERIALS AND METHODS: M. Pudica (Linn) is a red brown prickly, crowded annual herb. It forms a dense ground cover, preventing reproduction of other species. It s a wide land fire hazard when dry. The healthy plant material was collected from local areas of Kanyakumari District in the month of August to September. Specimens at voucher number 4214 was deposited in the herbarium of the botany department at the American college Madurai. Samples are dried in shade for 15 days and reduced to moderately coarse powder mechanically. EXTRACTION PROCEDURE: Powdered material of M.Pudica was subjected to extraction using 95% of ethyl alcohol. The extract was concentrated under reduced pressure according to qualitative chemical tests. Extract was further fractionated into pet ether (60-80º) all the extracts are concentrated under vacuum evaporation (Roteva, Equitran). Percentage yield of alcoholic (9.6%) was reported. Phytochemical Investigation: The quatitative chemical test of alcoholic extract of M.Pudica was carried out using standard procedure to determine the presence of various phytochemicals. Alcoholic extract was subjected to quantitive estimation of glycosides and sapponin content [6-8]. Tests show the presence of flavanoids and absence of glycosides. Experimental protocols Male wistar rats ( g) were used for the activity. They were housed under controlled conditions of light dark cycle (12:12) and temperature (25 ± 2 c) prior approval by institutional ethics committee was obtained for conduction of experiment. Acute toxicity study The acute toxicity study of alcoholic extract was evaluated in male wistar rats. The animals were fasted overnight prior to the acute toxicity study. Different groups containing 2 rats in each were orally administered with alcoholic extract at 0.5, 1, 1.5, 2 and 3 mg/kg p.o respectively. Mortality and general behaviour of the animals were observed continuously for initial four hour and then at 24 hours and 48 hours after dosing. The parameter observed and recorded were sedation, grooming, loss of righting reflex, respiratory rate and convulsion. 1/10 th of lethal dose was taken as the screening dose [8, 9]. Antigen SRBC The blood was withdrawn from the external jugular vein of sheep. It was mixed with alsevier s solution in 1:1 proportion and was stored at 4 c. Cell mediated immune response to SRBC delayed type hyper sensitivity Animals were divided into 4 groups each containing 6 animals immunized on day 0 by i.p administration of 0.5 x 10 9 SRBC/ml. The suspensions of solvent dried extracts of alcoholic were prepared in gum acacia solution (0.5%) and administered orally to rats from day 1 to day 13 at a dose at 200 mg/kg bodyweight. At the same time control group received 0.5% gum acacia solution. Animals from all groups were antigenically challenged by subcutaneous admisintration of 42 P a g e

3 0.25X10 9 SRBC /ml into the right hind food pad of the rats. On day 13, DTH response was measured after 48 hrs with respect to increase in paw volume by plethysmometer (UGO Basile, Italy). The change in the volume of the left hind paw injected similarly with phosphate buffered saline served as control. The percentage increase in the volume of the paw in alcoholic treated group was considered as an index of cell mediated immune [11, 12]. response Humoral immune response [13, 14] (haemagglutination antibody titer) Humoral immume response was measured by haemagglutination antibody titre method on days 13 and 20. Animals were divided into 5 groups, each group containing 6 animals. Group I served as control and group II and III were treated with alcoholic extract of mimosa pudica (linn) at a dose of 200mg/kg and 400 mg/kg for 13 days and each animal was immunised with 0.5 x10 9 SRBC/ml, by i.p route including control group on day 0. On day 13 and 20, blood samples were collected from the retro-orbital plexus of rats of all the groups and serum was separated. In the wells of micro-titrating plate, 25μl of serum was serially diluted with the same amount of phosphate suffer saline (PH 7.4) to get successive 2 fold dilution so that antibody concentration in any of the wells was half that of previous. The minimum dilution of serum (1/2) was ranked as 1. Each dilution with 25μl of SRBC suspension (0.025 x 10 9 cells/ml) was incubated at 37ºc for one hour. The highest rank number of the serum dilution, that exhibited agglutination was considered as antibody titre. The initially observed antibody titre was considered as primary humoral immune response while the observation on secondary considered as secondary humoral immune respose. Results and Discussion Administration of Mimosa pudica plant extract has been reported to increase production of circulating antibody titre. Alcholic extract of mimosa pudica linn was evaluated at a dose of 200mg/kg and 400 mg/kg for immunomodulatory activity. The DTH response to SRBC corresponding to cell mediated immunity & haemagglutination antibody titre for humoral immunity were measured respectively as shown in Table No 1 and 2. The results indicate that daily treatment of rats with alcoholic extract showed a significant elevation in the humoral as well as cell mediated immunity. The humoral immune response has been assessed by estimating the antibody levels in the rats. The humoral immunity involves interaction of B cells with antigen and their subsequent proliferation differentiation into antibody secreting plasma cells. SRBC have been used as antigenic material for initiating the antibody formation and subsequently the same was titrated with SRBC by haemagglutination method. The reaction resulted into an increase in the paw volume which was considered as an index for DTH or cell Mediated immune response on the other hand alcoholic extract on cell mediated immune response produced slightly significant decrease in the paw volume. Conclusion In the presence study the alcoholic extract of Mimosa pudica plant on cell mediated 43 P a g e

4 Mean Increase in paw vol. (%±SEM) immune response to SRBC and Humoral immune response showed positive immune modulatory activity. It is important to note that Mimosa pudica may have immune stimulant and immune suppressant activity due to other phytochemicals. The present investigation demonstrate immune modulatory activity of alcoholic extract of mimosa pudica thus have tremendous future potential for developing new pharmaceutical product with specific pharmacological activity. Acknowledgements Authors are greatly thankful to the department of pharmacology for providing free access to their facilities to carry out research work. Table No. 1- Effect of the alcoholic extract of mimosa pudica linn. On Delayed type Hypersensitivity reaction in the rats Group Substance Dose Mean Paw volume (mm) (mg/kg) 24h 48h 72h 96h I Control (Normal saline) - 5± ± ± ±0.13 II Test extract I ± ± ± ± 0.24* III Test extract II ± ±0.36* 5.8± ±0.24 The values are mean± SEM, P<0.01* when compared with control group CONTROL TEST EXTRACT I 200 mg/kg TEST EXTRACT II 400 mg/kg 0 24 HOURS 48 HOURS 72 HOURS 96 HOURS Fig. -1 Graphical representation of DTH reaction of alcoholic extract of Mimosa pudica linn. In rats 44 P a g e

5 Humoral Immune Response. (Mean Antibody Titer Values) Table No.-2 Effect of Alcoholic extracts of Mimosa pudica linn. On hameagglutination titre in rats Groups Humoral Immune response mean antibody titer Primary Secondary Control 08.70± ±0.16 Test extract I-200mg/kg 10.61±0.09* * Text extract II-400mg/kg 12.3±3 0.21* 14.52±0.29* The values are Mean ± SEM, P<0.01*, when compared with control group. Primary Secondary Control Test extract I 200mg/kg Text extract II 400mg/kg Fig -2 Graphical representation of alcoholic extract of Mimosa pudica linn. on Haemagglutination Titer in rats 45 P a g e

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