Effects of extract from Clerodendron trichotomum on blood pressure and renal function in rats and dogs

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Journal of

ETHNOPHARMACOLOGY" ELSEVIER SCIENCE

IRELAND

Journal of Ethnopharmacology42 (1994) 77-82

Effects of extract from Clerodendron trichotomum on blood pressure and renal function in rats and dogs Guang-Wei Lut, Katsuyuki Miura*, Tokihito Yukimura, Kenjiro Yamamoto Department of Pharmacology, Osaka City University Medical School, 1-4-54 Asahimaehi. Abeno-ku, Osaka 545. Japan (Received 15 June 1993; revision received 26 January 1994; accepted 28 January 1994}

Abstract Clerodendron trichotomum Thunb. is a herb which has been used for treatment of hypertension in Chinese medicine. To assess the action of the herb on blood pressure and renal function, the extract was administered to rats and dogs. Intravenous administration of the extract elicited renal vasodilation and increased urine flow and urinary sodium excretion. Acute oral administration of the extract reduced blood pressure of spontaneously hypertensive rats but not of normotensive control rats. Chronic daily administration for 6 weeks prevented the increase in blood pressure of spontaneously hypertensive rats. These results may give some support to the claim on the clinical efficacy of the extract of Clerodendron trichotomum Thunb. Key words: Clerodendron trichotomum Thunb.; Blood pressure; Renal blood flow; Urine flow; Spontaneously hypertensive rat

1. Introduction Clerodendron trichotomum Thunb. (CTT) is a Chinese traditional medicine which has been used for the treatment of headache and rheumatism for hundreds of years. It has been reported that CTT produces a fall in blood pressure (BP) in dogs and rats by oral or intravenous administration (Wang et al., 1960; Hsu, 1962; Hsu and Hsing, 1962). In the present experiments the effects of the extract * Corresponding author. tPresent address: The Institute of Herbal Chinese Drugs, Shanghai College of Traditional Chinese Medicine. Shanghai. 200032 P.R.C.

from CTT on blood pressure were studied in normotensive and hypertensive rats and in dogs. The effects of CTT extract on the kidney, the organ primarily involved in body fluid and electrolytes homeostasis, were also assessed.

2. Materials and methods 2.1. Preparation o f water and ethanol-soluble water extract o f C T T The dried leaves of CTT were purchased from Shanghai Company of Traditional Crude Drug (Shanghai, China) and kindly identified by Zhaoyun Wu, a lecturer in the Department of

0378-8741/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved. SSDI 0378-8741(94)01106-A

78 Herb Identification, Shanghai College of Traditional Chinese Medicine. Ten grams of CTT were added to 100 ml of distilled water and left for 30 min at room temperature. The mixture of the crude drug and water was heated at 80°C for 5 h. After filtration through filter paper (Hardened 4Aa Toyo, Japan), the filtrate was concentrated using a rotary evaporator. Before the intravenous injection in acute experiments with rats, the water extract was diluted with saline and centrifuged at 5000 rev./min for 5 min. To remove the ethanol-insoluble component, the water extract from 100 g of CTT was diluted with distilled water to the final volume of 100 ml and then 300 ml of ethanol was added to the solution. After mixing, the solution was left for 24 h at room temperature. The supernatant was filtered through a filter paper. The filtrate was concentrated in vacuo to give a brown mass. Five grams of the ethanol-soluble water extract were obtained from 100 g of the crude drug. Before the experiments, the extract were resuspended in saline for intravenous injection into dogs. 2.2. Effects o f intravenous administration o f the water extract on blood pressure and renal function in anesthetized rats. SD rats weighing 300-400 g were anesthetized with inactin (thiobutabarbital) (100 mg/kg, i.p.). After cannulation of the trachea, polyethylene catheters were placed in both sides of the femoral arteries for measurement of BP with a pressure transducer (PD231D, Gould) and blood sampling, and in both sides of the femoral veins for infusion of inulin and drug administration. The left kidney was exposed through a retroperitoneal flank incision and renal nerves around the renal artery were ablated with 5'V,, phenol in ethanol. A fiowprobe was placed on the left renal artery for continuous measurement of renal blood flow (RBF) by an electromagnetic flowmeter (MFV-2100, Nihon Kohden, Japan). The left ureter was catheterized with a catheter (PEI0 connected to PE50) for collection of urine. A priming dose of inulin (100 mg/kg i.v.) was followed by a sustained infusion (20 mg/ml) at a rate of 0.5 ml/kg per min for the measurement of glomerular filtration rate (GFR). After an equilibration period of 60 min. urine

G,-W. Lu et a l . / J. Ethnopharmacol. 42 (1994) 77-82

was collected twice at 20 min intervals. At the midpoint of each period, 0.2 ml of arterial blood samples were collected. Thereafter, the water extract of CTT, diluted by saline, was injected intravenously over 1 min at a dose of either 0.1 g CTT equivalent/kg or 0.5 g CTT equivalent/kg. Five minutes later, 4 consecutive urine collections were made at 20 min intervals. Inulin concentration in plasma and urine was determined by Walser's method (Walser et al., 1955) and G F R was estimated by the clearance of inulin. Concentrations of sodium and potassium in plasma and urine were measured by a flame photometer (205D, Hitachi, Japan). 2.3. Effects o f intravenous administration o f the ethanol-soluble water extract on blood pressure and renal function in anesthetized dogs Mongrel dogs of either sex weighing 7-12 kg were anesthetized with sodium pentobarbital (30 mg/kg, i.v.) to monitor the effects on renal function (Miura et al., 1990). Briefly, the left kidney was exposed through a retroperitoneal incision and renal nerves around the renal artery were surgically dissected. RBF was measured by an electromagnetic flowmeter and systemic BP was measured through a catheter inserted into the right femoral artery. Saline was infused into the left brachial vein at a rate of 0.35 ml/kg per min throughout the experiments. A polyethylene tube was inserted into the left ureter for timed urine collections. After completion of surgery, the dog was left for 60-90 min to allow for stabilization of BP, RBF and urine flow (UF). Experiments were started with 2 consecutive 10 min interval urine collections, and the ethanol-soluble water extract of C'IT was administered intravenously over 1 min, at a dose of 0.24 g CTT equivalent/kg and timed urine collections were followed at 10, 20, 50 and 70 min after the injection. 2.4. Effects of acute oral administration o f water extract on hypertensive and normotensive conscious rats Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) aged 19 months were anesthetized with pentobarbital (40 mg/kg), and

79

G.-I¥. Lu et al./J. Ethnopharmacol. 42 (1994) 77-82

polyethylene catheters (PE50) were inserted in the femoral artery and vein. Both catheters were exteriorized through a cutaneous tunnel at the back of the neck. The rats were allowed to recover for 24 h, and then BP was measured before and after oral administration of the water extract (1.8 g C T T equivalent/kg) or saline.

2.5. Effects of chronic oral administration of the ethanol-soluble water extract on elevation of BP in SHR Female SHR aged 10 weeks were divided into two groups; 16 controls and 15 treated. After training for BP measurement with the tail-cuff method for 1 week, 15 rats were orally ad-

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Fig. 1. Effects of water extract from Clerodendron trichotomum Thunb. on renal function in anesthetized Sprague-Dawley rats. MBP, mean blood pressure: HR. heart rate: RBF, renal blood flow: RVR. renal vascular resistance: GFR, glomerular filtration rate: UF. urine flow rate: UNaV, urinary excretion of sodium: Uy, V. urinary excretion of" potassium: FEN~~, fractional excretion of sodium: FE K, fractional excretion of potassium. The water extract was administered at a dose of 0. I g crude drug/kg (i.v.). Data represent mean -*- S.E.M. Asterisks indicate statistically significant difference from control, n = 10 for MBP, HR, RBF and RVR, n = 5 for other parameters

G.-W. Lu et al./.!. Ethnopharmacol. 42 (1994) 77-82

80

ministered the ethanol-soluble water extract, at a dose of 0.5 g CTT equivalent/kg. The BP was measured I h after oral administration every week by the tail-cuff method. All values are presented as means ± S.E.M. Time course data were analyzed by a randomized block analysis of variance and statistical significance of the difference between the control and each intervention was determined by LSD test. Comparison between two groups was made by the Student's t-test. 3. Results 3.1. Effects. of intravenous administration of the water extract on BP and renal function in anesthetized rats There was no significant change in BP and heart rate (HR) in anesthetized SD rats by intravenous injection of the water extract of CTT (0.1-0.5 g CTT equivalent/kg). As shown in Fig. 1, the water extract (0.1 g CTT equivalent/kg, i.v.) induced a significant increase in RBF. The calculated renal vascular resistance (RVR) was significantly decreased. The effects on RBF and RVR appeared from 10 to 20 min after the intravenous injection of the extract and were sustained for about 60 min. G F R remained unchanged while the apparent increase observed in the urine flow (UF) from 17.9 ± 7.7 to 32.2 ± 6.5 #l/min was not statistically significant. However, urinary excre-

tion of sodium and potassium was significantly increased from 2.16 to 5.74 ~eq/min and from 1.33 to 1.99 tLeq/min, respectively. The fractional excretion of sodium increased by 114% above the control value. A higher dose of the water extract produced a significant increase in RBF from 9.28 ± 0.72 to 10.5 ± 0.62 ml/min and a decrease in RVR from 13.25 ± 0.79 to 11.5 ± 0.57 mmHg/ml per rain. The G F R did not change throughout the experiments. 3.2. Effects of intravenous administration of the ethanol-soluble water extract on BP and renal function in anesthetized dogs The intravenous administration of the ethalaolsoluble extract resulted in a biphasic response in BP. The initial fall started immediately after intravenous injection and reached a minimum at 3 min after the injection, with a decrease of 40% in BP (from 127 ± 10 to 77 ± 12 mmHg), and rapidly reverted. Similarly, RBF decreased transiently by 30% from 3.65 ± 0.31 to 2.56 -4- 0.41 ml/min. The effects of the extract of CTT on BP, RBF and U F with a dose of 0.24 g CTT equivalent/kg during the second phase are shown in Table 1. The second fall in BP started 10 min after the injection and was well maintained over 50 min. During this period, the BP decreased by 13.8%, from 127 ± 10 to 109 ± 10 mmHg (Table 1). The administration of the extract also resulted in a significant increase in UF. Although the increase in RBF was not

Table 1 Effects of intravenous injection of ethanol-soluble water extract from Clerodendron trichotomum Thunb. on renal function in anesthetized dogs Time (rain)

MBP (mmHg)

RBF (ml/g per min)

RVR (mmHg/g per min/ml)

UF (/~l/g per min)

0 10 20 50 70 Maximum response % change

127 ~: 10 114 ± 12" 115 ~: 9* 113 .4- 10" 120 ± 10 II0 ~ 11 -13.8

3.65 4.16 4.00 3.85 3.66 4.42 21.1

35.3 27.5 29.1 30.3 33.6 24.9 29.5

3.18 • 1.02 6.63 -4- 2.01" 4.97 ± 1.05 4.92 ~ 0.98 5.20 ± 1.21" 7.02 ± 2.00 120.3

± ± ± ± ± ±

0.31 0.21 0.34 0.37 0.36 0.17

± ± ± ± ± ±

2.3 2.8* 2.0* 3.1" 3.1 2.3

Injection of extract caused a transient fall in blood pressure which was reverted within 10 min. See details in the text. MBP, mean blood pressure; RBF, renal blood flow; RVR, renal vascular resistance; UF, urine flow rate. Data represent mean ± S.E.M., n = 6. *P < 0.05 compared to time 0.

G..W. Lu et al./J. Ethnopharmacol. 42 (1994) 77-82 statistically significant, significantly decreased.

the

81

calculated

RVR

3.3. Effects o f acute oral administration o f the water extract on hypertensive and normotensive conscious rats A n oral a d m i n i s t r a t i o n o f the extract had n o effect on BP in W K Y , while a significant reduction in BP was observed in conscious S H R with a dose of 1.8 g C T T equivalent/kg. The BP in S H R decreased from the control values of 172 ± 12 to 154 ± 10 m m H g (Fig. 2).

MBP (mmHg) 160 140

WKY

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-

saline c l - r extract

120

Duration

(weeks)

Systolic blood pressure (mmHg) Control (n = 16)

Treated (n = 15)

0

189 ± 7

190 ± 6

I

180 ± 6

179 ± 7

2 3 4 5 6 Withdrawal 7 8

196 ± 194 ± 205 ± 205 ± 208 ±

183 ± 5** 181 4- 5* 186 ± 6** 188 ± 6** 196±7"

7 7 8 7 7

209 ± 7 211 ± 8

208 ± 7 205 ± 6

*P < 0.05, **P < 0.01 compared to control (Student's t-test) Data are shown as mean ± S.E.M.

3.4. Effects o f a long-term oral administration o f the ethanol-soluble water extract on elevation o f BP in SHR

100 80 60

Table 2 Effects of chronic oral administration of ethanol-soluble water estract from Clerodendron trochotomum Thunb. on systolic blood pressure of spontaneously hypertensive rats

I

I

control

I

1

I

I

I

I

I

40 80 120 T i m e after administration (rain)

MBP (mmHg) 220 -

SHR

T

2O0 180

Before the a d m i n i s t r a t i o n o f the C T T extract, there was no difference in BP between both groups (190 ± 6 vs. 189 ± 7 mmHg). In the control group, BP gradually increased, while there was n o change in the treated group. Thus, there was a significant difference in BP between the treated a n d control groups, from 2 weeks to 6 weeks after the start of daily oral a d m i n i s t r a t i o n o f the C T T extract (Table 2). Once the extract was withdrawn, the BP in the treated rats increased to close to that of the control rats.

160

4. Discussion

140

The present study shows that oral a d m i n i s t r a tion of the C T T extract decreased BP in S H R both acutely and chronically. Moreover, the extract prevented the increase in systemic BP in S H R by oral a d m i n i s t r a t i o n d u r i n g the experimental period of 6 weeks. Since the extract did not affect BP in n o r m o t e n s i v e W K Y at the oral dose used in the experiments, the hypotensive action of C T T may be related to the m e c h a n i s m by which high BP was developed or m a i n t a i n e d in SHR. Therefore, the withdrawal of the extract a p p a r e n t l y elevated

120

I control

I

I

40

I

I

80

I

I

I

I

120

T i m e after administration (min)

Fig. 2. Effects of acute oral administration of water extract from Clerodendron trichotomum Thunb. on mean blood pressure in conscious Wistar-Kyoto rats and spontaneously hypertensive rats. The water extract was orally administered at a dose of 1.8 g CTT equivalent/kg: MBP, mean blood pressure. Data represent mean ± S.E.M. * P < 0.05 when compared with the preceding control value.

and conclusions

82 BP in the treated group of S H R but the difference in the values was not statistically significant when compared to those in the non-treated animals. These results are consistent with the surmise that the extract may be effective in primary hypertensive patients by oral administration (Wang et al., 1960). When the water extract was subjected to ethanol precipitation, the supernatant fraction caused a reduction in systemic BP of dogs when given intravenously and also prevented the elevation of BP in SHR when chronically administered orally. Therefore, the active component(s) of C T T which can lower BP were retained in the ethanolsoluble water extract. Intravenous administration of the C T T extract increased R B F or decreased R V R with no change in systemic BP in anesthetized dogs and rats, clearly indicating a prominent vasodilating action of the extract in the kidney. Increased blood flow in the kidney may partially explain the hypotensive action of CTT. The diuretic and natriuretic actions of the water extract were observed in anesthetized rats, following intravenous administration. Similarly the C T T extract pretreated with ethanol caused a diuretic action in anesthetized dogs, while G F R remained unchanged throughout the entire course of the experiments. These results demonstrated the inhibition of tubular transport of water and solute by the extract, which increases in U F and urinary sodium excretion. Since RBF was increased by the extract with no change in G F R , decreased plasma oncotic pressure in the postglomerular capillary may lead to the suppression of tubular reabsorption. The diuretic and natriuretic actions of the extract may also partially explain the pharmacological action of CTT. Certain antihypertensive agents, such as hydralazine and clonidine, can produce retention of sodium and water, but the extract of C T T produced an increase in RBF, U F and excretion of urinary electrolytes. It is possible that treatment with C T T in hypertensives may, like diuretics, prevent retention of electrolytes and water but this remains to be tested clinically. From CTT, clerodendrin-a flavone glycoside was isolated and was shown to possess hypotensive property (Chen et al., 1988). Trichotomine and its plausible precursors were also isolated from the plant (lwadare et al., 1974; Irikawa et al.,

G.-W. Lu et al./J. Ethnopharmacol. 42 (1994) 77-82

1989). In the present study, we did not test these compounds. The use of these compounds would give us a clearer picture of the profile of pharmacological action of this herb. In conclusion, C T T is a herb that is effective in lowering BP, increasing R B F and promoting urinary water and sodium excretion. These results support the claims of therapeutic efficacy of extract of Clerodendron t r i c h o t o m u m Thunb. in Chinese medicine. 5. Acknowledgments

The authors thank Mr. Jiandong Meng and Yong Wang in Shanghai College of Traditional Chinese Medicine for their help with some of the animal experiments. 6. References

Chen, Z.N., Xu, P.J. and Yao, T.R. (1988) Spectral analysis of clerodendrin isolated from Clerodendron trichotomum Thunb. Acta Pharmaceutica Sinica 23, 789-791. Hsu, S.-Y. (1962) Studies on the pharmacology of Chinese medicinal plant Chou-Wu-Tung(Clerodendron trichotomum Thunb.). I1. The mechanism of its hypotensiveeffects. Acta Physiologica Sinica 25, 272-277. (Chinese) Hsu, S.-Y. and Hsing, W.-R. (1962) Studies on the pharmacology of Chinese drug Chou-Wu-Tung (aerial plant of Clerodendron trichotomum Thunb.). I. The toxicity and hypotensive effects of infusion and crude extract of ChouWu-Tung. Acta Pharmaceutica Sinica IX, 734-740. (Chinese) lrikawa, H., Toyoda, Y., Kumagai, H. and Okumura, Y. (1989) Isolation of four 2,3,5,6,11,1 Ib-hexahydro-3-oxoHindolizino[8,7-b]indole-5-carboxylic acids from Clerodendron trichotomum Thunb. and properties of their derivatives. Bulletin of the Chemical Society of Japan 62, 880-887. lwadare, S., Shizuri, Y., Sasaki, K. and Hirata, Y. (1974) Isolation and structure of trichotomine and trichotomine G~. Tetrahedron 30, 4105-4111. Miura, K., Yukimura, T., Yamashita, Y., Shichino, K., Shimmen, T., Saito, M., Okumura, M., Imanishi, M., Yamanaka, S. and Yamamoto, K. (1990) Effects of endothelin on renal hemodynamics and renal function in anesthetized dogs. American Journal of ttyperwnsion 3, 632-634. Walser, M., Davidson, D.G. and Orloff, J. (1955) The renal clearance of alkali-stable inulin. Journal of Clinical Investigation 34, 1520-1523. Wang, X.-L., Li, H.-Y. and Ting, K.-S. (1960) Studies on drugs for treatment of hypertension. XI. Therapy with the decoction of Clerodendron trichotomum in dogs. Acta Pharmaceutica Sinica VIii, 88-94. (Chinese)