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Diuretic activity of the aqueous extracts of Carum carvi and Tanacetum vulgare in normal rats
Journal of Ethnopharmacology 110 (2007) 458–463
Diuretic activity of the aqueous extracts of Carum carvi and Tanacetum vulgare in normal rats Sanaa Lahlou a , Adil Tahraoui a , Zafar Israili b , Badiaˆa Lyoussi a,∗ a
UFR Physiology-Pharmacology, Laboratory of Animal Physiology, Department of Biology, Faculty of Sciences Dhar El Mehraz, Fez, Morocco b Department of Medicine, Emory University School of Medicine, Atlanta, GA, USA
Received 17 February 2006; received in revised form 10 October 2006; accepted 12 October 2006 Available online 19 October 2006
Abstract In the Moroccan traditional medicine, the ripe fruits of Carum carvi L. (Apiaceae) and the leaves of Tanacetum vulgare L. (Asteraceae/Compositae), two widely available plant materials, are used as diuretics. Since, the diuretic activity of these substances has not been investigated in scientifically controlled studies, the aim of the present study was to evaluate the diuretic potential of aqueous extracts of Carum carvi fruit (caraway) and the leaves of Tanacetum vulgare (tansy) in normal rats after acute and sub-chronic oral administration. Water extracts of Carum carvi and Tanacetum vulgare (100 mg/kg) or the reference drug, furosemide (10 mg/kg) were administrated orally to male Wistar rats and their urine output was quantitated at several intervals of time after the dose. After single doses of the extracts of both caraway seeds and tansy leaves, urine output was significantly increased at all time points, and at 24 h after the dose, the total volume of urine excreted was similar for the plant extracts and furosemide. Both extracts increased urinary levels of Na+ and K+ , to about the same extent, while furosemide increased urinary levels of only Na+ and decreased urinary K+ . Despite changes in urinary excretion of the electrolytes, plasma Na+ and K+ levels were not affected by any of the three substances. In the 8-day sub-chronic study, all three substances induced significant diuresis and natriuresis; only tansy increased urinary potassium excretion. The plant extracts did not appear to have renal toxicity or any other adverse effects during the study period. In conclusion, water extracts of both Carum carvi and Tanacetum vulgare have strong diuretic action confirming their ethnopharmacological use. From the pattern of excretion of water, sodium and potassium, it may be deduced that there are atleast two types of active principals present in these extracts, one having a furosemide-like activity and the other a thiazide-like activity. © 2006 Published by Elsevier Ireland Ltd. Keywords: Carum carvi L.; Tanacetum vulgare L.; Caraway; Tansy; Diuretic activity; Urine output; Aqueous extract; Apiaceae; Asteraceae/Compositae
1. Introduction Plant medicine is commonly used in the traditional treatment of some renal diseases, and many plants are reported to possess significant diuretic activity (Bellakhdar, 1997; Maksimovic et al., 2004; Tanira et al., 1988). The diuretic activity of a number of plants used in ethnomedicine as diuretic agents has been confirmed in experimental animals (Benjumea et al., 2005; Maksimovic et al., 2004). The dried ripe fruits of Carum carvi L. Apiaceae (Umbelliferae) (caraway) are used in folk medicine as a carminative, ∗ Corresponding author at: UFR Physiology-Pharmacology, Universit´ e Sidi Mohamed Ben Abdellah, Facult´e des Sciences Dhar El Mehraz, Morocco. Fax: +212 55733059. E-mail address: [email protected] (B. Lyoussi).
0378-8741/$ – see front matter © 2006 Published by Elsevier Ireland Ltd. doi:10.1016/j.jep.2006.10.005
found to be effective against spasmodic gastrointestinal complaints, flatulence, irritable stomach, indigestion, lack of appetite, and dyspepsia in adults (Holtmann et al., 2003; Madisch et al., 2004; Thompson Coon and Ernst, 2002), and in relieving flatulent colic of infants (Reynolds, 1993). In Moroccan traditional medicine, an aqueous extract of caraway is used as an aperitif, tranquilizer, carminative, diuretic, emenagogue, galactagogue, spasmolytic, gastric stimulant and as an aphrodisiac (Bellakhdar, 1997). The plant extract and the volatile oils from Carum carvi have also been used as an antiulcerogenic agent (Khayyal et al., 2001). Furthermore, experimental studies have shown its antitumor (Zheng et al., 1992), antiproliferative (Nakano et al., 1998), antihyperglycemic (Eddouks et al., 2004), and antimicrobial (Iacobellis et al., 2005) activities. Tanacetum vulgare L. (tansy), an herbaceous plant belonging to the family of Asteraceae/Compositae, is found world-
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wide (Keskitalo et al., 1998; Global Compendium of Weeds home page, http://www.hear.org/gcw/html/index.html, accessed February 2006). In folk medicine, tansy has been used as panacea for diverse health problems, such as an anthelminthic, carminative, antispasmodic, abdominal viscera stimulant, tonic, emenagogue, antidiabetic, diuretic and antihypertensive agent. Aqueous extracts of tansy have been reported to exhibit antiinflammatory (Williams et al., 1999), antioxidant (Bandoniene et al., 2000), antimicrobial (Holopainen and Kauppinen, 1989) and hypoglycemic (Takagi, 2002) activities. It has also been reported to have a healing effect on experimental gastric ulcer (Tournier et al., 1999). Furthermore, it has been shown that tansy extract is used in a treatment of increased bone resorption (Muehlbauer, 2002). Diuretics, either alone or in combination with other drugs, are valuable in the treatment of hypertension, congestive heart failure, ascites, and pulmonary edema (Gupta and Neyses, 2005; Hughes, 2004; Jackson, 1996; Morganti, 2005; O’Brien et al., 2005). Two widely used diuretics, thiazides and the high ceiling loop diuretic, furosemide, have been associated with a number of adverse effects, such as, electrolyte imbalance, metabolic alterations, development of new-onset diabetes, activation of the rennin–angiotensin–neuroendocrine systems, and impairment of sexual function (Gupta and Neyses, 2005; Morganti, 2005). Hence, there is a need for new diuretics with lower potential for adverse effects, such as the plant-based substances which are considered to be relatively safe. Although, both caraway and tansy are well recognised in Moroccan traditional medicine as having a diuretic effect at a dose of 100 mg/kg, no scientific data have been published supporting the claimed ethnomedical use. Therefore, the aim of this study was to evaluate the acute and sub-chronic diuretic, saliuretic, and kaliuretic effects of orally administered aqueous extracts of caraway seeds and tansy leaves in normal rats and thus, to confirm their ethnomedical use. 2. Materials and methods 2.1. Experimental animals Adult male Wistar rats weighing between 150 and 200 g procured from our animal house were housed under standard environmental conditions (25 ± 1 ◦ C, 55 ± 5% humidity and 12 h/12 h light/dark cycle). The animals were allowed free access to tap water and standard laboratory rat food. The care and handling of rats were in accordance with the internationally accepted standard guidelines for use of animals, and the protocol was approved by our institutional committee on animal care following the French Technical Specifications for the Production, Care and Use of the Laboratory Animals.
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Professor Bel Kamel, Facult´e des Siences Techniques (F.S.T., Marrakech, Morocco). Both caraway fruits and tansy leaves were dried at room temperature in the shade, and then ground in an electric grinder to give a coarse powder. Fifty grams of the dried powder of caraway fruits was mixed with 500 mL of water, and the mixture was boiled at 100 ◦ C under reflux for 30 min. The decoction obtained was centrifuged, filtered, frozen at −20 ◦ C, and then lyophilised (FreeZone® Dry 4.5, USA), yielding approximately 13.6% (w/w) of dry extract of Carum carvi L. The same procedure was used to extract tansy leaves (50 g dry powder extracted with 500 mL of water); the yield of the product was approximately 13.5% (w/w) of the dry leaves of Tanacetum vulgare L. For each study, the lyophilized aqueous extract was carefully prepared under the same conditions used throughout the studies (time, temperature and the amount of plant material and water used for extraction under reflux, and lyophilization), and each time the quality of extraction was checked by the yield of the lyophilized material. For assuring stability, the lyophilized material was stored at −20 ◦ C until used. The two lyophilized products (caraway and tansy) were dissolved in water (10 mg/mL) before administration at the stated doses. 2.3. Reference drug Furosemide (Lasilix, Pharma 5, Morocco), a high-ceiling loop diuretic, was used as the reference drug (positive control). It was dissolved in water prior to administration. 2.4. Biochemical methods Blood was collected in capillary tubes containing ethylenediamine tetraacetic acid by retro-orbital puncture under light diethyl ether anesthesia. Plasma was obtained by centrifugation (600 × g at 4 ◦ C), and stored at −20 ◦ C until analyzed. Plasma and urinary levels of sodium and potassium were quantitated by flame spectrophotometry. Concentration of creatinine in plasma and urine was determined by the Jaffe alkaline picrate method. Creatinine clearance, as a measure of renal function, was calculated from plasma and urinary creatinine levels. 2.5. Assessment of diuretic activity: experimental design
2.2. Plant material and preparation of the extracts
The diuretic activity was evaluated as follows: each animal was placed in an individual metabolic cage 24 h prior to commencement of the experiment for adaptation. The animals were divided into four groups of five rats each for the acute (single dose) study and another four groups of five rats each for the sub-chronic (repeated dose) study. Rats were fasted overnight with free access to water, and subjected to the stated treatment as described below. The rats were observed occasionally for apparent toxicity.
The ripe fruits of the common caraway [Carum carvi L. (Asteraceae)] were collected from the Fes region and tansy leaves from the Marrakech region in Morocco. Tansy was identified as Tanacetum vulgare L. (Asteraceae/Compositae) by
2.5.1. Acute diuretic activity Before treatment, all animals received physiological saline (0.9% NaCl) at an oral dose of 5 mL/100 g body weight (BW), to impose a uniform water and salt load (Benjumea et al., 2005).
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The first group received orally distilled water 10 mL/kg BW, and served as the control group. The second and third groups were administered orally 100 mg/kg BW of caraway fruit extract and tansy leaf extract, respectively. The fourth group was treated with an oral dose of 10 mg/kg BW of furosemide. Urine was collected and measured at 1, 2, 4, 6, and 24 h after the dose. Sodium and potassium concentrations were determined in the 24 h urine samples as well as in the plasma of rats. 2.5.2. Chronic diuretic activity Daily oral doses of caraway fruit extract (100 mg/kg BW), tansy leaf extract (100 mg/kg BW), and furosemide (10 mg/kg BW) were administered to three groups of rats for 8 days; the control animals received water (10 mL/kg) every day. For each rat, 24 h urine was collected daily and its volume measured. Urinary sodium and potassium concentrations were measured in each urine specimen. Sodium, potassium and creatinine levels were measured in plasma of rats on Day 8. Urinary creatinine excretion was also determined and its clearance was calculated for Day 8. The rats were observed daily for apparent toxicity. 2.6. Statistical analysis Results are expressed as mean ± S.E.M. Statistical analysis of the data was performed with one-way analysis of variance (ANOVA) (Graph Pad Prism version 3.00, USA) or by Student’s t-test. Significant differences were indicated by P values lower than 0.05. 3. Results 3.1. Diuretic activity of a single dose of the plant extracts and furosemide 3.1.1. Effect on urine volume Treatment with a single dose of the caraway extract increased diuresis (Fig. 1), which became significantly higher than in the control rats at 4 h after the dose (caraway 5.7 ± 0.3 mL versus controls 3 ± 0.5 mL; P < 0.05). Urine output continued to be
stimulated throughout the study period, such that the cumulative urinary excretion was significantly higher at 6 and 24 h after the dose of the caraway extract compared to the controls (caraway 12.8 ± 0.1 mL versus controls 7.7 ± 0.7 mL at 24 h after dose; P < 0.01) (Fig. 1). The effect of a single dose of tansy extract was more rapid and slightly higher than that of caraway extract (Fig. 1). The effect became significantly higher at 1 h after the dose compared to controls (tansy 4.0 ± 0.6 mL versus 0.7 ± 0.1 mL for controls; P < 0.05). The diuretic effect remained significantly higher for the tansy-treated rats as compared to control rats at all time points (Fig. 1), such that the cumulative 24 h urinary excretion for the tansy-treated rats was 14.3 ± 0.4 mL as compared to 7.7 ± 0.7 mL (P < 0.01) for the controls. The effect of a single dose of the reference diuretic, furosemide, was also rapid and higher than that of the plant extracts (Fig. 1), however, the 24 h cumulative urinary excretion was not too different for the three substances (caraway 12.8 ± 0.1 mL, tansy 14.3 ± 0.4 mL, and furosemide 18.5 ± 0.6 mL). 3.1.2. Effect on urinary electrolyte excretion The effect of single doses of furosemide and the aqueous extracts of caraway and tansy on electrolyte (Na+ and K+ ) excretion in the 24 h urine is presented in Table 1. Both plant extracts enhanced the excretion of the electrolytes [Na+ (P < 0.001) and K+ (P < 0.05)], which was greater than produced by furosemide, especially that of K+ . Furosemide actually decreased K+ excretion as compared to the controls, such that the Na+ /K+ excretion ratio (2.51) was higher than for the plant extracts (caraway 1.85, tansy 1.88) (Table 1). 3.1.3. Effect on plasma electrolyte levels There was no effect of any of the three substances, furosemide, caraway and tansy extracts on plasma levels of Na+ and K+ (Table 1). 3.2. Diuretic activity of the plant extracts and furosemide after sub-chronic administration 3.2.1. Effect on urine volume Administration of daily doses of both caraway and tansy extracts produced significant diuresis starting on Day 1 (caraway 9.3 mL ± 1.2; tansy 9 mL ± 0.6; controls 5.4 mL ± 0.7; P < 0.05), which became increasingly significant until Day 6 (caraway 20.2 mL ± 0.4; tansy 23 mL ± 1.6; controls 5.8 mL ± 1.0; P < 0.001), after that urinary output levelled off (Fig. 2). The diuretic effect of furosemide was initially higher than produced by the plant extracts, and then it was similar to that of the plant extracts after Day 4 (Fig. 2).
Fig. 1. Acute diuretic effect: diuretic activities of single oral doses (100 mg/kg BW) of aqueous extracts of Carum carvi L. seeds, Tanacetum vulgare L. leaves, and furosemide (10 mg/kg BW). The volume of excreted urine was measured at 1, 2, 4, 6 and 24 h after the treatment; cumulative values are reported as mean ± S.E.M. for five rats in each group. * P < 0.05; ** P < 0.01; *** P < 0.001 compared with controls using Student’s t-test.
3.2.2. Effect on urinary electrolyte excretion Both plant extracts (caraway and tansy) significantly induced urinary excretion of Na+ (P < 0.01) starting on Day 4, which became more pronounced (P < 0.001) from Day 5 to Day 8. The excretion of K+ increased significantly (P < 0.01) throughout the treatment [with maximum effect (P < 0.001) from Day 4 to
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Table 1 Effect of a single oral dose of the aqueous extracts of caraway and tansy, and furosemide on 24 h urinary electrolyte excretion and plasma Na+ and K+ levels in normal rats Treatmenta
Water, 10 mL/kg BW (control) Furosemide Carum carvi extract Tanacetum vulgare extract
Dose (mg/kg BW)
10 100 100
Urinary electrolyte concentration
Plasma electrolyte level
Na+ (mmol/L)
Na+ (mmol/L)
89.7 127.7 138.7 148.0
± ± ± ±
1.8 1.5** 1.5*** 1.7***
K+ (mmol/L) 62.9 50.8 75.0 78.4
± ± ± ±
1.1 0.9** 2.0* 1.8*
145.7 147.5 142.3 143.3
± ± ± ±
1.2 1.6 1.3 1.6
K+ (mmol/L) 5.3 5.1 5.8 5.2
± ± ± ±
0.4 0.3 0.3 0.5
Values are expressed as mean ± S.E.M. of five rats in each group. * P < 0.05; ** P < 0.01; *** P < 0.001 compared to controls using Student’s t-test. a All rats were pre-treated with an oral dose of 5 mL/100 g BW of normal saline prior to the administration of the test substances.
Day 8], by the tansy extract, but not by the caraway extract or furosemide (Fig. 4). 3.2.3. Effect on plasma electrolyte levels As was the case with the acute dose, there was no effect of sub-chronic administration of any of the three substances, furosemide [Na+ (149.2 mmol/L ± 1.3) and K+ (5.2 mmol/ L ± 0.3)], caraway [Na+ (147.1 mmol/L ± 0.8) and K+ (5.6 mmol/L ± 0.7)] and tansy extracts [Na+ (147.3 mmol/ L ± 0.6) and K+ (6.3 mmol/L ± 0.5)] on plasma levels of Na+ and K+ . 3.2.4. Effect on creatinine clearance Creatinine clearance, measured on the last day of treatment, was not affected by any of the treatments (caraway 2.97 mL/min ± 0.11; tansy 3.37 mL/min ± 0.18; furosemide 2.48 mL/min ± 0.09; controls 3.13 mL/min ± 0.06). 4. Discussion and conclusions In the present study, the diuretic effect of orally administered aqueous extracts of caraway fruit and tansy leaves was evaluated in normal rats after one dose and after daily dosing for 8 days. The pharmacological response was compared with that produced by furosemide, a widely used diuretic in clin-
Fig. 2. Sub-chronic diuretic effect: diuretic activities of daily oral doses (100 mg/kg BW) of aqueous extracts of Carum carvi L. seeds, Tanacetum vulgare L. leaves, and furosemide (10 mg/kg BW) administered for 8 days. The volume of 24 h urine was measured on Days 1 through 8 of treatment and reported as mean ± S.E.M. for five rats in each group. * P < 0.05; ** P < 0.01; *** P < 0.001 compared with controls using Student’s t-test.
ical practice. The two plant materials selected for the study, caraway fruits and tansy leaves, are commonly available and used in Moroccan traditional medicine to induce diuresis. Oral route was chosen because that is the way people use these plants in traditional medicine. The effect on electrolyte balance and creatinine clearance was also determined. The mechanism of action by which diuresis was induced by these two extracts was also assessed by comparing the effect with that of furosemide, a high-ceiling loop diuretic (Jackson, 1996), and that of hydrochlorothiazide (from published reports; Benjumea et al., 2005). Diuresis has two components: increase in urine volume (water excretion) and a net loss of solutes (i.e. electrolytes) in the urine (Jackson, 1996). These processes result from suppression of renal tubular reabsorption of water and electrolytes into the blood stream. The reference drug, furosemide, increases urine output and urinary excretion of sodium by inhibiting Na+ /K+ /2Cl− symporter (co-transporter system) in the thick ascending limb of the Loop of Henley (Jackson, 1996), while the thiazide diuretics inhibit the Na+ /Cl− symporter (co-transporter system) in the distal convoluted tubule, by competing for the Cl− binding site, and increasing the excretion of Na+ and Cl− (Jackson, 1996). In the saline primed rats, a dose of the tansy extract caused a significant increase in urine output beginning from the first hour while the caraway extract took 4 h after the dose to increase urine output significantly. In comparison, a single dose of furosemide induced a brisk and significant diuresis within 60 min of administration (Fig. 1). The difference in the time of onset of the diuretic action of these substances may be related to the gastrointestinal absorption characteristics of the active principle(s). Stimulation of diuresis by single doses of all three substances continued for atleast 24 h (Fig. 1). Both caraway and tansy extracts induced significant increases in urinary excretion of Na+ (154% and 164% of controls, respectively) and to a lesser extent of K+ (119% and 123% of controls, respectively); furosemide increased Na+ excretion (142% of controls) but not that of K+ : actually there was a small decrease in urinary concentration of K+ . Hydrochlorothiazide has been reported to increase the urinary excretion of both Na+ and K+ by 50–60% over controls after a single oral dose in normal rats (Benjumea et al., 2005). In this respect, the two plant extracts appear to have several active components, with atleast one having a mechanism of natriuresis similar to that of the thiazide
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Fig. 3. Sub-chronic natriuretic activity: effect of daily oral doses (100 mg/kg BW) of aqueous extracts of Carum carvi L. seeds, Tanacetum vulgare L. leaves, and furosemide (10 mg/kg BW) administered for 8 days on urinary excretion of Na+ . The reported Na+ levels (mean ± S.E.M.) are in pooled 24 h urine for each day for each group of five rats each group. * P < 0.05; ** P < 0.01; *** P < 0.001 compared with controls using Student’s t-test.
diuretic, and another with a mechanism of kaliuresis similar to that of furosemide. In the sub-chronic study, all three substances, furosemide and caraway and tansy water extracts, induced significant diuresis from Day 1 (without priming with saline); urine output continued to increase until Day 5 (maximum effect), after that it stayed stable until the last day of the study (Fig. 2). Although, the effect of furosemide was initially greater than that of the plant extracts, the 24 h urine output became similar for the three substances after Day 6, indicating that all the three substances had reached their maximum effect. Tolerance does not seem to develop to the diuresis stimulating activity of the plant extracts. The plant extracts and furosemide did not influence urinary excretion of Na+ for the first 2–3 days of repeated administration in these rats, which were not primed with saline (Fig. 3), probably because of homeostasis mechanisms. After that period, repeated administration of all three substances caused significant increase (by overcoming the homeostasis mechanisms) in saliuresis (Fig. 2). On the other hand, repeated administration of furosemide and caraway extract virtually had no effect on urinary K+ excretion for the entire 8 days. Contrarily, tansy extract caused a significant increase in urinary excretion of K+ beginning on Day 1 and continuing throughout the study period. The results of the present studies suggest that the effect of one or more of the active components of caraway on electrolyte excretion is furosemide-like, while the active substance(s) in tansy have a thiazide-like action. Further, despite increased urinary electrolyte (Na+ and K+ ) excretion (Figs. 3 and 4), there was no effect of the plant extracts and furosemide on plasma electrolyte levels both in the acute (Table 1) and sub-chronic studies, suggesting that the active principle(s) in both caraway and tansy do not act like potassium-sparing diuretics. There was no effect of repeated administration of the plant extracts on renal creatinine clearance, suggesting that the plant extracts have no renal toxicity at the studied dose. In addition, no apparent toxicity was observed in the rats during the 8 days of repeated dosing with the plant extracts. Nevertheless, additional
Fig. 4. Sub-chronic kaliuretic activity: effect of daily oral doses (100 mg/kg BW) of aqueous extracts of Carum carvi L. seeds, Tanacetum vulgare L. leaves, and furosemide (10 mg/kg BW) administered for 8 days on urinary excretion of K+ . The reported K+ levels (mean ± S.E.M.) are in pooled 24 h urine for each day for each group of five rats each group. * P < 0.05; ** P < 0.01; *** P < 0.001 compared with controls using Student’s t-test.
studies have to be carried out to confirm the lack of renal toxicity and to rule out other organ toxicity, especially after chronic administration. A number of compounds have been identified in caraway, including carvone and other monoterpenoids and their glucosides (Zheng et al., 1992), menthanetriols and glucosides (Matsumura et al., 2001), and limonene (Zheng et al., 1992), and in tansy, such as flavonoids (Williams et al., 1999), mono, tri-, and sesqui-terpine lactones, and isoprenoids (Chandler et al., 1982; Umlauf et al., 2004), saponins, polyphenols, and plant sterols (Chandler et al., 1982), parthenolide (Tournier et al., 1999), polysaccharides (Polle et al., 2002), thujones (Dragland et al., 2005), and chrysanthenyl analogs (Umlauf et al., 2004). At present, it is not known which compounds are responsible for the diuretic, natriuretic and kaliuretic activities of caraway and tansy. In conclusion, the present studies support the ethnomedical use of both caraway and tansy for their diuretic effect. The plant extracts do not seem to have renal toxicity in rats at the dose studied. Although, the active components remain unidentified, based on the pattern of excretion of water, sodium and potassium, it appears that that there are atleast two types of active principals present in these extracts, one having a furosemide-like activity and the other a thiazide-like activity. These findings suggest, for the first time, mechanism(s) of diuretic action of caraway and tansy used in traditional medicine by the Moroccan population. References Bandoniene, D., Pukalskas, A., Venskutonis, P.R., Gruzdiene, D., 2000. Preliminary screening antioxidant activity of some plant extracts in rapeseed oil. Food Res. Int. 33, 785–791. Bellakhdar, J., 1997. La Pharmacop´ee Marocaine Traditionnelle, M´edecine Arabe Ancienne et Savoirs Populaires. Edition Ibis Press, p. 150. Benjumea, D., Abdala, S., Hernandez-Luis, F., P´erez-Paz, P., Martin-Herrera, D., 2005. Diuretic activity of Artemisia thuscula, an endemic canary species. J. Ethnopharmacol. 100, 205–209. Chandler, R.F., Hooper, S.N., Hooper, D.L., Jamieson, W.D., Lewis, E., 1982. Herbal remedies of the Maritime Indians: sterols and triterpenes of Tanacetum vulgare L. (tansy). Lipids 17, 102–106.
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Diuretic activity of the aqueous extracts of Carum carvi and Tanacetum vulgare in normal rats Sanaa Lahlou a , Adil Tahraoui a , Zafar Israili b , Badiaˆa Lyoussi a,∗ a
UFR Physiology-Pharmacology, Laboratory of Animal Physiology, Department of Biology, Faculty of Sciences Dhar El Mehraz, Fez, Morocco b Department of Medicine, Emory University School of Medicine, Atlanta, GA, USA
Received 17 February 2006; received in revised form 10 October 2006; accepted 12 October 2006 Available online 19 October 2006
Abstract In the Moroccan traditional medicine, the ripe fruits of Carum carvi L. (Apiaceae) and the leaves of Tanacetum vulgare L. (Asteraceae/Compositae), two widely available plant materials, are used as diuretics. Since, the diuretic activity of these substances has not been investigated in scientifically controlled studies, the aim of the present study was to evaluate the diuretic potential of aqueous extracts of Carum carvi fruit (caraway) and the leaves of Tanacetum vulgare (tansy) in normal rats after acute and sub-chronic oral administration. Water extracts of Carum carvi and Tanacetum vulgare (100 mg/kg) or the reference drug, furosemide (10 mg/kg) were administrated orally to male Wistar rats and their urine output was quantitated at several intervals of time after the dose. After single doses of the extracts of both caraway seeds and tansy leaves, urine output was significantly increased at all time points, and at 24 h after the dose, the total volume of urine excreted was similar for the plant extracts and furosemide. Both extracts increased urinary levels of Na+ and K+ , to about the same extent, while furosemide increased urinary levels of only Na+ and decreased urinary K+ . Despite changes in urinary excretion of the electrolytes, plasma Na+ and K+ levels were not affected by any of the three substances. In the 8-day sub-chronic study, all three substances induced significant diuresis and natriuresis; only tansy increased urinary potassium excretion. The plant extracts did not appear to have renal toxicity or any other adverse effects during the study period. In conclusion, water extracts of both Carum carvi and Tanacetum vulgare have strong diuretic action confirming their ethnopharmacological use. From the pattern of excretion of water, sodium and potassium, it may be deduced that there are atleast two types of active principals present in these extracts, one having a furosemide-like activity and the other a thiazide-like activity. © 2006 Published by Elsevier Ireland Ltd. Keywords: Carum carvi L.; Tanacetum vulgare L.; Caraway; Tansy; Diuretic activity; Urine output; Aqueous extract; Apiaceae; Asteraceae/Compositae
1. Introduction Plant medicine is commonly used in the traditional treatment of some renal diseases, and many plants are reported to possess significant diuretic activity (Bellakhdar, 1997; Maksimovic et al., 2004; Tanira et al., 1988). The diuretic activity of a number of plants used in ethnomedicine as diuretic agents has been confirmed in experimental animals (Benjumea et al., 2005; Maksimovic et al., 2004). The dried ripe fruits of Carum carvi L. Apiaceae (Umbelliferae) (caraway) are used in folk medicine as a carminative, ∗ Corresponding author at: UFR Physiology-Pharmacology, Universit´ e Sidi Mohamed Ben Abdellah, Facult´e des Sciences Dhar El Mehraz, Morocco. Fax: +212 55733059. E-mail address: [email protected] (B. Lyoussi).
0378-8741/$ – see front matter © 2006 Published by Elsevier Ireland Ltd. doi:10.1016/j.jep.2006.10.005
found to be effective against spasmodic gastrointestinal complaints, flatulence, irritable stomach, indigestion, lack of appetite, and dyspepsia in adults (Holtmann et al., 2003; Madisch et al., 2004; Thompson Coon and Ernst, 2002), and in relieving flatulent colic of infants (Reynolds, 1993). In Moroccan traditional medicine, an aqueous extract of caraway is used as an aperitif, tranquilizer, carminative, diuretic, emenagogue, galactagogue, spasmolytic, gastric stimulant and as an aphrodisiac (Bellakhdar, 1997). The plant extract and the volatile oils from Carum carvi have also been used as an antiulcerogenic agent (Khayyal et al., 2001). Furthermore, experimental studies have shown its antitumor (Zheng et al., 1992), antiproliferative (Nakano et al., 1998), antihyperglycemic (Eddouks et al., 2004), and antimicrobial (Iacobellis et al., 2005) activities. Tanacetum vulgare L. (tansy), an herbaceous plant belonging to the family of Asteraceae/Compositae, is found world-
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wide (Keskitalo et al., 1998; Global Compendium of Weeds home page, http://www.hear.org/gcw/html/index.html, accessed February 2006). In folk medicine, tansy has been used as panacea for diverse health problems, such as an anthelminthic, carminative, antispasmodic, abdominal viscera stimulant, tonic, emenagogue, antidiabetic, diuretic and antihypertensive agent. Aqueous extracts of tansy have been reported to exhibit antiinflammatory (Williams et al., 1999), antioxidant (Bandoniene et al., 2000), antimicrobial (Holopainen and Kauppinen, 1989) and hypoglycemic (Takagi, 2002) activities. It has also been reported to have a healing effect on experimental gastric ulcer (Tournier et al., 1999). Furthermore, it has been shown that tansy extract is used in a treatment of increased bone resorption (Muehlbauer, 2002). Diuretics, either alone or in combination with other drugs, are valuable in the treatment of hypertension, congestive heart failure, ascites, and pulmonary edema (Gupta and Neyses, 2005; Hughes, 2004; Jackson, 1996; Morganti, 2005; O’Brien et al., 2005). Two widely used diuretics, thiazides and the high ceiling loop diuretic, furosemide, have been associated with a number of adverse effects, such as, electrolyte imbalance, metabolic alterations, development of new-onset diabetes, activation of the rennin–angiotensin–neuroendocrine systems, and impairment of sexual function (Gupta and Neyses, 2005; Morganti, 2005). Hence, there is a need for new diuretics with lower potential for adverse effects, such as the plant-based substances which are considered to be relatively safe. Although, both caraway and tansy are well recognised in Moroccan traditional medicine as having a diuretic effect at a dose of 100 mg/kg, no scientific data have been published supporting the claimed ethnomedical use. Therefore, the aim of this study was to evaluate the acute and sub-chronic diuretic, saliuretic, and kaliuretic effects of orally administered aqueous extracts of caraway seeds and tansy leaves in normal rats and thus, to confirm their ethnomedical use. 2. Materials and methods 2.1. Experimental animals Adult male Wistar rats weighing between 150 and 200 g procured from our animal house were housed under standard environmental conditions (25 ± 1 ◦ C, 55 ± 5% humidity and 12 h/12 h light/dark cycle). The animals were allowed free access to tap water and standard laboratory rat food. The care and handling of rats were in accordance with the internationally accepted standard guidelines for use of animals, and the protocol was approved by our institutional committee on animal care following the French Technical Specifications for the Production, Care and Use of the Laboratory Animals.
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Professor Bel Kamel, Facult´e des Siences Techniques (F.S.T., Marrakech, Morocco). Both caraway fruits and tansy leaves were dried at room temperature in the shade, and then ground in an electric grinder to give a coarse powder. Fifty grams of the dried powder of caraway fruits was mixed with 500 mL of water, and the mixture was boiled at 100 ◦ C under reflux for 30 min. The decoction obtained was centrifuged, filtered, frozen at −20 ◦ C, and then lyophilised (FreeZone® Dry 4.5, USA), yielding approximately 13.6% (w/w) of dry extract of Carum carvi L. The same procedure was used to extract tansy leaves (50 g dry powder extracted with 500 mL of water); the yield of the product was approximately 13.5% (w/w) of the dry leaves of Tanacetum vulgare L. For each study, the lyophilized aqueous extract was carefully prepared under the same conditions used throughout the studies (time, temperature and the amount of plant material and water used for extraction under reflux, and lyophilization), and each time the quality of extraction was checked by the yield of the lyophilized material. For assuring stability, the lyophilized material was stored at −20 ◦ C until used. The two lyophilized products (caraway and tansy) were dissolved in water (10 mg/mL) before administration at the stated doses. 2.3. Reference drug Furosemide (Lasilix, Pharma 5, Morocco), a high-ceiling loop diuretic, was used as the reference drug (positive control). It was dissolved in water prior to administration. 2.4. Biochemical methods Blood was collected in capillary tubes containing ethylenediamine tetraacetic acid by retro-orbital puncture under light diethyl ether anesthesia. Plasma was obtained by centrifugation (600 × g at 4 ◦ C), and stored at −20 ◦ C until analyzed. Plasma and urinary levels of sodium and potassium were quantitated by flame spectrophotometry. Concentration of creatinine in plasma and urine was determined by the Jaffe alkaline picrate method. Creatinine clearance, as a measure of renal function, was calculated from plasma and urinary creatinine levels. 2.5. Assessment of diuretic activity: experimental design
2.2. Plant material and preparation of the extracts
The diuretic activity was evaluated as follows: each animal was placed in an individual metabolic cage 24 h prior to commencement of the experiment for adaptation. The animals were divided into four groups of five rats each for the acute (single dose) study and another four groups of five rats each for the sub-chronic (repeated dose) study. Rats were fasted overnight with free access to water, and subjected to the stated treatment as described below. The rats were observed occasionally for apparent toxicity.
The ripe fruits of the common caraway [Carum carvi L. (Asteraceae)] were collected from the Fes region and tansy leaves from the Marrakech region in Morocco. Tansy was identified as Tanacetum vulgare L. (Asteraceae/Compositae) by
2.5.1. Acute diuretic activity Before treatment, all animals received physiological saline (0.9% NaCl) at an oral dose of 5 mL/100 g body weight (BW), to impose a uniform water and salt load (Benjumea et al., 2005).
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The first group received orally distilled water 10 mL/kg BW, and served as the control group. The second and third groups were administered orally 100 mg/kg BW of caraway fruit extract and tansy leaf extract, respectively. The fourth group was treated with an oral dose of 10 mg/kg BW of furosemide. Urine was collected and measured at 1, 2, 4, 6, and 24 h after the dose. Sodium and potassium concentrations were determined in the 24 h urine samples as well as in the plasma of rats. 2.5.2. Chronic diuretic activity Daily oral doses of caraway fruit extract (100 mg/kg BW), tansy leaf extract (100 mg/kg BW), and furosemide (10 mg/kg BW) were administered to three groups of rats for 8 days; the control animals received water (10 mL/kg) every day. For each rat, 24 h urine was collected daily and its volume measured. Urinary sodium and potassium concentrations were measured in each urine specimen. Sodium, potassium and creatinine levels were measured in plasma of rats on Day 8. Urinary creatinine excretion was also determined and its clearance was calculated for Day 8. The rats were observed daily for apparent toxicity. 2.6. Statistical analysis Results are expressed as mean ± S.E.M. Statistical analysis of the data was performed with one-way analysis of variance (ANOVA) (Graph Pad Prism version 3.00, USA) or by Student’s t-test. Significant differences were indicated by P values lower than 0.05. 3. Results 3.1. Diuretic activity of a single dose of the plant extracts and furosemide 3.1.1. Effect on urine volume Treatment with a single dose of the caraway extract increased diuresis (Fig. 1), which became significantly higher than in the control rats at 4 h after the dose (caraway 5.7 ± 0.3 mL versus controls 3 ± 0.5 mL; P < 0.05). Urine output continued to be
stimulated throughout the study period, such that the cumulative urinary excretion was significantly higher at 6 and 24 h after the dose of the caraway extract compared to the controls (caraway 12.8 ± 0.1 mL versus controls 7.7 ± 0.7 mL at 24 h after dose; P < 0.01) (Fig. 1). The effect of a single dose of tansy extract was more rapid and slightly higher than that of caraway extract (Fig. 1). The effect became significantly higher at 1 h after the dose compared to controls (tansy 4.0 ± 0.6 mL versus 0.7 ± 0.1 mL for controls; P < 0.05). The diuretic effect remained significantly higher for the tansy-treated rats as compared to control rats at all time points (Fig. 1), such that the cumulative 24 h urinary excretion for the tansy-treated rats was 14.3 ± 0.4 mL as compared to 7.7 ± 0.7 mL (P < 0.01) for the controls. The effect of a single dose of the reference diuretic, furosemide, was also rapid and higher than that of the plant extracts (Fig. 1), however, the 24 h cumulative urinary excretion was not too different for the three substances (caraway 12.8 ± 0.1 mL, tansy 14.3 ± 0.4 mL, and furosemide 18.5 ± 0.6 mL). 3.1.2. Effect on urinary electrolyte excretion The effect of single doses of furosemide and the aqueous extracts of caraway and tansy on electrolyte (Na+ and K+ ) excretion in the 24 h urine is presented in Table 1. Both plant extracts enhanced the excretion of the electrolytes [Na+ (P < 0.001) and K+ (P < 0.05)], which was greater than produced by furosemide, especially that of K+ . Furosemide actually decreased K+ excretion as compared to the controls, such that the Na+ /K+ excretion ratio (2.51) was higher than for the plant extracts (caraway 1.85, tansy 1.88) (Table 1). 3.1.3. Effect on plasma electrolyte levels There was no effect of any of the three substances, furosemide, caraway and tansy extracts on plasma levels of Na+ and K+ (Table 1). 3.2. Diuretic activity of the plant extracts and furosemide after sub-chronic administration 3.2.1. Effect on urine volume Administration of daily doses of both caraway and tansy extracts produced significant diuresis starting on Day 1 (caraway 9.3 mL ± 1.2; tansy 9 mL ± 0.6; controls 5.4 mL ± 0.7; P < 0.05), which became increasingly significant until Day 6 (caraway 20.2 mL ± 0.4; tansy 23 mL ± 1.6; controls 5.8 mL ± 1.0; P < 0.001), after that urinary output levelled off (Fig. 2). The diuretic effect of furosemide was initially higher than produced by the plant extracts, and then it was similar to that of the plant extracts after Day 4 (Fig. 2).
Fig. 1. Acute diuretic effect: diuretic activities of single oral doses (100 mg/kg BW) of aqueous extracts of Carum carvi L. seeds, Tanacetum vulgare L. leaves, and furosemide (10 mg/kg BW). The volume of excreted urine was measured at 1, 2, 4, 6 and 24 h after the treatment; cumulative values are reported as mean ± S.E.M. for five rats in each group. * P < 0.05; ** P < 0.01; *** P < 0.001 compared with controls using Student’s t-test.
3.2.2. Effect on urinary electrolyte excretion Both plant extracts (caraway and tansy) significantly induced urinary excretion of Na+ (P < 0.01) starting on Day 4, which became more pronounced (P < 0.001) from Day 5 to Day 8. The excretion of K+ increased significantly (P < 0.01) throughout the treatment [with maximum effect (P < 0.001) from Day 4 to
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Table 1 Effect of a single oral dose of the aqueous extracts of caraway and tansy, and furosemide on 24 h urinary electrolyte excretion and plasma Na+ and K+ levels in normal rats Treatmenta
Water, 10 mL/kg BW (control) Furosemide Carum carvi extract Tanacetum vulgare extract
Dose (mg/kg BW)
10 100 100
Urinary electrolyte concentration
Plasma electrolyte level
Na+ (mmol/L)
Na+ (mmol/L)
89.7 127.7 138.7 148.0
± ± ± ±
1.8 1.5** 1.5*** 1.7***
K+ (mmol/L) 62.9 50.8 75.0 78.4
± ± ± ±
1.1 0.9** 2.0* 1.8*
145.7 147.5 142.3 143.3
± ± ± ±
1.2 1.6 1.3 1.6
K+ (mmol/L) 5.3 5.1 5.8 5.2
± ± ± ±
0.4 0.3 0.3 0.5
Values are expressed as mean ± S.E.M. of five rats in each group. * P < 0.05; ** P < 0.01; *** P < 0.001 compared to controls using Student’s t-test. a All rats were pre-treated with an oral dose of 5 mL/100 g BW of normal saline prior to the administration of the test substances.
Day 8], by the tansy extract, but not by the caraway extract or furosemide (Fig. 4). 3.2.3. Effect on plasma electrolyte levels As was the case with the acute dose, there was no effect of sub-chronic administration of any of the three substances, furosemide [Na+ (149.2 mmol/L ± 1.3) and K+ (5.2 mmol/ L ± 0.3)], caraway [Na+ (147.1 mmol/L ± 0.8) and K+ (5.6 mmol/L ± 0.7)] and tansy extracts [Na+ (147.3 mmol/ L ± 0.6) and K+ (6.3 mmol/L ± 0.5)] on plasma levels of Na+ and K+ . 3.2.4. Effect on creatinine clearance Creatinine clearance, measured on the last day of treatment, was not affected by any of the treatments (caraway 2.97 mL/min ± 0.11; tansy 3.37 mL/min ± 0.18; furosemide 2.48 mL/min ± 0.09; controls 3.13 mL/min ± 0.06). 4. Discussion and conclusions In the present study, the diuretic effect of orally administered aqueous extracts of caraway fruit and tansy leaves was evaluated in normal rats after one dose and after daily dosing for 8 days. The pharmacological response was compared with that produced by furosemide, a widely used diuretic in clin-
Fig. 2. Sub-chronic diuretic effect: diuretic activities of daily oral doses (100 mg/kg BW) of aqueous extracts of Carum carvi L. seeds, Tanacetum vulgare L. leaves, and furosemide (10 mg/kg BW) administered for 8 days. The volume of 24 h urine was measured on Days 1 through 8 of treatment and reported as mean ± S.E.M. for five rats in each group. * P < 0.05; ** P < 0.01; *** P < 0.001 compared with controls using Student’s t-test.
ical practice. The two plant materials selected for the study, caraway fruits and tansy leaves, are commonly available and used in Moroccan traditional medicine to induce diuresis. Oral route was chosen because that is the way people use these plants in traditional medicine. The effect on electrolyte balance and creatinine clearance was also determined. The mechanism of action by which diuresis was induced by these two extracts was also assessed by comparing the effect with that of furosemide, a high-ceiling loop diuretic (Jackson, 1996), and that of hydrochlorothiazide (from published reports; Benjumea et al., 2005). Diuresis has two components: increase in urine volume (water excretion) and a net loss of solutes (i.e. electrolytes) in the urine (Jackson, 1996). These processes result from suppression of renal tubular reabsorption of water and electrolytes into the blood stream. The reference drug, furosemide, increases urine output and urinary excretion of sodium by inhibiting Na+ /K+ /2Cl− symporter (co-transporter system) in the thick ascending limb of the Loop of Henley (Jackson, 1996), while the thiazide diuretics inhibit the Na+ /Cl− symporter (co-transporter system) in the distal convoluted tubule, by competing for the Cl− binding site, and increasing the excretion of Na+ and Cl− (Jackson, 1996). In the saline primed rats, a dose of the tansy extract caused a significant increase in urine output beginning from the first hour while the caraway extract took 4 h after the dose to increase urine output significantly. In comparison, a single dose of furosemide induced a brisk and significant diuresis within 60 min of administration (Fig. 1). The difference in the time of onset of the diuretic action of these substances may be related to the gastrointestinal absorption characteristics of the active principle(s). Stimulation of diuresis by single doses of all three substances continued for atleast 24 h (Fig. 1). Both caraway and tansy extracts induced significant increases in urinary excretion of Na+ (154% and 164% of controls, respectively) and to a lesser extent of K+ (119% and 123% of controls, respectively); furosemide increased Na+ excretion (142% of controls) but not that of K+ : actually there was a small decrease in urinary concentration of K+ . Hydrochlorothiazide has been reported to increase the urinary excretion of both Na+ and K+ by 50–60% over controls after a single oral dose in normal rats (Benjumea et al., 2005). In this respect, the two plant extracts appear to have several active components, with atleast one having a mechanism of natriuresis similar to that of the thiazide
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Fig. 3. Sub-chronic natriuretic activity: effect of daily oral doses (100 mg/kg BW) of aqueous extracts of Carum carvi L. seeds, Tanacetum vulgare L. leaves, and furosemide (10 mg/kg BW) administered for 8 days on urinary excretion of Na+ . The reported Na+ levels (mean ± S.E.M.) are in pooled 24 h urine for each day for each group of five rats each group. * P < 0.05; ** P < 0.01; *** P < 0.001 compared with controls using Student’s t-test.
diuretic, and another with a mechanism of kaliuresis similar to that of furosemide. In the sub-chronic study, all three substances, furosemide and caraway and tansy water extracts, induced significant diuresis from Day 1 (without priming with saline); urine output continued to increase until Day 5 (maximum effect), after that it stayed stable until the last day of the study (Fig. 2). Although, the effect of furosemide was initially greater than that of the plant extracts, the 24 h urine output became similar for the three substances after Day 6, indicating that all the three substances had reached their maximum effect. Tolerance does not seem to develop to the diuresis stimulating activity of the plant extracts. The plant extracts and furosemide did not influence urinary excretion of Na+ for the first 2–3 days of repeated administration in these rats, which were not primed with saline (Fig. 3), probably because of homeostasis mechanisms. After that period, repeated administration of all three substances caused significant increase (by overcoming the homeostasis mechanisms) in saliuresis (Fig. 2). On the other hand, repeated administration of furosemide and caraway extract virtually had no effect on urinary K+ excretion for the entire 8 days. Contrarily, tansy extract caused a significant increase in urinary excretion of K+ beginning on Day 1 and continuing throughout the study period. The results of the present studies suggest that the effect of one or more of the active components of caraway on electrolyte excretion is furosemide-like, while the active substance(s) in tansy have a thiazide-like action. Further, despite increased urinary electrolyte (Na+ and K+ ) excretion (Figs. 3 and 4), there was no effect of the plant extracts and furosemide on plasma electrolyte levels both in the acute (Table 1) and sub-chronic studies, suggesting that the active principle(s) in both caraway and tansy do not act like potassium-sparing diuretics. There was no effect of repeated administration of the plant extracts on renal creatinine clearance, suggesting that the plant extracts have no renal toxicity at the studied dose. In addition, no apparent toxicity was observed in the rats during the 8 days of repeated dosing with the plant extracts. Nevertheless, additional
Fig. 4. Sub-chronic kaliuretic activity: effect of daily oral doses (100 mg/kg BW) of aqueous extracts of Carum carvi L. seeds, Tanacetum vulgare L. leaves, and furosemide (10 mg/kg BW) administered for 8 days on urinary excretion of K+ . The reported K+ levels (mean ± S.E.M.) are in pooled 24 h urine for each day for each group of five rats each group. * P < 0.05; ** P < 0.01; *** P < 0.001 compared with controls using Student’s t-test.
studies have to be carried out to confirm the lack of renal toxicity and to rule out other organ toxicity, especially after chronic administration. A number of compounds have been identified in caraway, including carvone and other monoterpenoids and their glucosides (Zheng et al., 1992), menthanetriols and glucosides (Matsumura et al., 2001), and limonene (Zheng et al., 1992), and in tansy, such as flavonoids (Williams et al., 1999), mono, tri-, and sesqui-terpine lactones, and isoprenoids (Chandler et al., 1982; Umlauf et al., 2004), saponins, polyphenols, and plant sterols (Chandler et al., 1982), parthenolide (Tournier et al., 1999), polysaccharides (Polle et al., 2002), thujones (Dragland et al., 2005), and chrysanthenyl analogs (Umlauf et al., 2004). At present, it is not known which compounds are responsible for the diuretic, natriuretic and kaliuretic activities of caraway and tansy. In conclusion, the present studies support the ethnomedical use of both caraway and tansy for their diuretic effect. The plant extracts do not seem to have renal toxicity in rats at the dose studied. Although, the active components remain unidentified, based on the pattern of excretion of water, sodium and potassium, it appears that that there are atleast two types of active principals present in these extracts, one having a furosemide-like activity and the other a thiazide-like activity. These findings suggest, for the first time, mechanism(s) of diuretic action of caraway and tansy used in traditional medicine by the Moroccan population. References Bandoniene, D., Pukalskas, A., Venskutonis, P.R., Gruzdiene, D., 2000. Preliminary screening antioxidant activity of some plant extracts in rapeseed oil. Food Res. Int. 33, 785–791. Bellakhdar, J., 1997. La Pharmacop´ee Marocaine Traditionnelle, M´edecine Arabe Ancienne et Savoirs Populaires. Edition Ibis Press, p. 150. Benjumea, D., Abdala, S., Hernandez-Luis, F., P´erez-Paz, P., Martin-Herrera, D., 2005. Diuretic activity of Artemisia thuscula, an endemic canary species. J. Ethnopharmacol. 100, 205–209. Chandler, R.F., Hooper, S.N., Hooper, D.L., Jamieson, W.D., Lewis, E., 1982. Herbal remedies of the Maritime Indians: sterols and triterpenes of Tanacetum vulgare L. (tansy). Lipids 17, 102–106.
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