Journal of Ethnopharmacology 75 (2001) 185 190 www.elsevier.

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Diuretic effects of selected Thai indigenous medicinal plants in rats

Bungorn Sripanidkulchai a,*, Varima Wongpanich b, Pisamai Laupattarakasem a, Jamsai Suwansaksri c,1, Dusit Jirakulsomchok a
a Faculty of Medicine, Khon Kaen Uni6ersity, Khon Kaen 40002, Thailand Faculty of Pharmaceutical Sciences, Khon Kaen Uni6ersity, Khon Kaen 40002, Thailand c Faculty of Associated Medical Sciences, Khon Kaen Uni6ersity, Khon Kaen 40002, Thailand b

Received 26 April 2000; received in revised form 18 September 2000; accepted 25 December 2000

Abstract Extracts of ve indigenous Thai medicinal having ethnomedical application in the treatment of dysuria were investigated for their diuretic activity. Root extracts of Ananas comosus and Carica papaya, given orally to rats at a dose of 10 mg/kg, demonstrated signicantly increased urine output (P B 0.01) which was 79 and 74%, respectively, of the effect of an equivalent dose of hydrochlorothiazide. Both plant extracts gave similar proles of urinary electrolyte excretion to that of the hydrochlorothiazide. The analyses of the urinary osmolality and electrolyte excretion per unit time suggest the observed effect of A. comosus was intrinsic, whereas that of C. papaya may have resulted from a high salt content of this extract. However, our experimental evidence on the diuretic activities of the other three plants did not parallel their local utilization for dysuria. It was found that the rhizome of Imperata cylindrica apparently inhibited the urination of rats whereas the rhizome of Cyperus rotundus and the stem of A6errhoa carambola failed to demonstrate any diuretic activities. These results indicate that two of the plants investigated exert their action by causing diuresis. The other three plants need further investigation to determine their effectiveness in the treatment of dysuria. 2001 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Herbal medicine; Medicinal plant; Diuretic activity; Diuresis; Dysuria

1. Introduction Sripanidkulchai et al. (2000) listed 13 plants employed by local practitioners in the district of Pon for the treatment of dysuria. This symptom is common to the Northeast of Thailand, and most of the cases are related to the medical ndings of renal stone. Chemotherapy is mainly symptomatic, and based on the use of diuretics, as well as antibacterial and/or anti-inammatory drugs. Certain plants with an ethnomedical reputation of diuresis, such as Tribulus terrestris and Pluchea indica, were previously reported to increase urine output in rats (Nilveses et al., 1988,
* Corresponding author. Tel./fax: +66-43-348386. E-mail address: bungorn@kkul.kku.ac.th (B. Sripanidkulchai). 1 Present Address: Faculty of Associated Medical Sciences, Chulalongkorn University, Bangkok 10330, Thailand.

1989). However, there was no experimental record on the diuretic activity of the 13 plants listed above. This present study aimed to evaluate the diuretic activity of the aqueous extracts of the top ve listed plants in order to relate the efcacy of those plants with their ethnomedical uses.

2. Materials and methods

2.1. Plant materials and preparation of the extracts

The top ve plants used as traditional medicines for the treatment of dysuria in Khon Kaen province were investigated for their diuretic activity in this study (Table 1). The plants were collected by local traditional practitioners and were veried for their botanical identities. Voucher specimens were deposited in the herbar-

0378-8741/01/$ - see front matter 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 1 ) 0 0 1 7 3 - 8

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ium of the Faculty of Pharmaceutical Sciences, Khon Kaen University. The collected parts were washed and dried at 5060C until the constant weights were obtained. After pulverizing, each material was passed through a 20-mm seive and the powder kept in an air-tight and light-protected container at 4C. As described by the local traditional practitioners, a decoction was generally made with plant materials by boiling the samples in hot water until the volume was decreased to one-third of the initial volume (Sripanidkulchai et al., 2000). The laboratory method of extraction was performed accordingly, with slight modications. To be more scientic and reliable, the powdered samples were soaked in distilled water at room temperature for 2 h. After boiling, the temperature was subsequently reduced and kept at 60 80C for an hour. The contents were gauze-ltered, and the ltrate was centrifuged at low-speed. The supernatant was collected, lyophilized and stored at 4C. The yields varied from 2.1 to 16.3% of the initial dried weight, as summarized in Table 1. The lyophilized extracts were reconstituted with distilled water prior to pharmacological studies. In this paper, all doses are expressed in terms of dried weight of plant samples used for extraction per body weight of experimental rats (g/kg).

2.3. Chemicals
Hydrochlorothiazide, from the Thai Government Pharmaceutical Organization, was used as a reference diuretic drug. All other chemicals were obtained for Sigma Chemical Company.

2.4. Urine 6olume, osmolality, and urinary electrolyte excretion

The method modied from Kawashima et al. (1985) was used for the determination of diuresis. All doses were given orally. Rats, fasted overnight with free access to drinking water, were given 40 ml/kg of bicarbonate saline solution (containing 110 mM NaCl and 30 mM NaHCO3) 30 min prior to using in the following experiment. The animals were divided into 12 groups, 8 animals/ group. Group 1 (control) was given distilled water. Group 2 was given hydrochlorothiazide, 10 mg/kg dissolved in distilled water. Groups 312 (test groups) were given plant extracts at doses equivalent to 5 and 10 g, of plant dried weight before extraction, per kg body weight of the animals. Each animal was then placed in an individual metabolic cage and its urine was collected and the volume measured every hour for 4 h. Urine osmolality was assessed by the freezing point depression method (Osmette, Precision System, Inc.). The content of urinary electrolytes, which included sodium (Na+), potassium (K+) and chloride (Cl), was determined by using ion selective electrodes (Electrolyte Analyzer system E4A, Beckman).

2.2. Animals
Adult male Sprague Dawley rats with a weigh range of 140 150 g were purchased from Mahidol University Animal Center at Salaya, Thailand. The animals were then housed in cages of ve, at 25C in the animal unit, Faculty of Medicine, KKU for a minimum of 3 days prior to pharmacological studies, with free access to pellet diet and water. The conditions were maintained on a 12 h light:12 h dark cycle, with an ambient temperature of 25C. Prior to the start of the experiment all animals were fasted overnight with water, which was available ad libitum.

2.5. Data and statistical analysis

Data are expressed as mean9standard error of mean (S.E.M.). Statistical comparisons within the same group were performed with Students t-test for paired observation. Differences between groups were evaluated, at PB0.01, by using the Students unpaired t-test.

Table 1 Plant names, part used, voucher specimen number, and their yields of extraction Plant name Family Part used Voucher Yield (% w/w) specimen number
a b

A. comosus (Linn.) Merr. (Pineapple, Nana of the Tupi Indians) C. rotundus Linn. (Nutgrass, Coco grass) C. papaya Linn. (Papaya, Pawpaw, Melon tree) I. cylindrica (Linn.) Pal. (Cogon grass, Cotton grass, Lalang, Kunai) A. carambola Linn. (Starfruit, Carambola, Caramba)
a b

Bromeliaceae Cyperaceae Caricaceae Gramineae Averrhoaceae

Root Rhizome Root Rhizome Stem

BS BS BS BS BS

32-016-03 32-021-01 32-019-01 32-010-03 32-016-04

56 23 17 60 23

16.3 10.1 10.2 2.1 13.4

Percentage of dried plants compared with fresh weights. Percentage of dried extracts after lyophilized compared with dried plant weights.

B. Sripanidkulchai et al. / Journal of Ethnopharmacology 75 (2001) 185190 Table 2 Effects of Thai medicinal plants on urine volume in rats Treatment Distilled water (control) Hydrochlorothiazide A. comosus C. rotundus C. papaya I. cylindrica A. carambola Dosea 10 5 10 5 10 5 10 5 10 5 10 Urine volumeb (4 h in ml/kg body weight) 26.3 91.62 43.09 92.13 32.42 92.66d 33.91 91.90d 28.02 92.29 26.43 90.85 28.31 93.46 31.95 91.40d 14.95 92.41d 15.58 9 2.48d 21.35 92.25 27.74 92.23 Diuretic actionc 1.00 1.64 1.23 1.29 1.07 1.00 1.08 1.21 0.57 0.59 0.81 1.05

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mg/kg of hydrochlorothiazide and g of dried plant before extracted per kg. Each value represents the mean 9S.E.M. of eight rats. c Diuretic action =(urinary excretion of treated group (4 h))/(urinary excretion of control group (4 h)). d PB0.01, statistically signicant relative to control.
b

3. Results

3.2. Urinary osmolality

The urinary osmolatily of the control group appeared to be constant, regardless of time, whereas the hydrochlorothiazide-treated group had a signicant and a time-dependent increase of the urinary osmolality, with a maximum increment during the second hour (Table 3). Animals treated with all ve plant extracts showed higher urinary osmolality than that of the control group. With an exception of C. papaya at a dose of 10 g/kg, p.o. and A. carambola at both 5 and 10 g/kg, p.o, the rest of the extracts caused a maximum urinary osmolality during the second hour. At 10 g/kg, p.o, C. papaya rendered the highest urinary osmolality during the rst hour. However, the highest urinary osmolality of A. carambola was observed at 3 h after treatment. These proles related to the proles of the excreted urine volume.

3.1. Urine 6olume

As shown in Table 2, Ananas comosus and Carica papaya were the only two plant extracts that exhibited diuretic activities. Both extracts increased a 4-h urine volume when administered at both 5 and 10 g/kg, p.o. However, the diuretic actions of these two extracts were less potent than that of hydrochlorothiazide. At 10 g/kg, A. comosus and C. papaya increased the urine output volume approximately 79 and 74% of the hydrochlorothiazide action, respectively. The extracts of Cyperus rotundus and A6errhoa carambola did not change the urine volume, whereas the extract of Imperata cylindrica decreased the urine volume at both 5 and 10 g/kg, p.o. The urinary excretion was compared at the end of each hour during the 4-h period of studies (Table 3). It was observed that the control rats urinated more during the rst and the second hours. The extracts from A. comosus (5 and 10 g/kg, p.o.) and C. papaya (5 g/kg, p.o.) provided maximum excreted volume during the second hour, which was a similar prole as that of hydrochlorothiazide. In contrast, a dose of 10 g/kg, p.o. of C. papaya gave maximum excretion during the rst hour. I. cylindrica, at both 5 and 10 g/kg, p.o., inhibited urination of rats throughout the period of studies, while a maximum inhibition was observed during the rst hour. The other two plant extracts did not increase the 4-h urine volume, but their patterns of urinary excretion in rats were interestingly different from that of the control. C. rotundus increased the output of urine during the second hour, whereas A. carambola delayed the urinary excretion and resulted in less excretion during the rst hour but more excretion during the third hour.

3.3. Urinary electrolytes

The amount of urinary electrolytes (Na+, Cl and K ) were measured every hour from the collected urine, as shown in Table 4. In general, the urine content patterns of the three electrolytes of the control animals were similar. Hydrochlorothiazide caused about two times more urinary Na+ and Cl excretion, during every hour, than those of the control. A slight increase of the urinary K+ excretion was also noted with the hydrochlorothiazide-treated group at the second hour. Among the ve extracts tested, a signicant higher amount of the urinary Na+ excretion was observed only in the animals treated with C. papaya and A. carambola. Carica papaya, at 5 and 10 g/kg, p.o. assumed the highest urinary Na+ excretion during the second and the rst hours, respectively. This amount
+

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Table 3 Urine volume and osmolarity after the treatment with the plant extracts over a period of 4 h Treatment Dosea Urine volumeb (ml/100 g body weight) hr1 Control Hydrochlorothiazide A. comosus C. rotundus C. papaya I. cylindrica A. carambola 10 5 10 5 10 5 10 5 10 5 10 0.969 0.41 0.58 9 0.13 0.759 0.20 0.839 0.17 0.789 0.16 0.629 0.12 0.649 0.15 1.339 0.13 0.159 0.10 0.339 0.14 0.359 0.14 0.659 0.13 hr2 0.83 9 0.16 1.62 9 0.21 1.09 9 0.11 1.23 9 0.17 1.23 9 0.10 0.80 9 0.16 1.22 9 0.15 0.95 9 0.18 0.66 9 0.15 0.33 9 0.18 0.65 9 0.20 0.75 9 0.19 hr3 0.43 90.10 0.83 9 0.19 0.92 90.11 0.68 9 0.13 0.40 90.10 0.71 90.12 0.60 9 0.10 0.67 90.14 0.36 90.11 0.39 90.14 0.85 90.09 1.05 90.13 hr4 0.41 90.10 0.77 90.16 0.48 90.11 0.66 90.11 0.40 90.14 0.52 90.06 0.38 90.12 0.26 90.11 0.32 90.13 0.51 90.13 0.29 9 0.08 0.33 9 0.10 Osmolalityb (mosm) hr1 168 930 325 972 177 948 200 929 190 956 217 914 431 933 594 936 196 925 227 9 58 383 9 73 413 9 57 hr2 211 937 765 990 378 9 46 376 9 35 430 9 67 269 9 23 670 9 82 392 9 77 291 952 366 9102 544 968 492 931 hr3 211 9 38 467 938 380 9 43 260 9 39 270 9 50 311 9 48 359 944 337 969 262 9 31 311 956 619 993 526 960 hr4 286 9 71 351 9 60 260 940 260 9 37 310 9 60 228 9 10 359 938 247 936 327 9 57 359 9 60 263 971 309 9 38

a b

mg/kg of hydrochlorothiazide and g of dried plant before extracted per kg. Each value represents the mean 9S.E.M. of eight rats.

appeared as the highest in the urine of both groups treated with A. carambola during the second and third hours. The extracts from A. comosus, C. rotundus, and I. cylindrica produced the reduced amount of urine sodium, compared with the control. The amount of urine potassium measured in animals treated with A. comosus, C. rotundus, and I. Cylindrica, were slightly higher than that of the control. Throughout the rst 3 h, A. carambola raised the urinary excretion of potassium, regarding the control and the hydrochlorothiazide-treated groups. Among these ve extracts, C. papaya resulted in the highest urinary K+ excretion. The maximum amounts were detected during the second and the rst hours at doses of 5 and 10 g/kg, p.o., respectively. The urinary Cl excretion proles of these extracts were similar to those of the sodium. With respect to the control, extracts from C. papaya and A. carambola were the only two that increased the urinary excretion of chloride. However, the urine chloride and sodium raised by these two extracts was less than that caused by the hydrochlorothiazide.

4. Discussion Results from the present study indicate diuretic activity in the aqueous extracts from the roots of A. comosus and C. papaya. Both plant extracts gave similar proles of urinary electrolyte excretion to that of the hydrochlorothiazide, regardless of having a lesser degree of potency. The extracts from the rhizomes of C. rotundus and the stem of A. carambola did not exhibit diuresis, but the extract from I. cylindrica rhizomes decreased the urine volume under the conditions of our study. The ndings of an antidiuretic effect of I. cylindrica is in agreement with the previous studies by Kanchanapee (1966, 1967). According to those studies, the antidiuretic activity of I. cylindrica was reported for rats but not for rabbits. However, these ve medicinal plants were selected from the top of the ethnomedical list for dysuria, as we aimed to evaluate the relevance and correlation of plant pharmacological activities with the practice of traditional herbal medicines. The plant extracts were singly prepared and tested for diuretic activity, not in combination as traditionally applied. However, our procedure for extract preparation emulated the method described by the traditional practitioners. These factors might have contributed to the fact that the experimental ndings did not totally agree with the ethnomedical information for those plants. Nevertheless, the results paralleled the utilization of A. comosus and C. papaya for dysuria, and also indicate that a more critical review of I. cylindrica is required before continuing to prescribe as a diuretic. For C. rotundus and A. carambola, more studies are also needed, especially on other pharmacological activities related to the treatment of dysuria, such as anti-inammation and antibacterial activity.

3.4. The amount of Na+, K+, and Cl in plant extracts

Since aqueous extraction was employed in this study, water soluble salts could be present in the extracts and subsequently interfere with the urinary excretion. The content of sodium, potassium, and chloride in the extracts was, therefore, determined and the results are shown in Table 5. The total content of salts were in the order of C. papaya\C. rotundus \I. cylindrica \A. comosus \A. carambola. C. rotundus contained the highest Na+ content, whereas C. papaya had the highest content of potassium and chloride. However, the order of these electrolyte contents was not correlated with the diuretic activities of the plant extracts.

Table 4 Total excretion over a period of 4 h of urinary electrolytes Cl (mol)b hr2 hr3 hr4 hr1 hr2 hr3 hr4 hr1 K+ (mol)b hr2 hr3 hr4

Treatment

Dosea

Na+ (mol)b

hr1

Control Hydrochlorothiazide A. comosus

C. rotundus

C. papaya

I. cylindrica

A. carambola

10 5 10 5 10 5 10 5 10 5 10

1079 42 1279 33 279 8 489 10 469 13 459 5 1299 14 1699 14 389 13 249 4 1289 21 1289 22

1249 34 352 941 739 9.5 93 98.8 115914 649 7.6 2309 41 91 919 429 8 68 923 184 9 15 169 9 16

102 927 209 9 18 91 911 72 9 13 70 9 9 78 99 105 916 66 9 15 48 97.6 48 9 21 187 923 174 9 19

92 924 173 929 74 913 79 911 87 9 15 699 6 132 9 17 61 98 82 916 72 919 65 96.5 106 911

79 92.4 106 9 27 43 96.1 46 9 11 34 9 11 3992.6 130 913 216 915 25 95.7 27 95.5 105 918 109 9 19

91 9 16 325 935 84 9 10 100 98 111 912 60 95.5 247 933 166 9 31 46 910 83 923 171 913 156 911

69 9 16 187 9 16 77 911 66 911 55 99 62 99 134 912 127 925 51 97.6 57 9 19 167 924 146 9 23

66 911 131 9 22 45 9 6.8 54 98.5 63 9 12 33 9 5.7 1099 17 71 918 55 9 10 75 9 14 69 9 11 77 9 9

33 9 6.7 19 9 3 33 9 6.5 28 9 4.5 22 95.3 33 9 3.8 93 9 10.6 190 9 9.2 27 91.4 32 9 6 39 919 54 9 8.9

33 93.8 61 9 7.4 52 9 5 45 9 2.5 57 9 7 36 9 3.8 124 9 13 126 925 42 9 6.8 53 914 68 9 15 57 93.3

229 5.7 32 9 3.3 41 95 29 9 4.5 259 5.7 36 9 7.4 61 9 6.8 109 9 20 339 3.1 45 9 8.5 769 20 53 9 8.5

28 9 10 17 92.3 219 3.4 39 9 15 25 95.4 14 9 2.1 45 9 9.4 59 920 319 3 52 910 659 6.5 106 9 11

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mg/kg of hydrochlorothiazide and g of dried plant before extracted per kg. Each value represents the mean 9S.E.M. of eight rats.

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Table 5 The content of electrolytes in plant extractsa Plant extract A. comosus C. rotundus C. papaya I. cylindrica A. carambola Na+ 141 1156 275 342 165 K+ 260 385 2798 816 435 Cl 471 1175 1660 638 136 Total 872 2716 4832 1796 736

lowest of the ve plants studied. More studies are needed to clarify the efcacy of this plant.

Acknowledgements This work was nancially supported by the Canadian International Development Agency (CIDA) and the Research and Development Institute (RDI), Khon Kaen University. The authors would like to thank Deans, Faculty of Medicine, Faculty of Pharmaceutical Sciences, and Faculty Associated Medical Sciences for the generous provision of research instruments and facilities. Our sincere thanks are due to Professor Dr R.M.E. Richards, Professor Dr Jit Sitteeamorn and Associate Professor Dr Nantawan Bunyapapatsara for their critical suggestions and encouragement.

a Values expressed in terms of mmol for extracts equivalent to a dose of 10 g/kg body weight of dried plant.

It was reported by Nilveses et al. (1989) that an increment of the urine output in rats might result from a high potassium content in the plant infusion. An analysis of the urinary osmolality and electrolyte excretion per unit time, together with the plant salt contents, may help to differentiate the mechanisms by which these plants act as diuretics. Our results indicated that the existing diuretic activity of A. comosus root extract was intrinsic, and not a result of the salt loading effect. In fact, this was not the case because its salt content was the second lowest of the ve extracts studied (see Table 5). The diuretic activity of C. papaya may have resulted from the high salt content of its extract. However, this activity appeared to correlate well with the maximum volume, the highest osmolality, and the amount of electrolytes excreted during the rst hour of urine collection, at a dose of 10 g/kg, p.o. In contrast to other extracts, we also observed an interesting effect with the stem extract of A. carambola. This delayed urine excretion and resulted in the highest urine volume and the maximum excretion of potassium during the third hour of urine collection. Furthermore, the observed diuretic effect of this plant was unlikely to be associated with its salt content, because this was the

References
Kanchanapee, P., 1966. Phytochemical and pharmacological studies of I. cylindrica beauv. rhizomes. Bulletin of the Department of Medical Science 8, 182 184. Kanchanapee, P., 1967. Medicinal plants in Thailand (II). Occurrence of arundoin and cylindrin in the rhizomes of I. cylindrica and the examination of the diuretic action of the extracts. Shoyakugaka Zasshi 21, 65 67. Kawashima, K., Miwa, Y., Kimura, M., 1985. Diuretic action of paeonol. Planta Medica 50, 187 189. Nilveses, N., Wammanachinda, W., Wanverakul, B., 1988. Pharmacological studies of extract from Tribulus terrestris L. Bulletin of the Department of Medical Science 30, 171 183. Nilveses, N., Wammanachinda, W., Wanverakul, B., Pidech, P., 1989. Diuretic effect of Pluchea indica. Thai Journal of Pharmacology 11, 1 7. Sripanidkulchai, B., Na-nakorn, S., Wongpanich, V., Tanyakupta, P., 2000. A behavioral investigation on utilization of medicinal plants for dysuria at Pon District, Khon Kaen. Khon Kaen University Research Journal 5 (1), 4 10.