MATERIALS AND METHODS

Collection and extraction of raw plant material: Authenticated seeds of Mimosa pudica were procured from a commercial herb supplier and coarsely ground with the help of an electric grinder. The coarse seed powder was placed in a porous cellulose thimble and extracted with methanol (1:5) in soxhlet extractor at a temperature 40±5°C continuously for 20-22 cycles. The extract (HMEMPS) was dried at moderate temperature (37°C) to obtain the residual material.

Collection of uterine tissues: Complete uterine along with the ovaries of adult nondescript buffaloes were collected from local abattoir, Mathura. Uterine tissue were collected from mid cornual region in case of early stage of pregnancy while in case of mid or late stage of pregnancy, complete corneal part of uterine tissue was collected. The gestation stage was determined by measuring Curved-crown Versus Rump (CVR) length of foetus by applying the equation (Soliman, 1970).

When CVR length was below 20 cm, Y = 28.66+4.498x
When CVR length was 20 cm or above, Y = 73.544+2.256x

where, Y is days of gestation and X is curve crown rump length in centimeters.

Uterine tissues were transported to laboratory in a thermos flask containing chilled (4±0.5°C) Ringer-Locke solution (RLS, pH 7.4) (Singh, 2006).

Solutions of chemicals/antagonist: Solutions of the chemicals, propranolol (10^-3 M; Sigma Aldrich) and verapamil (10^-9 M; Sigma Aldrich) used for evaluation of uterotonic activity in the present study, were prepared as stock solutions in triple distilled water and stored at 4°C. Further dilutions of the required concentration were made in freshly prepared RLS on the day of use.

Fig. 1:	Representative physiographic recording of the effect of cumulative concentrations of HMEMPS (10-6000 μg) on myometrial strip of pregnant buffalo

Uterotonic/oxytocic studies
Preparation and mounting of uterine tissue: Uterine strips were dissected out from the midcornual region and a perimetrial strip of about 3.0x0.5 cm was prepared by carefully removing the endometrial and myometrial tissues. Both the ends of strip were threaded and mounted in a thermostatically controlled (37.0±0.5°C) organ bath of 10 mL capacity containing continuously aerated RLS.

Calibration of physiograph and recording of responses: The change in tension of tissue was measured using a high sensitivity isometric force transducer and recorded in a PC using Chart V5.4.1 software programme (Powerlab, AD Instruments, Australia). Sensitivity of the instrument was set as required and the sampling rate was adjusted to 5 samples sec^-1. The bridge amplifier was calibrated at two points-point 1:0 mV = 0 g and Point 2:1 mV = 1 g and signal range was kept at 10 mV.

After calibration of the equipment, myometrial strip was set under an initial constant tension of 2 g and allowed to equilibrate for 90-120 min till the tissue developed regular phasic contractions (Fig. 1) before recording isometric muscle tension or response to plant extracts or drugs. During the equilibration period, the bath fluid was changed every 10-15 min.

Qualitative studies on the effect of M. pudica root extract on buffalo uterus: Working solutions of M. pudica extracts (10, 30, 100, 300, 600, 1000, 3000, 6000 μg mL^-1) were prepared a fresh on the day of experiment. Different concentrations of M. pudica seed extracts were added in organ bath to record the responses of uterine tissues and also to record the minimum threshold concentration required for producing uterotonic effect.

Contact period of different plants extracts: Different concentrations of extracts of M. pudica seeds (10, 30, 100, 300, 600, 1000, 3000, 6000 μg mL^-1) were added to the tissue bath in an increasing manner. Each concentration was allowed an initial contact period of 5-10 min and the consecutive concentration was added only after the maximum effect of the previous concentration was evident. Once the maximal possible response was achieved, the tissue was washed. A minimum washout period of 30 min was allowed between two successive recordings. Then, the uterine strips were incubated with different antagonist (Propranolol or Verapamil) in the organ bath for 10 min and again the different concentrations of the extract were added. After recording the tissue response(s), bathing fluid in organ bath was changed every 10 min till the base line was achieved.

Statistical analysis: Statistically analysis of data was performed using SPSS 10 software applying Duncan’s test. Mean of various parameters for contractions between different treatments groups were compared using ANOVA with Duncan Multiple Range Test. A value of p<0.05 was considered as statically significant.

RESULTS

HMEMPS in the concentration range of 10-1000 μg mL^-1 produced concentration-dependent inhibitory effect on amplitude and frequency of spontaneous rhythmic contractions (Fig. 1 and 2). It relaxed the uterine strip completely with an EC₅₀ of 222.4 μg mL^-1. However, at higher concentrations (3000 and 6000 μg mL^-1), the base line was somewhat elevated.

Following propranolol (10-6 M) pretreatment, there was an apparent and marked increase in amplitude of spikes of normal spontaneity. An apparent increase in amplitude of spikes up to 1000 μg mL^-1 followed by decrease with further increase in concentration of HMEMPS was also recorded (Fig. 3a). The tension following exposure to HMEMPS was less in propranolol-treated tissues compared to that in its absence. There was parallel shift to left of the dose-response curve, thereby suggesting the inhibitory effect of propranolol on HMEMPS-induced myometrial relaxation. Verapamil apparently failed to markedly alter the HMEMPS induced inhibitory effect on buffalo myometrium (Fig. 3b).

Fig. 2:	Cumulative concentrations response curve of HMEMPS on isolated myometrial strips of pregnant buffaloes (n = 5)

Fig. 3(a-b):	Cumulative concentration response curves of HMEMPS alone and in the presence of an antagonist (a) Propranolol (10-6 M) and (b) Verapamil (10-12 M) on pregnant buffaloes isolated myometrial strips (n = 4)

DISCUSSION

Role of ethnoveterinary medicine in livestock development is beyond dispute (Martin et al., 2001). Because of their holistic nature, traditional remedies more often offer better efficacy combined with safety and economy than single cosmopolitan/conventional drugs (Varier, 1996). Mimosa pudica has been used for stomachache, womb cleaning, to stop menstruation and for gonorrhea (Lans, 2006). In the present study, an attempt had been made to work out the possible mechanism(s) of tocolytic effect of Mimosa pudica seeds extract using isolated uterine strips of pregnant buffaloes.

HMEMPS produced consistent, rapid and sharp concentration-dependent inhibitory and excitatory effect in some of the tissues i.e., biphasic effect. The uterine contractibility is determined by increase in intracellular free calcium ion concentration in the myometrial cells and calcium channel blocker have been reported to inhibit uterine contraction by decreasing Ca²⁺-influx (Longo et al., 2003). The bovine myometrial tone and spontaneity has previously been reported to be extra-cellular Ca²⁺-dependent (Singh, 2006).

Pretreatment of uterine tissue with propranolol (10^-6 M), the cumulative concentration response curve of HMEMPS shifted leftwards indicating the blockade of relaxation produced by the extract. These observations suggested that HMEMPS-induced myometrial effects are possibly mediated through β-adrenergic receptors. To explore the possibility of involvement of other inhibitory mechanisms including ion-channels, NO-pathways, second messengers etc., further studies are required to be conducted.

Verapamil (10^-12 M), a Ca⁺²-channel blocker, failed to produce marked decrease in tissue tension or the dose response curve was not shifted either to left or to the right. These observations are in contrast to earlier suggestions from this laboratory (Singh, 2006) that the normal spontaneity of buffalo myometrium is Ca²⁺ dependent. Verapamil has also been reported to have inhibitory effect on many other smooth muscles including uterus (Forman et al., 1982; Janis and Triggle, 1986) preferentially by inhibiting Ca⁺²-influx through potential-dependent Ca⁺²-channel (Fleckenstein, 1977; Bolton, 1979; Janis and Triggle, 1986). Several studies show the presence of NOS in the uterus from animal species and the human with differences with respect to the predominant form and Ca²⁺ dependence (Sladek et al., 1993; Ramsay et al., 1996; Sladek and Roberts, 1997). In pregnant rat myometrium, the activity of NOS has been reported to be quite insensitive to Ca²⁺ (Sladek and Roberts, 1997).

M. pudica total plant extract is reported to produce depressant effect on isolated rabbit duodenum and the percent decrease in either amplitude or frequency of duodenal contraction was found to be only marginally different from that found after a similar dose of atropine sulphone. The observations apparently suggest that HMEMPS-induced myometrial relaxation was regulated through Ca⁺²-channels-independent mechanism; however, further studies are indicated.

From the results of the present study, it may be inferred that M. pudica seeds extracts produced concentration-dependent inhibitory effect on buffalo myometrium and it seemed to be mediated through inhibitory β receptors. Calcium channels did not seem to regulate tocolytic effect of Mimosa pudica seeds extracts. Further studies need to be done on to explore the involvement of Ca²⁺ and K⁺ channels, NO and other signaling mechanisms including involvement of second messengers.

ACKNOWLEDGMENT

The authors are grateful to Hon’ble Vice Chancellor of the University (DUVASU) for providing all necessary facilities.