Evaluation of Diuretic Activity of Ipomoea aquatica (Kalmisak) in Mice Model Study

MATERIALS AND METHODS

The experiment was conducted at Pharmaceutical Biotechnology Laboratory, Biotechnology Discipline, Khulna University, Khulna B9208, Bangladesh.

Plant material

The plant Ipomoea aquatica was collected from Khulna during March 2002 and identified by Bangladesh National Herbarium, Dhaka (Sample # DACB.NO.29, 636). After collection, whole plant was sun-dried for eight days and made into a coarse powder by grinding. 500 g powder of Ipomoea aquatica was extracted with 800 ml of methanol (40-55°C) in soxhlet apparatus. The extraction was carried out until the process was completed. The methanolic extract was concentrated to one third of its initial volume and a dark greenish colored mass was obtained. The concentrated mass was kept in room temperature to remove methanol by evaporation and finally heated it at 45-50^oC until it became methanol free and highly dense.

Table 1:	Design of the diuretic activity screening of methanol extract of Ipomoea aquatica
TS: Test sample

Experimental animal

Post weaning Swiss albino mice (20-25 g) of either sex were obtained from the animal house of International Center for Diarrhoeal Disease and Research, Bangladesh (ICDDR, B). The animals were housed in standard metal cages and provided with food and water ad libitum. They were deprived of any food and water for the preceding 12 h and were divided into five groups containing 5 animals in each group. They were weighed before the experiment.

Preparation of sample solution

Urea (62.5 mg) was accurately weighed and taken into a graduated test tube. Then it was dissolved slowly in saline by gentle shaking. Finally the volume was adjusted up to 2.5 ml with saline solution. To prepare suspension of the methanol extract of Ipomoea aquatica 31.5 mg of the extract was measured accurately and suspended in saline solution using 0.1% tween-80 as the suspending agent. The final volume was adjusted to 2.5 ml so that each animal of group IV received 0.5 ml, which was equivalent to a dose of 250 mg kg^-1 body weight. Similarly 63 mg of the extract was measured accurately and suspended in saline solution using 0.1% tween-80 as the suspending agent. The final volume was adjusted to 2.5 ml so that each animal of group V received 0.5 ml, which was equivalent to a dose of 500 mg kg^-1 body weight.

Screening of diuretic activity

The test animal was divided into five groups, containing five mice in each group. Group-I was provided only with saline solution and 0.1% tween-80 i.e. control. Group-II was given urea at a dose of 500 mg kg^-1 body weight and was considered as positive control group. Group-III was provided with standard diuretic drug Fursemide at a dose of 500 mg kg^-1 body weight. Group-IV and Group-V received the test materials, obtained by methanolic extract, at a dose of 250 and 500 mg kg^-1 of body weight, respectively. These preparations were given by oral route (Table 1). Twenty-four hours prior to the experiment, the test animals were placed into metabolic cages with total withdrawal of food and water. After oral administration of test samples, the urinary output of each group was recorded at different time intervals from the graduated urine chamber of metabolic cage. Urine samples, which are collected from metabolic case, were analyzed for Na⁺ and K⁺ concentration by flame photometric method (Mukherjee et al., 1997) while Cl^- concentration was determined by the Argentometric titration method (Ramesh and Anbu, 1996).

The volume of the urine excreted in 5 h of study by each group was expressed as percent of the liquid administered giving rise to a measure of AUrinary Excretion@(U. E.)- independent of group weight.

The ratio of urinary excretion (U.E) in test group and control group was denoted Diuretic Action which was used as the measure of degree of diuresis.

RESULTS AND DISCUSSION

The hot extraction of coarse powder (500 gm) of Ipomoea aquatica was carried out with distilled methyl alcohol which yielded 3.8 gm of methanolic extract after evaporation of solvent The research was launched on the basis of its folkloric use as a diuretic.

Since the plant Ipomoea aquatica has traditional use as a diuretic, the effect of methanol extract of the plant on urination was investigated in Swiss albino mice. The urinary output at different hours of study has been presented in Table 2. The result of the experiment (Table 3) revealed that the diuretic activity of methanol extract at a dose of 250 g kg^-1 body weight was comparable to that of the standard drug fursemide at a dose of 500 mg kg^-1 body weight. However the diuretic activity of the extract at a dose of 500 mg kg^-1 body weight was much higher than that observed with the standard drug fursemide. The methanol extract of Ipomoea aquatica at an oral dose of 250 g kg^-1 and 500 mg kg^-1 body weight showed maximum diuretic activity at 2.89 and 3.38, respectively at the first hour of the study. This indicated the rapid onset of diuretic action, which is dose dependent, of the test material. The diuretic activity of a drug is considered to be good if it is above 1.50, moderate if it is with in 1.00-1.50, little if it is between 0.72-1.00 and nil if it is less then 0.72. In this respect after fifth hour of drug administration, the methanol extract of Ipomoea aquatica showed good diuretic activity of 1.93 and 2.44 at doses of 250 mg kg^-1 and 500 mg kg^-1 body weight respectively till the fifth hour of study.

The diuretic responses with its electrolyte excretion potency of the extract were highly moderate in comparison with control animals. The extract, at doses of 250 mg kg^-1 and 500 mg kg^-1 show a dose dependent increase in volume of urine with remarkable increase in Na⁺ and Cl^B excretion, accompanied by the excretion of K⁺. In all cases the excretion of electrolytes and volume of urine increased was higher than the standard diuretic fursemide (Table 4).

Table 2:	Urinary output of mice at different time intervals after oral administration of methanol extract of Ipomoea aquatica
Vo= Total Urinary output; V1 = Total Fluid input (2.5 ml); TS= Test sample

Table 3:	Diuretic action and diuretic activity at different time intervals after oral administration of methanol extract of Ipomoea aquatica
TS = Test sample UEt = Urinary excretion in test group; UEc = Urinary excretion in control group DAt = Diuretic action of the test sample; DAu = Diuretic action of the urea ● Diuretic activity > 1.50 indicates good diuretic activity; ● Diuretic activity between 1.00 to 1.50 indicates moderate diuretic activity; ● Diuretic activity between 0.72 to 1.00 indicates little diuretic activity; ● Diuretic activity < 0.72 indicates no diuretic activity

Table 4:	Dose response study of diuretic effects of Ipomoea aquatica extract through electrolyte excretion in urine in mice
TS= Test sample

From the above observation it can be suggested that the methanolic extract of Ipomoea aquatica is an effective hypernatraemic, hyperchloremic and hyperkalaemic diuretic; which supports the claim about the Ipomoea aquatica being used as a diuretic.

A large number of indigenous drugs have been claimed to have a diuretic effect in the Ayurvedic system of medicine. Gujral et al. (1955) found that the flowers of Viola odorata, Nymphaea alba, Nelumbium speciosum; roots of Asparagus recemosus, Arundo karka, Portulaca oleracea posses diuretic effect in different animal models. Diuretic activity has also been observed in extract of the rhizomes of Nelumbo nucifera Gaertn (Mukherjee et al., 1997). Malalavidhane et al. (2000 and 2001) found that an aqueous extract of Ipomoea aquatica is as effective as the oral hypoglycemic drug tolbutamide in reducing the blood sugar levels of Wistar rats.

Though the extract appeared to cause good diuresis at both dose levels, the actual mode of action, which may be due to its effect either on loop permeability or reduction of antidiuretic hormone (ADH) secretion or inhibition of carbonic anhydrase enzyme, is not clear from the test result. The exact mechanism of diuretic activity exhibited by the extract can only be established after more investigation of the extract and screening of diuretic activity of isolated pure constituents.