Diuretic activity may be very useful in a number of conditions like hypertension, hypercalciuria, and cirrhosis of liver. Since diuretics are employed clinically in the treatment of edema, it would be highly important to demonstrate effectiveness in the presence of electrolyte and water . Thus, it is presumed to be advantageous to ‘pre-treat’ or ‘prime’ the test animals with various fluids in screening agents for potential diuretic activity.
The aqueous and 80% methanol extracts of the leaves of Ajuga remota showed an increase in urine volume that appeared to vary with dose and time as well as the nature of the extract. Compared to the 80% mehanol extract, the aqueous extract produced a better diuretic effect, particularly with increasing dose in the early hours (p < 0.05 for AA1000 vs. AM250, AM500, & AM7500) (Table 1). The lower doses of both extracts did not produce an appreciable effect, but, whilst the medium dose of the aqueous extract was able to produce significant effect beginning from the fourth hour, the same dose of the 80% extract was devoid of any effect until the end of the observation period. This could probably suggest that the lower doses might represent subthreshold doses. The higher dose of both the aqueous and 80% methanol extracts produced comparable effect to that of furosemide. Even better effect was observed with the aqueous extract, as it had a diuretic activity even more than the standard drug. It is therefore possible to suggest that the ingredient (s) of the plant material responsible for increasing urine output could probably be polar and hence better extracted in water than 80% methanol.
The diuretic activity of the extracts of Ajuga remota at their higher respective doses was a moderate type for the aqueous and mild for the hydroalcoholic extract, since their values were 1.01 and 0.98 for AA1000 and AM750, respectively. Diuretic activity is considered to be good if it is more than 1.50, moderate if it is 1.00-1.50, little if it is between 0.72-1.00 and nil if it less than 0.72 .
The effect of the extracts on water excretion was accompanied by urinary electrolyte excretion effect, since there appeared to be an increased salt excretion as compared to the control group, which supports the idea that the diuretic effect of Ajuga remota was of the saluretic type in contrast to aquaretic type, which is a typical feature of most phytodiuretic agents . The larger doses of both extracts did have an interesting natriuretic effect and thus could have a beneficial effect in different edematous conditions. The ratio Na+/K+ was calculated as indicator of natriuretic activity and resulted in values of 2.66, 1.90 and 1.1 for AA1000, AM750, and F10 respectively. This indicates that the extracts increase sodium excretion more than potassium, which is considered as a very good safety profile of diuretic agents, as hypokalemia is one of the potential adverse effects of synthetic diuretics, such as furosemide.
In contrast to previous studies of aqueous and methanol extracts of some plants which showed an interesting K+-saving effect at low and intermediate doses , in the present study this effect was observed at the higher doses. It is probable that at low dosages of the aqueous and methanol extracts of Ajuga remota, the substances responsible for K+- sparing effect might not reach sufficient concentration to exert the effect. On the other hand, aqueous and 80% methanol extracts of some plants such as Rumex abyssinicus J. have been demonstrated to be devoid of K+- sparing effect . These observations point to the fact that there are at least two different mechanisms by which diuersis could be achieved, one of which produces notable diuresis with a sparing of potassium and another with very strong diuresis in which there is a clear tendency to lose the K+ - conservative effect .
Moreover, onset of the diuretic action of AA1000 was sufficiently rapid and had a fairly long duration of action as it produced significant effect from the first hour (p < 0.01) to the fifth hour (p < 0.01) of the experiment. This is an appealing diuretic profile, as it would curtail the frequency of administration. This coupled to the little or no risk of hypokalemia makes the extract a potential herb worth investigating for treatment of edematous conditions.
It is possible that Ajuga remota extracts exerted diuretic effect by inhibiting tubular reabsorption of water and electrolytes, since such action has been suggested for some other plants. The possibility of direct action of potassium content of the plant extract on diuretic effect could be excluded, since the K+ content of the extract was very low in comparison with the salt concentration obtained from other plants .
Loop diuretics like furosemide increase urinary flow rate and urinary excretion of sodium, potassium and chloride by inhibiting Na+–K+–2Cl− symporter in the thick ascending loop and by inhibiting carbonic anhydrase enzyme. The larger doses of aqueous and 80% methanol extracts of Ajuga remota used in the present study produced similar Na+ and Cl− excretion profile to that of furosemide. However, there is a difference between the extracts and the standard when K+ excretion is considered. This could possibly suggest that the mechanism by which the extract produces diuresis is not exactly the same to that of loop diuretics. It is also possible to exclude the thiazide-like type of mechanism either, as the extracts, depending on dose, relatively increased the urinary K+ level more and alter the urinary Na+/K+ ratio.
The Cl−/ Na++K+ ratio was calculated and shows the extent of carbonic anhydrase inhibitory effect. Carbonic anhydrase inhibition can be excluded at ratios between 0.8 and 1.0 and with decreasing ratios; slight to strong inhibition can be assumed . The Cl−/ Na++K+ ratio was calculated for both extracts and the intermediate doses, AA500 and AM500, showed the strongest inhibitory effect with values of 0.38 and 0.29, respectively. Thus, it appears likely that the strongest carbonic anhydrase inhibition effect at these middle doses might have contributed to the highest K+ loss compared to the other doses. Hence, it is plausible to assume that one of the possible mechanisms of action of these extracts could be carbonic anhydrase inhibition.
In determination of urinary pH, the extracts showed a relative increase in pH values as compared to controls, reinforcing the notion that carbonic anhydrase inhibition as one of the possible mechanism of action of the plant. In addition, the reduction of potassium excretion at the maximum doses of the extracts along with the resulted alkalinization of urine might give a clue on the possibility of the plant acting as a modest potassium-saving diuretic.
The exact nature of the active principle/s responsible for the diuretic effects of the hydroalcoholic and aqueous extracts of the plant is/are, so far, not known. However, preliminary phytochemical analysis carried out with both extracts revealed the presence of bioactive molecules, including flavonoids and steroids. Previous studies have demonstrated that there are several compounds which could be responsible for the plants diuretic effects such as flavonoids, saponins or organic acids . The effect may be produced by stimulating regional blood flow or initial vasodilatation, or by producing inhibition of tubular reabsorption of water and anions, with the result in both cases being diuresis .