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Doi:10.1016/j.jep.2007.07.005Journal of Ethnopharmacology 113 (2007) 487–491 Diuretic activity of Withania aristata: An endemic Canary Island species D. Mart´ın-Herrera , S. Abdala , D. Benjumea , P. P´erez-Paz a Unidad de Farmacolog´ıa y Farmacognosia, Facultad de Farmacia, Universidad de La Laguna, 38207 La Laguna, Islas Canarias, Spain b Departamento de Biolog´ıa Vegetal, Universidad de La Laguna, Tenerife, Islas Canarias, Spain Received 28 February 2007; received in revised form 6 June 2007; accepted 3 July 2007 Abstract
This study reports on the pharmacological evaluation of the diuretic activity of an infusion and a methanol extract of Withania aristata Ait. in laboratory rats. Water excretion rate, pH, density, conductivity, and content of Na+, K+ and Cl− were measured in the urine of rats subjected tohypersaline conditions. Both the infusion and the methanol extract showed a significant diuretic effect compared with non-treated controls, withnotable increases in the rates of water and sodium excretion. There was also a potassium retention effect. The diuretic effect did not appear to berelated to the potassium content in the material tested, but did have some relation to its content of active polar compounds. The results justify theuse of Withania aristata as a diuretic agent in folk medicine of the Canary Islands.
2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: Withania aristata; Diuretic activity; Medicinal plant; Urinary excretion; Rats; Folk medicine 1. Introduction
the fruit strongly stimulates urine production, making it usefulagainst hydropesia ( Withania aristata Ait. (Solanaceae) is an endemic species of the Canary Islands. It is popularly known as “orobal” or Partial studies on the chemical composition of Withania “s´aquido” (The plant grows wild on many aristata have isolated withanolides – types of steroid lactones of the central islands, and is common in ravines and at the bases – including withaferine A and withanolide D, among other of mountains, in soils which are somewhat nitrified and humid.
It is common in the thickets of thermophilic forests found were phytosterols, oleoresins and withaminol ( Other Withania species from other parts This species forms a bush with variegated foliage which can of the world such as Withania somnifera and Withania coag- be tree-like in size, although not reaching over 4 m in height. Its ulans have been submitted to numerous chemical studies, also bark is coarse and grey in color, and its fragile branches form a encountering withanolides which are compounds characteristic dense mass. The plant bears greenish flowers which arise from of the Solanaceae and in particular the genus Withania the axillae of the leaves on peduncles. The fruit is globose and These products have demonstrated interesting orange-colored (initially green), fleshy, and enveloped in a thin, properties such as anti-inflammatory, immunosuppressive, anti- stress, anxiolytic, cicatrizant, fungicidal and trypanosomacidal This species has wide use in folk medicine practice on the islands due to the wide variety of medicinal properties attributed to it; including its use as a scarring agent, antispasmodic, for rheumatic problems, eye problems and otitis, as well as for Until the present, however, no formal studies had insomnia, constipation, and urinary pathologies. Ingestion of been made on the biological activities and medicinal propertiesof Withania aristata.
The present study, using laboratory mice and rats, is thus the first formal attempt to demonstrate the diuretic efficacy of hot Corresponding author. Tel.: +34 922318494; fax: +34 922318514.
water infusion and methanol extract of the plant.
0378-8741/$ – see front matter 2007 Elsevier Ireland Ltd. All rights reserved.
doi: D. Mart´ın-Herrera et al. / Journal of Ethnopharmacology 113 (2007) 487–491 2. Materials and methods
before the start of the experiment. The mice were observed forsymptoms of toxicity for 15 days in terms of weight loss, and autonomic and neurobehavioral alterations. On the 15th day, theanimals were sacrificed and their vital organs were individually Withania aristata was harvested from the Santa Cruz Coast in a place called Taganana in Tenerife, Canary Islands (Spain)at 75 m altitude above sea level, in March 2003, and labeled Exp. NE. UTM E381093-N3160004. Voucher specimens weredeposited in the La Laguna University Herbarium (TFC Diuretic activity was determined following the methods of with minor modifications. Male rats weredivided into seven groups of eight animals each, in laboratory cages. They were fed laboratory diet ad libitum and allowedfree access to drinking water. They were exposed to a 12/12 h The leaves of flowering and immature fruiting Withania aris- light–dark cycle at 22 ◦C. Eighteen hours before testing, the ani- tata were air-dried in an oven at 40 ◦C for 4 days and then the mals were fasted overnight, with free access to tap water only.
dry plant was cut and ground to a powder mechanical milling.
Then all animals were given an oral loading of normal saline Then three aqueous extracts at 5, 10 and 15% from the (5% bw). Subsequently, three groups of rats were orally admin- dried powdered plant material were prepared by mean traditional istered 5 ml/kg bw of the 5, 10 and 15% infusions of Withania method applied in Canaries. Amounts of 5, 10 and 15 g, respec- aristata, two groups of rats were orally administered 5 ml/kg bw tively of pulverized plant material were each placed in 100 ml of the methanol extract at doses 100 and 200 mg/kg of weight, distilled boiling water and left at room temperature 15 min to respectively, and other two groups of rats were orally admin- infuse, and then were filtered. Five millilitres per kilogram body istered 5 ml/kg bw p.o. of HCTZ at doses 10 and 25 mg/kg, weight (bw) of each infusion was then given orally to indi- respectively. Control rats received the same amount of deionised vidual rats (equivalent to doses of 0.25, 0.50 and 0.75 g/kg).
water (5 ml/kg bw). Immediately after administration, the rats The infusions were freshly prepared just prior to administration.
were paired and placed in metabolism cages. Urine was col- In a second test procedure, the dried powdered plant material lected in a graduated cylinder and its volume was recorded was submitted to a continuous extraction in a soxhlet extrac- at 2 h intervals for 8 h. Cumulative urine excretion was calcu- tor for 5 days using 100% methanol as a solvent. The solvent lated in relation to body weight and expressed as ml/100 g bw.
was then eliminated by vacuum distillation in a rotary vac- Electrolyte (Na+, K+, Cl−) concentrations, pH, density and con- uum evaporator (Buchler Corp.), representing a yield of 10.39% ductivity were estimated from a pooled urine sample of each of the dry material extracted. The methanol residue obtained pair of rats at the end of the experiment (8 h) and expressed as was dissolved in distilled water just before administration, and administered at doses of 100 and 200 mg/kg bw in a volume of5 ml/kg bw.
Na+ and K+ concentrations were measured using a Jen- way Corp. model PFP7 flame photometer. The instrument was Male albino Sprague-Dawley rats (180–210 g) and male and calibrated with standard solutions containing different concen- female albino Swiss mice (20–24 g) obtained from the Cen- trations of Na+ and K+. Cl− concentrations were determined tral Animal House, University of La Laguna, were used for by direct potentiometry, using an ion-selective chloride elec- the experiments, according with the guidelines of the European trode (Orion 9417B) and an Ag/AgCl reference electrode with a double junction (Orion 90-02). The potentials were measuredwith an Orion Ionalyzer 901. KNO3 2 M was used as a standard in all the determinations; pH and conductivity were directlydetermined on fresh urine samples using a HI-8424 Hanna Hydrochlorothiazide (HCTZ; Sigma Chemical Co.) was used Instruments pH-meter and a LF-320 WTF conductivity meter, respectively. Density estimation was made by weighing with aMettler AE163 (±0.1 mg) analytical balance on urine volume measured with a Nichiryo micropipette.
Groups of 10 mice, 5 male and 5 female weighing 20–24 were used for administration of the infusion and MeOH extractof Withania aristata. The animals had free access to standard Results are expressed as the mean values ± S.E. (standard commercial diet and water ad libitum in a 12/12 h light–dark error of mean) of four pairs of rats. The statistical evaluation cycle at 22 ◦C. The test infusion at 2.5 g/kg bw (0.4 ml/20 g bw) was carried out by analysis of variance. The difference between and MeOH extract at 1 g/kg bw, respectively, were administered the means of treated groups and the non-treated control groups orally by means of a gastric catheter. Food was withdrawn 16 h was evaluated by the Student’s unpaired t-test.
D. Mart´ın-Herrera et al. / Journal of Ethnopharmacology 113 (2007) 487–491 Table 1Effect of oral administration of the infusion and the methanol extract of Withania aristata on urinary volume excretion Withania aristata (MeOH) 100 mg/kg Withania aristata (MeOH) 200 mg/kg The results show the mean values and standard errors; n = number of pairs used in each group. *p < 0.01 and **p < 0.05 compared with the control group (Student’sunpaired t-test).
a Diuretic index = volume problem group/volume control group.
Table 2Effects of oral administration of the infusion and the methanol extract of Withania aristata on urinary electrolyte excretion Withania aristata (MeOH) 100 mg/kg Withania aristata (MeOH) 200 mg/kg The results show the mean values and standard errors; n = number of pairs used in each group. *p < 0.001, **p < 0.01 and ***p < 0.05 compared with the controlgroup (Student’s unpaired t-test).
a Saluretic index = mequiv. problem group/mequiv. control group.
The results in the tables showed that the reference diuretic HCTZ induced excretion values for water of nearly 40%, and between 30 and 50% for the excretion of Na+ and K+, when com-pared with the untreated control group. It should also be noted The different parameters analyzed for the infusion and for the that maximum excretion was observed in animals receiving the methanol extract of Withania aristata in the test animals, as well as the HCTZ and control groups, are included in Excretion followed a dose-dependent relation in tests of the lists the urinary volume results (ml/100 g/8 h) Withania aristata infusions, with values from between 15 and and the electrolyte (Na+, K+, Cl−) content 40% compared with the control group, suggesting that this phy- (mequiv./100 g/8 h) in the urine of animals treated with Withania todiuretic had important effects on the excretion of water. This aristata infusion, methanol extract, HCTZ and control groups.
effect was repeated in rats receiving the methanol extract, where Other parameters related to excretion such as the density, pH, values of 40 and 45% were obtained for the 100 and 200 mg/kg and conductivity of the urine samples are also presented in doses, respectively, and which were comparable to the effects of Table 3Effects of oral administration of the infusion and the methanol extract of Withania aristata on the conductivity, pH, and density of the urine Withania aristata (MeOH) 100 mg/kg Withania aristata (MeOH) 200 mg/kg The results show the mean values and standard errors; n = number of pairs used in each group. *p < 0.05 and **p < 0.01 compared with the control group (Student’sunpaired t-test).
D. Mart´ın-Herrera et al. / Journal of Ethnopharmacology 113 (2007) 487–491 Electrolyte excretion induced by Withania aristata showed less than that of the HCTZ group (0.29 mequiv.); this suggested that both the infusion and the methanol extract gave equivalent an interesting K+-saving effect with the use of Withania aristata.
results for the excretion of water. These results also demon- This effect disappeared when a higher concentration (15%) of strated a dose-dependent relation for the excretion of Na+ and the infusion was employed, causing 0.39 mequiv. of potassium to K+, although the results were lower than those produced by the be excreted, and also exceeding the results obtained with HCTZ.
HCTZ, with the only exception noted when using the 15% infu- Nevertheless, it should be noted that the 15% Withania aristata sion which produced results higher than those obtained from the infusion level was unusually high, and not normally achieved group receiving the HCTZ. Here, it was observed that both the in typical household preparations. In fact, typical preparation of infusion and the methanol extract showed significantly reduced the infusion in Canary Island households is about 2.5–5%.
potassium excretion, both inferior to that induced by the HCTZ Quantitative determinations of the ions present in the Witha- and less than or equal to values in the control group. The nia aristata infusion revealed the presence of very low amounts only exception was the result from the 15% infusion ( of potassium salts, suggesting that diuretic effect does not seem Reduced potassium excretion was noted from the saluretic index to be due to the potassium content in the infusion samples. It is of the infusion and the methanol extract, the values of which for well known that potassium overloading, which occurs when the the experimental groups were between 0.96 and 1.37, with 1.0 kidney tubules are incapable of absorbing it, produces urinary for the control group and 1.42 for the HCTZ groups.
excretion of the osmotic type (Our (unpub.) The conductivity, which is an indirect measure of ion content data has shown that an aqueous solution of KCl (1.06 mmol/l), of the urine, showed a dose-dependent increase in all the treated of similar potassium concentration as that occurring in the 15% groups in comparison with the control group. In all cases the Withania aristata infusion, did not increase diuresis in the test response was less for the Withania aristata treatments than with Regarding to the methanol extract, we should point out that, The pH values were higher in the treated groups than in the in contrast to the infusion in whose water preparation it occurs controls, showing a decrease in effect with increase in the dosage a removal of salts, with the methanol this salts removal does of the Withania aristata materials. There were no statistically not generate. Thus the notable diuretic effect produced by the valid differences in urine density among treated and control methanol extract reaffirmed the concept that the diuretic activity of Withania aristata was not due to its content of potassium salts.
For this reason it is more appropriate to assume the occurrence of a diuretic effect which was not of the osmotic type; this wasevident with the methanol extract and even more probable for Neither the infusions nor the methanol extract used in the tests produced acute toxicity in the mice tested, as evidenced by the We therefore suggest that the diuretic effects of Withania aris- absence of mortality in the animals during the study period. No tata are fundamentally due to the presence of naturally active macroscopic alterations were noted in the viscera of the treated polar compounds, among which we have cited the withano- lides these are the main activecomponents in this species, although until now there has been 4. Discussion and conclusions
no literature found containing conclusive data concerning thediuretic activity in these products.
HCTZ produced its maximum diuretic effect at a dose of There was a significant increase in the conductivity in the 10 mg/kg, in agreement with values given in the literature urine of the rats in both treatment with the Withania infusion and methanol extract in relation to the data from the controls. Since tory effect on both water and ions, typical of saluretic diuretics the conductivity value is an indirect measure of the electrolyte concentration in the urine, the diuretic effect of the Withania Results from the Withania aristata infusion and extract aristata was again concluded to be (as above) saluretic rather showed a clear and significative dose-dependent diuretic effect, than aquaretic, the latter being the typical effect of diuretics with values very similar to those of the HCTZ. These results con- of plant origin. This was to be expected, as it coincides with firmed therefore the popular use as diuretic agent of this species the above-mentioned concept of the lightly saluretic charac- teristic of Withania aristata products, although these results Both the infusion and methanol extract showed an electrolyte are not comparable with the important saluretic effect of the excretion clearly in proportion to the water excretion, in a dose- dependent manner. Thus it seems reasonable to think that the The absence of acute toxicity confirmed the safe nature of diuretic effect of Withania aristata was of the saluretic type, and the ingestion of this plant since doses of up to 10× the typically similar to that produced by HCTZ, in contrast to the aquaretic used dosage in folk medicine failed to elicit any toxic symptoms type typical of most phytodiuretic agents.
On the other hand, it was also noted that, beginning with the In summary, Withania aristata produces a notable effect of initial dose and both for the infusion and the methanol extract, the saluretic type, not due to an osmotic mechanism related to the there was a weak excretion of potassium (0.18 mequiv.), which salts contained within the plant, and with a diuretic profile dif- was lower than in the control group (0.19 mequiv.) and notably ferent from the HCTZ, due to the interesting potassium-saving D. Mart´ın-Herrera et al. / Journal of Ethnopharmacology 113 (2007) 487–491 effect showed at 5 and 10% infusion and 100 and 200 mg/kg Choudhary, M.I., Dur-e-Shahwar, Parveen, Z., Jabbar, A., Ali, I., Atta-Ur- methanol extract. Also, Withania aristata was very safe, and Rahman, 1995. Antifungal steroidal lactones from Withania coagulance.
within the rodent model, failed to exhibit any toxicity.
Darias, V., Bravo, L., Barqu´ın, E., Mart´ın-Herrera, D., Fraile, C., 1986. Contri- The present results provide a quantitative basis explaining the bution to the ethnopharmacological study of the Canary Island. Journal of traditional folk medicine use of Withania aristata as a diuretic agent by the Canary Island population. The fact that the simple Darias, V., Bravo, L., Rabanal, R., S´anchez-Mateo, C., Gonz´alez-Luis, R.M., infusions provided diuretic effects comparable to the more dif- Hern´andez, A.M., 1989. New contribution to the ethnopharmacological ficult to obtain methanol extract, suggested that the traditional study of the Canary Island. Journal of Ethnopharmacology 25, 77–92.
Darias, V., Mart´ın-Herrera, D., Abdala, S., de la Fuente, D., 2001. Plants used folk medicine infusions need not be replaced by more costly in urinary pathologies in the Canary Islands. Pharmaceutical Biology 39, Additionally, this species may be of use in treatment of Ganzera, M., Choudhary, M.I., Khan, I.A., 2003. Quantitative HPLC analysis bacterial urinary infections through the action of “therapeutic of withanolides in Whitania somnifera. Fitoterapia 74, 68–76.
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Habtemariam, S., 1997. Cytotoxicity and immunosuppressive activity of with- In the future more studies are required to further define the anolides from Discopodium penninervium. Planta Medica 63, 15–17.
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