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The cardiovascular effects of green beans (Phaseolus aureus), common rue (Ruta graveolens), and kelp (Laminaria japonica) in rats
ISSN 0306-3623/97 $17.00 + .00 PIl S0306-3623 (97)00001-3 All rights reserved
Gen. Pharmac. Vol. 29, No. 5, pp. 859-862, 1997 Copyright @ 1997 Elsevier Science Inc. Printed in the USA. ELSEVIER
The Cardiovascular Effects of Green Beans (Phaseolus aureus), Common Rue (Ruta graveolens), and Kelp (Laminaria japonica) in Rats K. W. Chiu and A. Y. L. Fung* DEPARTMENT OF
BIOLOGY,THE
CHINESE UNIVERSITY OF HONC
KONO,SHATIN,
N.
T.,
HONG KONG
ABSTRACT. 1. The hypotensive effect of green beans, common rue and kelp was recently shown in normotensive rats in vivo. A number of mechanisms of action of these aqueous extracts was identified. The present study examined these actions at the tissue level in vitro with possible interactions of these extracts. 2. Rue showed positive chronotropic and inotropic effects on isolated right atria. Green beans and kelp alone showed negative chronotropic effects on isolated right atria but no effect on atrial tension (AT). A combination of green beans and kelp showed no additive effect on the decrease in atrial rate (AR) nor any negative inotropic responses. Combinations of rue and green beans and of rue and kelp showed responses that were either positive or negative chronotropically, were not dose dependent and were less than the sum total of their individual responses (i.e., subtractive). A combination of all three showed subtractive effects on the decrease in AR that were dose related. No change in AT was observed upon treatment with a combination of the three plant extracts in spite of the positive inotropic effect of rue. 3. Rue and kelp alone relaxed KC1 preconstricted rat tail artery strips probably by a direct effect of vascular smooth muscle. Green beans had no effect. The combination of rue and kelp exerted a subtractive relaxation effect. These plants therefore contained cardiovascular active substances that had a direct effect on the cardiovascular system. These substances further interacted to modify their cardiovascular effects. 4. Data explained why herbs, as in herbal medicine, should be used together therapeutically. GEN PHARMAC29;5:859--862, 1997. © 1997 Elsevier Science Inc. KEY WORDS. Interactions of plant extracts, cardiovascular effect, Phaseolus sp., Ruta sp., Laminaria sp. INTRODUCTION Many plants are described as antihypertensive (Chang and Miles, 1986; Chang and Quimio, 1984; Huang, 1987; Sham, 1983) because concoction preparations of these plant materials from either a single plant or a mixture of plants have been found to lower the blood pressure (BP) in animals and human beings. Aqueous extracts of green beans, rue and kelp, whether given alone or in various combinations, have been reported to be hypotensive in normotensive rats (Chiu and Fung, 1997) and in spontaneously hypertensive rats (SHR) (Chiu and Fung, unpublished). It had been suggested that the hypotensive effects were due to the stimulatory/inhibitory actions of these extracts on various receptors of the autonomic nervous system in particular in various tissues. Further, these plant extracts showed interactions in their hypotensive effects when administered as a mixture of two or three extracts. The present study was to examine whether these extracts act directly on the cardiovascular tissues and, at this tissue level, whether interaction occurred, should a mixture of two or three extracts be given. MATERIALS AND METHOD
Preparative procedures PREPARATION OF RIGHT ATRIA FOR IN VITRO STUDIES. Male
Sprague-Dawley (SD) rats weighing between 250 and 300 g were *To whom correspondenceshould be addressed. Received 1 July 1996; revised 14 December 1996; accepted 9 January 1997.
killed by cervical dislocation. An incision was made on the skin of the thorax and the underlying muscle layer. The ribs were cut open to expose the heart. The entire heart was excised rapidly and placed in K-H solution (NaCl, 115 mM; KC1, 25 mM; NaH2PO4.2H20, 1.2 mM; NaHCO3, 25 raM; CaCI>2H20, 2.1 raM; MgSO4.7H20, 1.2 mM; glucose, 11 mM) aerated with 95% O2 and 5% CO2. The right atrium was isolated and transferred to another petri dish containing fresh K-H solution, which was aerated continuously. The ends of the contraction axis of the atrial tissue were tied by silk threads and suspended inside a tissue chamber containing 10 ml of K-H solution aerated continuously at 37°C. In tying up the silk thread, a round loop was made at one end to hook the tissue to a fixed position in the organ bath. The other end of the silk thread was connected to a force displacement transducer (Narco Biosystem, Myograph F-60). Isometric contractions were recorded on a physiograph (Narco BiGsystem, MK-III). After the atrial tissue was equilibrated for 30 min under a resting tension of 1 g, plant extracts were added directly into the tissue chamber, and cumulative dose responses were obtained (Lam, 1992). PREPARATION OF ARTERY STRIPS FOR IN VITRO STUDIES. Male Sprague-Dawley rats weighing between 250 and 300 g were killed by cervical dislocation. The ventral tail artery was isolated and placed in a petri dish containing K-H solution that was aerated with 95% 02 and 5% CO> Connective and fat tissues were removed. A piece of the artery (2 cm) was cut under a dissecting microscope at 45° to produce a helical strip. The helical strip was tied at both ends and then suspended in a 10-ml tissue chamber, which was aerated continuously with 95% 02 and 5% CO2 at 37°C. One end of the he-
860
K. W. Chiu and A. Y. L. Fung
T A B L E 1. Effect of green beans, rue and kelp on atrial rate of rats Dosage (mg/L) 3 30 100 300 1,000 3,000
Atrial rate (beats/min) Green beans -5.17 -5.67 -15.67 -17.50 -49.67 -100.17
-+ 1.23" -+ 1.02" -+ 4.98* -+ 1.02" -+ 14.65" +- 24.27*
Rue +0.33 +7.00 +11.86 +14.38 +22.86 -0.50
Kelp
-+ 1.49 -+ 2.36* + 3.52* + 4.44* + 8.17" + 9.35*
+2.40 -2.20 -13.80 -31.60 -59.20 -115.20
+ 3.67 -+ 6.50* -- 4.79* + 6.15" -+ 9.60* + 16.40"
Basal atrial rates of green beans, me and kelp were 273.20 + 4.42, 299.17 -+ 8.42, and 269.67 + 12.39, respectively (n = 6). * Significant difference from the control (P < 0.05).
lical strip was hooked near the bottom of the organ bath, and the other end was connected to a force displacement transducer. Isometric contractions were recorded on a physiograph. The helical strip was equilibrated for 60 min under a resting tension of 0.7 g. KC1 (60 raM) was used to test the helical strip. The use of the preconstrictor was to verify the suitability of the vessels isolated (Sham, 1983). A n acceptable strip should develop tension between 0.5 g and 1.0 g, and testing with KC1 should be performed three times. W h e n a strip was found acceptable, plant extract was added. Any tension changes were recorded to test whether the extract had any effect on the unstimulated, basal tension of the strip. The strip was then contracted with 60 mM KCI. W h e n the tension was steady, plant extracts were added to the tissue chamber as before. Changes in tension were recorded to test the relaxation effect of plant extracts on KCl-stimulated tension increases. After each test, the strip was washed with K-H solution several times and given 10 min for equilibration. Six successful preparations were used in each test unless specified otherwise (Chiu et al., 1995).
ophilized to dried powder form. About 16 g of dried powder was obtained from 1,200 g of green beans. The same preparative procedure was done for common rue (Rum graveolens) and kelp (Laminaria japonica). Rue was grown in the green house of the Chinese University of Hong Kong and kelp was bought at a supermarket. Dried powder of rue weighing 2.6 g and of kelp weighing 6.2 g was obtained from 100 g of rue and 300 g of kelp. The dried powder was stored in a desiccator and was dissolved in 0.9% saline for the experiment.
Experiments STUDIES ON CHRONOTROPIC AND INOTROPIC EFFECTSON ISOLATED RIGHT ATRIA
Effect of individual plant extract. Right atria of male SD rats were prepared. The basal beating rate and tension were tested. Six doses (3, 30, 100, 300, 1,000 and 3,000 mg) of plant extract per liter of K-H solution in an organ bath were tested, beginning with the smallest dose. W h e n the atrial beat (AR) and atrial tension (AT) were steady after the first dose, the next higher dose was applied immediately, and so on. Chronotropic and inotropic responses were then obtained.
Effect of combination of plant extracts. Right atria of SD rats were prepared. Various combinations of two plant extracts and three plant extracts were prepared and added to the organ bath. W h e n the A R and A T were steady, the next combination of plant extracts was added immediately. Chronotropic and inotropic responses were then obtained. These were compared with responses after treatment with a single extract, as well as with data obtained by a summation of responses with single extracts. If the effect of the combined extracts was equal to that of the sum of individual extracts put together, the effect was described as additive. If the effect was lesser or greater, it was referred to as subtractive or synergistic, respectively. CONTRACTILE RESPONSES OF RAT TAIL ARTERY STRIPS
PREPARATION OF PLANT EXTRACTS. Green beans (Phaseolus aureus) were bought at a supermarket, weighed and washed. Green beans weighing 1,200 g were boiled with 6 liters of distilled water for 2 h. The aqueous extract was then centrifuged at 8,000 rpm (Sorvall instrument at 4°C) for 30 min. The supernatant was then concentrated to 200 ml by rotary evaporator. The concentrated solution was then put inside cellulose benzoated dialysis tubing (Sigma, at a cut off point of 2,000) with dimensions of 32 m m × 150 ram. Both ends of the tubing were tied firmly with clips to prevent leakage. The tube was then put into 2 liters of distilled water for 24 h inside a cold room. Stirring was provided to make sure dialysis was completed. The retente inside the dialysis tubing was collected and ly-
T A B L E 2. Effects of green beans, rue and kelp on atrial tension Dosage (mg/L) 3 30 100 300 1,000 3,000
Atrial tension (g)
Green beans +0.03 +0.02 +0.09 0.00 -0.02 -0.13
-+ 0.02 -+ 0.03 + 0.03 -+ 0.00 -+ 0.03 -+ 0.03*
Rue +0.01 +0.06 +0.06 +0.13 +0.18 +0.09
-+ + + -+ + -+
prepared. Four doses (100, 300, 1,000 and 3,000 rag) of plant extract per liter of K-H solution in an organ bath were added, beginning with the smallest dose. Changes in tension were recorded.
Effect of combination of plant extracts. Male SD rat tail artery strips were prepared. Because green bean extract had no effect on rat tail artery strips, different combinations of rue and kelp were prepared, and the effects on rat tail artery strips were noted. (See section on chronotopic and inotropic effects on isolated right atria for ways of assessing interactions of extracts.)
Statistics All data obtained were grouped as mean+SEM. Statistical tests were done to identify significant differences between groups. RESULTS
Kelp 0.01 0.03 0.02 0.02* 0.03* 0.05
Effect of individual plant extract. Male SD rat tail artery strips were
-0.06 0.00 +0.02 -0.01 -0.08 -0.15
+ 0.01 + 0.00 -+ 0.02 -+ 0.03 +- 0.04 --+ 0.06*
Basal atrial tensions from green beans, me and kelp were 0.43 --- 0.03, 0.45 -+ 0.05 and 0.43 -+ 0.08 respectively (n = 5). * Significant difference from the control (P < 0.05).
Isolated right atria EFFECT OF INDIVIDUAL PLANT EXTRACT. Both green beans and kelp extracts produced a significant decrease in A R compared with the control (basal rate) (P<0.05). The negative chronotropic effect of these two extracts on isolated right atria was dose dependent (Table 1). There was a large variation at low doses of kelp. Rue produced a significant increase in A R except at 3,000 mg/kg, which was dose dependent. Both green beans and kelp produced no significant effect on A T compared with the control, except at the dose of 3,000 mg/L, which
861
Cardiovascular Effects of Plant Extracts TABLE 3. Interaction of green beans, kelp and rue on atrial rates of rats Dosage (mg/L) kelp, green beans, rue (each)
3 30 100 300
Atrial rate (beats/min) Green beans + rue
-2.44 +3.30 -6.22 -9.29
+ ± ± ±
0.70 5.09* 5.70* 10.52"
Green beans + kelp
-2.67 -13.40 -14.40 -99.20
± ± ± ±
Kelp + rue
1.22 2.66 2.98 13.11"
-1.00 +0.57 +6.71 -22.00
+ ± ± +
0.25 0.66* 2.40* 8.60*
Green beans + kelp + rue
-2.70 -6.80 -10.89 -38.83
_+ 1.86 ± 1.80 + 2.96 ± 7.39
n~6.
* Significantdifference from the control (P < 0.05).
registered a significant decrease in AT. Rue showed a graded increase in AT from 3 to 1,000 mg/L (P<0.05) (Table 2). In all three extracts, when the dose was raised from 1,000 to 3,000 mg/L, there was an abrupt change in AR and AT. This indicated too high a dose. Thus, in rue, there was a change from a+22.86+8.17 beats/rain increase to a-0.5+9.35 beats/rain decrease in AR and a 50% decrease in AT (Tables 1 and 2). EFFECT OF C O M B I N A T I O N OF P L A N T EXTRACTS. Tables 3 and 4 show data from some of the combinations of plant extracts on AR and AT changes. When different doses of green beans and rue were mixed together, the decreases in AR were significantly less than when green beans were used alone (P<0.05) (Tables 1 and 3). In the presence of rue, the decrease in AR induced by kelp was less than that caused by kelp alone. The combination of green beans and kelp showed a decrease in AR: the decrease in AR of green beans and kelp mixture showed increases or a decrease compared with the summation from individual decreases in AR of green beans and kelp, at 300 mg/L of green beans and kelp (i.e., 600 mg extract/L). The precipitous drop in AR due to the mixture was significantly greater than the sum of responses to individual green beans and kelp extracts (P<0.05) (Tables 1 and 3). Whether this was due to a synergistic effect of these two extracts at this concentration or the concentration was too high to be toxic was unknown. When green beans, rue and kelp were mixed together at different doses, all produced a significant dose-dependent decrease in AR compared with the control (P<0.05). The decreases in AR of the three plant mixtures were significantly less than those of green beans and kelp combinations but greater than those of green beans and rue and of kelp and rue (Table 3). This was easily understood, because the positive chronotropic effect of rue reduced this decrease in the AR of green beans and kelp. There was no change in AT in the presence of any two of the three extracts at 3 and 10 mg/L. Further increases in the concentration of these extracts showed that, when green beans and rue or when kelp and rue were mixed together, there was a decrease in AT. When kelp and green beans were mixed together, there was an un-
expected but higher variable decrease in AT at 300 mg/L. Mixture of green beans, kelp and rue produced no change in AT (Table 4).
T a i l artery
strips
EFFECTOF PLANTEXTRACTSON CONTRACTILERESPONSES. None of these extracts had any effect on the basal tension of the strips. With KC1 preconstricted strips, green beans produced no significant change in contractile force. Rue significantly reduced the contractile force of KC1 preconstricted strips. The initial tension was 0.50+0.01 g. Kelp also produced a significant change in contractile force of the strips. The initial tension was 0.53+0.01 g. The decrease in contractile force produced by kelp was greater than that produced by rue (Table 5). The reduction in tension by extracts of rue or kelp in KCI preconstricted strips was dose dependent. When different concentrations of kelp and rue were mixed together, these mixtures produced dose-dependent changes in contractile force of tail strips (Table 5). These changes were identical with those from rue alone. Kelp apparently produced no effect in the mixture. No explanation could be offered. DISCUSSION Data from the present study showed that each of these aqueous plant extracts had a direct effect on the cardiovascular system: the heart or the vessels or both. These plant species therefore added to the list of plants that we have reported showing cardiovascular activities-Desmodium sp. and Clematis sp. (Ho et al., 1989), Salvia sp. (Li et al., 1990), Solanum sp. (Shum and Chiu, 1991), Narcissus sp. (Chiu et al., 1992) and Volvariella sp. (Chiu et al., 1995). Further, the interactions between the plant extracts on the cardiovascular system in the rat could be demonstrated at the tissue/organ level, as shown earlier in the whole animal (Chiu and Fung, 1997). Their effects and interactions might account for the hypotensive effects and changes therein in the rat. In their study, aqueous extracts of green beans, rue and kelp were shown to reduce the BP of normotensive rats. Administratration of combinations of these extracts produced additive or subtractive effects on the BP that were ascribed to their interactions on the receptors modulating BP.
TABLE 4. Interaction of green beans, kelp and rue on atrial rates of rats (n = 5) Dosage (mg/L) kelp, green beans, rue (each)
3 30 100 300
Green beans + rue
-0.01 0.00 -0.05 -0.12
__ 0.01 + 0.00 + 0.01 _+ 0.02
Green beans + kelp
0.00 0.00 -0.01 -0.08
_+ 0.00 + 0.00 _+ 0.02 _+ 0.05
Atrial tension Kelp + rue
0.00 0.00 -0.07 -0.05
_+ 0.00 + 0.00 _+ 0.02 + 0.04
Green beans + kelp + rue
-0.07 0.00 0.00 0.00
+ 0.03 _+ 0.00 _+ 0.00 _+ 0.00
862
K . W . Chiu and A. Y. L. Fung TABLE 5. Effect of rue and kelp on contractile force of rat tail artery strips Change in contractile force (g) Dosage (mg/L) 100 300 1000 3000
Rue 0.00 -0.07 -0.13 -0.20
Kelp
-+ 0.00 -+ 0.01 - 0.08* _+ 0.11"
-0.07 -0.13 -0.23 -0.33
m +-+ -+
0.01 0.05* 0.10" 0.11"
Rue + kelp 0.00 ± 0.00 - 0 . 0 6 -+ 0.00 -0.15 -+ 0.07 --
n~5.
* Significant difference from the control (P < 0.05). Green beans and kelp alone showed negative dose-dependent chronotropic effects on isolated atria without any effect on AT, whereas rue increased both AR and AT. Alone, green beans did not act on the vascular smooth muscle cells, but kelp and rue relaxed KCI preconstricted vessels. It came as no surprise that kelp was the most effective hypotensive substance among the three (Chiu and Fung, 1997), reducing the heart rate and being vasorelaxant. Of interest was the interaction on the cardiovascular tissues observed among these plant extracts. Green beans and kelp did not produce any additive effect on AR despite the fact that both were negative chronotropic. Changes in AR that were dose dependent spread all over. Responses to kelp were increased or synergized by green beans. Green beans and rue as well as kelp and rue exhibited a synergistic increase and a subtractive cardiac effect, respectively. As expected, rue decreased responses to green beans and kelp. All three extracts together produced an additive effect on AR. This effect was ascribed to a balance of the negative chronotropic effects of green beans and kelp and the positive chronotropic effect of rue. The positive inotropic effect of rue also explained the changes in A T when mixtures of rue and kelp or of rue and green beans were attempted. Although green beans or kelp alone did not have an effect on AT, when given together, surprisingly, they decreased A T so much that the positive effect of rue was nullified. That kelp or rue alone, but not green beans, relaxed KCI preconstricted tail artery strips of rats indicated a direct effect of these plant extracts on the vasculature. None of them had any effect on the basal tension of the strips (i.e., the unstimulated vasculature). Kelp and rue were likely to be antihypertensives. They produced a subtractive effect on the decrease in contractility when used together. This finding suggested that at some point, these two plant extracts have a common pathway of action in smooth muscle relaxation. Despite the fact that the mechanism of action of these plant extracts on cardiovascular tissue was not examined in the present study and therefore remains unknown, the present findings provide data on why these extracts should be used together therapeutically, as herbal medicines often are (Zhou, 1995). These extracts interacted when used together: synergism, summation or subtraction. Whenever an herb is chosen to treat a disease, a concoction preparation is made that is an "aqueous" extract of the herb for easy administration and perhaps absorption. The amount varies with the seriousness of the disease, because a dose-dependent relation usually exists. However, being a "crude" extract, it contains many active substances with diverse effects (including the one treating or relieving a particular symptom of the disease). Any effects other than those related to the symptom in question are considered "side" el-
fects. These side effects are of prime concern when the dose is high. For example, kelp used as an antihypertensive has been shown to be very effective in lowering BP through a number of mechanisms (Chiu and Fung, 1997). It slowed the heart rate in a dose-dependent manner, as observed in the present study. Used alone, kelp might not be desirable as an antihypertensive. If kelp is used together with rue, which also is hypotensive, their combined effect is an additive one (Chiu and Fung, 1997). Importantly, the decrease in heart activity due to kelp is attenuated, and indeed the contractility of the myocardium becomes stronger owing to rue. Kelp's effect on the vasculature is also compromised by rue. However, it is not good to use rue in the absence of kelp, because it disturbs the balance by increasing the cardiac activity. Trying to strike a balance and checking the physiological or pharmacological activity of different dosages as well as different plant combinations instead of a single plant, because plants in combination may share certain pharmacological properties with varying efficacy toward a symptom, provides a better way of achieving the same objective with less "side" or undesirable effect. The data obtained in this study supported this contention.
References Chang S. T. and Miles P. G. (1986) Edible Mushrooms and Their Cultivation. pp. 225-253. CRC Press Inc, Boca Raton, FL. Chang S. T. and Quimio T. H. (1984) A new look at cultivated mushroom. BioScience 34, 358-362. Chiu K. W. and Fung A. Y. L. (1997) The hypotensive effects of green bean (Phaseolus aureus ), common rue (Ruta graveolens), kelp (Laminaria japonica) in rats. Phytother. Res. 11,203-206. Chiu K. W., Lam A. H. W. and Pang P. K. T. (1995) Cardiovascular active substances from the straw mushroom, VolvarieUa volvacea. Phytother. Res. 9, 93-99. Chiu K. W., Lee Y. C. and Yung K. H. (1992) Bioactive substances from the Chinese daffodil, Narcissis tazetta. Phytother. Res. 6, 231-236. Ho C. S., Wong Y. H. and Chiu K. W. (1989) The hypotensive action of Desmodium styricifolium and Clematis chinesis. Am. J. Chin. Med., 17, 189-202. Huang L. L. (1987) Edible Mushroom for Self Study. pp. 2-58. Nan Jing University Press, Nan Jing. Lam A. H. W. (1992) The cardiovascular effects of straw mushroom, VolvarieUa volvaceae, in rats. A M. Phil. Thesis, Chinese University of Hong Kong, Hong Kong. Li C. P., Yung K. H. and Chiu K. W. (1990) Hypotensive action of Salvia miltiorrhiza cell culture extract. Am. J. Chin. Med. 18, 157-166. Sham S. K. (1983) Hypotensive actions of some Chinese medicinal plants: with emphasis on Acacia catechu. A M. Phil. Thesis. pp. 12-68. Chinese University of Hong Kong, Hong Kong. Shum O. L. and Chiu K. W. (1991) Hypotensive action of Solanum melongena on normotensive rats. Phytother. Res. 5, 76-81. Zhou J. H. (1995) Perspectives on the development of traditional Chinese drugs (TCD) in the new century. An opening speech at the 10th Annual Meeting of the Chinese Med. Pharmac. Soc. Chongqing, People's Republic of China. Abstract, pp. 1--4.
Gen. Pharmac. Vol. 29, No. 5, pp. 859-862, 1997 Copyright @ 1997 Elsevier Science Inc. Printed in the USA. ELSEVIER
The Cardiovascular Effects of Green Beans (Phaseolus aureus), Common Rue (Ruta graveolens), and Kelp (Laminaria japonica) in Rats K. W. Chiu and A. Y. L. Fung* DEPARTMENT OF
BIOLOGY,THE
CHINESE UNIVERSITY OF HONC
KONO,SHATIN,
N.
T.,
HONG KONG
ABSTRACT. 1. The hypotensive effect of green beans, common rue and kelp was recently shown in normotensive rats in vivo. A number of mechanisms of action of these aqueous extracts was identified. The present study examined these actions at the tissue level in vitro with possible interactions of these extracts. 2. Rue showed positive chronotropic and inotropic effects on isolated right atria. Green beans and kelp alone showed negative chronotropic effects on isolated right atria but no effect on atrial tension (AT). A combination of green beans and kelp showed no additive effect on the decrease in atrial rate (AR) nor any negative inotropic responses. Combinations of rue and green beans and of rue and kelp showed responses that were either positive or negative chronotropically, were not dose dependent and were less than the sum total of their individual responses (i.e., subtractive). A combination of all three showed subtractive effects on the decrease in AR that were dose related. No change in AT was observed upon treatment with a combination of the three plant extracts in spite of the positive inotropic effect of rue. 3. Rue and kelp alone relaxed KC1 preconstricted rat tail artery strips probably by a direct effect of vascular smooth muscle. Green beans had no effect. The combination of rue and kelp exerted a subtractive relaxation effect. These plants therefore contained cardiovascular active substances that had a direct effect on the cardiovascular system. These substances further interacted to modify their cardiovascular effects. 4. Data explained why herbs, as in herbal medicine, should be used together therapeutically. GEN PHARMAC29;5:859--862, 1997. © 1997 Elsevier Science Inc. KEY WORDS. Interactions of plant extracts, cardiovascular effect, Phaseolus sp., Ruta sp., Laminaria sp. INTRODUCTION Many plants are described as antihypertensive (Chang and Miles, 1986; Chang and Quimio, 1984; Huang, 1987; Sham, 1983) because concoction preparations of these plant materials from either a single plant or a mixture of plants have been found to lower the blood pressure (BP) in animals and human beings. Aqueous extracts of green beans, rue and kelp, whether given alone or in various combinations, have been reported to be hypotensive in normotensive rats (Chiu and Fung, 1997) and in spontaneously hypertensive rats (SHR) (Chiu and Fung, unpublished). It had been suggested that the hypotensive effects were due to the stimulatory/inhibitory actions of these extracts on various receptors of the autonomic nervous system in particular in various tissues. Further, these plant extracts showed interactions in their hypotensive effects when administered as a mixture of two or three extracts. The present study was to examine whether these extracts act directly on the cardiovascular tissues and, at this tissue level, whether interaction occurred, should a mixture of two or three extracts be given. MATERIALS AND METHOD
Preparative procedures PREPARATION OF RIGHT ATRIA FOR IN VITRO STUDIES. Male
Sprague-Dawley (SD) rats weighing between 250 and 300 g were *To whom correspondenceshould be addressed. Received 1 July 1996; revised 14 December 1996; accepted 9 January 1997.
killed by cervical dislocation. An incision was made on the skin of the thorax and the underlying muscle layer. The ribs were cut open to expose the heart. The entire heart was excised rapidly and placed in K-H solution (NaCl, 115 mM; KC1, 25 mM; NaH2PO4.2H20, 1.2 mM; NaHCO3, 25 raM; CaCI>2H20, 2.1 raM; MgSO4.7H20, 1.2 mM; glucose, 11 mM) aerated with 95% O2 and 5% CO2. The right atrium was isolated and transferred to another petri dish containing fresh K-H solution, which was aerated continuously. The ends of the contraction axis of the atrial tissue were tied by silk threads and suspended inside a tissue chamber containing 10 ml of K-H solution aerated continuously at 37°C. In tying up the silk thread, a round loop was made at one end to hook the tissue to a fixed position in the organ bath. The other end of the silk thread was connected to a force displacement transducer (Narco Biosystem, Myograph F-60). Isometric contractions were recorded on a physiograph (Narco BiGsystem, MK-III). After the atrial tissue was equilibrated for 30 min under a resting tension of 1 g, plant extracts were added directly into the tissue chamber, and cumulative dose responses were obtained (Lam, 1992). PREPARATION OF ARTERY STRIPS FOR IN VITRO STUDIES. Male Sprague-Dawley rats weighing between 250 and 300 g were killed by cervical dislocation. The ventral tail artery was isolated and placed in a petri dish containing K-H solution that was aerated with 95% 02 and 5% CO> Connective and fat tissues were removed. A piece of the artery (2 cm) was cut under a dissecting microscope at 45° to produce a helical strip. The helical strip was tied at both ends and then suspended in a 10-ml tissue chamber, which was aerated continuously with 95% 02 and 5% CO2 at 37°C. One end of the he-
860
K. W. Chiu and A. Y. L. Fung
T A B L E 1. Effect of green beans, rue and kelp on atrial rate of rats Dosage (mg/L) 3 30 100 300 1,000 3,000
Atrial rate (beats/min) Green beans -5.17 -5.67 -15.67 -17.50 -49.67 -100.17
-+ 1.23" -+ 1.02" -+ 4.98* -+ 1.02" -+ 14.65" +- 24.27*
Rue +0.33 +7.00 +11.86 +14.38 +22.86 -0.50
Kelp
-+ 1.49 -+ 2.36* + 3.52* + 4.44* + 8.17" + 9.35*
+2.40 -2.20 -13.80 -31.60 -59.20 -115.20
+ 3.67 -+ 6.50* -- 4.79* + 6.15" -+ 9.60* + 16.40"
Basal atrial rates of green beans, me and kelp were 273.20 + 4.42, 299.17 -+ 8.42, and 269.67 + 12.39, respectively (n = 6). * Significant difference from the control (P < 0.05).
lical strip was hooked near the bottom of the organ bath, and the other end was connected to a force displacement transducer. Isometric contractions were recorded on a physiograph. The helical strip was equilibrated for 60 min under a resting tension of 0.7 g. KC1 (60 raM) was used to test the helical strip. The use of the preconstrictor was to verify the suitability of the vessels isolated (Sham, 1983). A n acceptable strip should develop tension between 0.5 g and 1.0 g, and testing with KC1 should be performed three times. W h e n a strip was found acceptable, plant extract was added. Any tension changes were recorded to test whether the extract had any effect on the unstimulated, basal tension of the strip. The strip was then contracted with 60 mM KCI. W h e n the tension was steady, plant extracts were added to the tissue chamber as before. Changes in tension were recorded to test the relaxation effect of plant extracts on KCl-stimulated tension increases. After each test, the strip was washed with K-H solution several times and given 10 min for equilibration. Six successful preparations were used in each test unless specified otherwise (Chiu et al., 1995).
ophilized to dried powder form. About 16 g of dried powder was obtained from 1,200 g of green beans. The same preparative procedure was done for common rue (Rum graveolens) and kelp (Laminaria japonica). Rue was grown in the green house of the Chinese University of Hong Kong and kelp was bought at a supermarket. Dried powder of rue weighing 2.6 g and of kelp weighing 6.2 g was obtained from 100 g of rue and 300 g of kelp. The dried powder was stored in a desiccator and was dissolved in 0.9% saline for the experiment.
Experiments STUDIES ON CHRONOTROPIC AND INOTROPIC EFFECTSON ISOLATED RIGHT ATRIA
Effect of individual plant extract. Right atria of male SD rats were prepared. The basal beating rate and tension were tested. Six doses (3, 30, 100, 300, 1,000 and 3,000 mg) of plant extract per liter of K-H solution in an organ bath were tested, beginning with the smallest dose. W h e n the atrial beat (AR) and atrial tension (AT) were steady after the first dose, the next higher dose was applied immediately, and so on. Chronotropic and inotropic responses were then obtained.
Effect of combination of plant extracts. Right atria of SD rats were prepared. Various combinations of two plant extracts and three plant extracts were prepared and added to the organ bath. W h e n the A R and A T were steady, the next combination of plant extracts was added immediately. Chronotropic and inotropic responses were then obtained. These were compared with responses after treatment with a single extract, as well as with data obtained by a summation of responses with single extracts. If the effect of the combined extracts was equal to that of the sum of individual extracts put together, the effect was described as additive. If the effect was lesser or greater, it was referred to as subtractive or synergistic, respectively. CONTRACTILE RESPONSES OF RAT TAIL ARTERY STRIPS
PREPARATION OF PLANT EXTRACTS. Green beans (Phaseolus aureus) were bought at a supermarket, weighed and washed. Green beans weighing 1,200 g were boiled with 6 liters of distilled water for 2 h. The aqueous extract was then centrifuged at 8,000 rpm (Sorvall instrument at 4°C) for 30 min. The supernatant was then concentrated to 200 ml by rotary evaporator. The concentrated solution was then put inside cellulose benzoated dialysis tubing (Sigma, at a cut off point of 2,000) with dimensions of 32 m m × 150 ram. Both ends of the tubing were tied firmly with clips to prevent leakage. The tube was then put into 2 liters of distilled water for 24 h inside a cold room. Stirring was provided to make sure dialysis was completed. The retente inside the dialysis tubing was collected and ly-
T A B L E 2. Effects of green beans, rue and kelp on atrial tension Dosage (mg/L) 3 30 100 300 1,000 3,000
Atrial tension (g)
Green beans +0.03 +0.02 +0.09 0.00 -0.02 -0.13
-+ 0.02 -+ 0.03 + 0.03 -+ 0.00 -+ 0.03 -+ 0.03*
Rue +0.01 +0.06 +0.06 +0.13 +0.18 +0.09
-+ + + -+ + -+
prepared. Four doses (100, 300, 1,000 and 3,000 rag) of plant extract per liter of K-H solution in an organ bath were added, beginning with the smallest dose. Changes in tension were recorded.
Effect of combination of plant extracts. Male SD rat tail artery strips were prepared. Because green bean extract had no effect on rat tail artery strips, different combinations of rue and kelp were prepared, and the effects on rat tail artery strips were noted. (See section on chronotopic and inotropic effects on isolated right atria for ways of assessing interactions of extracts.)
Statistics All data obtained were grouped as mean+SEM. Statistical tests were done to identify significant differences between groups. RESULTS
Kelp 0.01 0.03 0.02 0.02* 0.03* 0.05
Effect of individual plant extract. Male SD rat tail artery strips were
-0.06 0.00 +0.02 -0.01 -0.08 -0.15
+ 0.01 + 0.00 -+ 0.02 -+ 0.03 +- 0.04 --+ 0.06*
Basal atrial tensions from green beans, me and kelp were 0.43 --- 0.03, 0.45 -+ 0.05 and 0.43 -+ 0.08 respectively (n = 5). * Significant difference from the control (P < 0.05).
Isolated right atria EFFECT OF INDIVIDUAL PLANT EXTRACT. Both green beans and kelp extracts produced a significant decrease in A R compared with the control (basal rate) (P<0.05). The negative chronotropic effect of these two extracts on isolated right atria was dose dependent (Table 1). There was a large variation at low doses of kelp. Rue produced a significant increase in A R except at 3,000 mg/kg, which was dose dependent. Both green beans and kelp produced no significant effect on A T compared with the control, except at the dose of 3,000 mg/L, which
861
Cardiovascular Effects of Plant Extracts TABLE 3. Interaction of green beans, kelp and rue on atrial rates of rats Dosage (mg/L) kelp, green beans, rue (each)
3 30 100 300
Atrial rate (beats/min) Green beans + rue
-2.44 +3.30 -6.22 -9.29
+ ± ± ±
0.70 5.09* 5.70* 10.52"
Green beans + kelp
-2.67 -13.40 -14.40 -99.20
± ± ± ±
Kelp + rue
1.22 2.66 2.98 13.11"
-1.00 +0.57 +6.71 -22.00
+ ± ± +
0.25 0.66* 2.40* 8.60*
Green beans + kelp + rue
-2.70 -6.80 -10.89 -38.83
_+ 1.86 ± 1.80 + 2.96 ± 7.39
n~6.
* Significantdifference from the control (P < 0.05).
registered a significant decrease in AT. Rue showed a graded increase in AT from 3 to 1,000 mg/L (P<0.05) (Table 2). In all three extracts, when the dose was raised from 1,000 to 3,000 mg/L, there was an abrupt change in AR and AT. This indicated too high a dose. Thus, in rue, there was a change from a+22.86+8.17 beats/rain increase to a-0.5+9.35 beats/rain decrease in AR and a 50% decrease in AT (Tables 1 and 2). EFFECT OF C O M B I N A T I O N OF P L A N T EXTRACTS. Tables 3 and 4 show data from some of the combinations of plant extracts on AR and AT changes. When different doses of green beans and rue were mixed together, the decreases in AR were significantly less than when green beans were used alone (P<0.05) (Tables 1 and 3). In the presence of rue, the decrease in AR induced by kelp was less than that caused by kelp alone. The combination of green beans and kelp showed a decrease in AR: the decrease in AR of green beans and kelp mixture showed increases or a decrease compared with the summation from individual decreases in AR of green beans and kelp, at 300 mg/L of green beans and kelp (i.e., 600 mg extract/L). The precipitous drop in AR due to the mixture was significantly greater than the sum of responses to individual green beans and kelp extracts (P<0.05) (Tables 1 and 3). Whether this was due to a synergistic effect of these two extracts at this concentration or the concentration was too high to be toxic was unknown. When green beans, rue and kelp were mixed together at different doses, all produced a significant dose-dependent decrease in AR compared with the control (P<0.05). The decreases in AR of the three plant mixtures were significantly less than those of green beans and kelp combinations but greater than those of green beans and rue and of kelp and rue (Table 3). This was easily understood, because the positive chronotropic effect of rue reduced this decrease in the AR of green beans and kelp. There was no change in AT in the presence of any two of the three extracts at 3 and 10 mg/L. Further increases in the concentration of these extracts showed that, when green beans and rue or when kelp and rue were mixed together, there was a decrease in AT. When kelp and green beans were mixed together, there was an un-
expected but higher variable decrease in AT at 300 mg/L. Mixture of green beans, kelp and rue produced no change in AT (Table 4).
T a i l artery
strips
EFFECTOF PLANTEXTRACTSON CONTRACTILERESPONSES. None of these extracts had any effect on the basal tension of the strips. With KC1 preconstricted strips, green beans produced no significant change in contractile force. Rue significantly reduced the contractile force of KC1 preconstricted strips. The initial tension was 0.50+0.01 g. Kelp also produced a significant change in contractile force of the strips. The initial tension was 0.53+0.01 g. The decrease in contractile force produced by kelp was greater than that produced by rue (Table 5). The reduction in tension by extracts of rue or kelp in KCI preconstricted strips was dose dependent. When different concentrations of kelp and rue were mixed together, these mixtures produced dose-dependent changes in contractile force of tail strips (Table 5). These changes were identical with those from rue alone. Kelp apparently produced no effect in the mixture. No explanation could be offered. DISCUSSION Data from the present study showed that each of these aqueous plant extracts had a direct effect on the cardiovascular system: the heart or the vessels or both. These plant species therefore added to the list of plants that we have reported showing cardiovascular activities-Desmodium sp. and Clematis sp. (Ho et al., 1989), Salvia sp. (Li et al., 1990), Solanum sp. (Shum and Chiu, 1991), Narcissus sp. (Chiu et al., 1992) and Volvariella sp. (Chiu et al., 1995). Further, the interactions between the plant extracts on the cardiovascular system in the rat could be demonstrated at the tissue/organ level, as shown earlier in the whole animal (Chiu and Fung, 1997). Their effects and interactions might account for the hypotensive effects and changes therein in the rat. In their study, aqueous extracts of green beans, rue and kelp were shown to reduce the BP of normotensive rats. Administratration of combinations of these extracts produced additive or subtractive effects on the BP that were ascribed to their interactions on the receptors modulating BP.
TABLE 4. Interaction of green beans, kelp and rue on atrial rates of rats (n = 5) Dosage (mg/L) kelp, green beans, rue (each)
3 30 100 300
Green beans + rue
-0.01 0.00 -0.05 -0.12
__ 0.01 + 0.00 + 0.01 _+ 0.02
Green beans + kelp
0.00 0.00 -0.01 -0.08
_+ 0.00 + 0.00 _+ 0.02 _+ 0.05
Atrial tension Kelp + rue
0.00 0.00 -0.07 -0.05
_+ 0.00 + 0.00 _+ 0.02 + 0.04
Green beans + kelp + rue
-0.07 0.00 0.00 0.00
+ 0.03 _+ 0.00 _+ 0.00 _+ 0.00
862
K . W . Chiu and A. Y. L. Fung TABLE 5. Effect of rue and kelp on contractile force of rat tail artery strips Change in contractile force (g) Dosage (mg/L) 100 300 1000 3000
Rue 0.00 -0.07 -0.13 -0.20
Kelp
-+ 0.00 -+ 0.01 - 0.08* _+ 0.11"
-0.07 -0.13 -0.23 -0.33
m +-+ -+
0.01 0.05* 0.10" 0.11"
Rue + kelp 0.00 ± 0.00 - 0 . 0 6 -+ 0.00 -0.15 -+ 0.07 --
n~5.
* Significant difference from the control (P < 0.05). Green beans and kelp alone showed negative dose-dependent chronotropic effects on isolated atria without any effect on AT, whereas rue increased both AR and AT. Alone, green beans did not act on the vascular smooth muscle cells, but kelp and rue relaxed KCI preconstricted vessels. It came as no surprise that kelp was the most effective hypotensive substance among the three (Chiu and Fung, 1997), reducing the heart rate and being vasorelaxant. Of interest was the interaction on the cardiovascular tissues observed among these plant extracts. Green beans and kelp did not produce any additive effect on AR despite the fact that both were negative chronotropic. Changes in AR that were dose dependent spread all over. Responses to kelp were increased or synergized by green beans. Green beans and rue as well as kelp and rue exhibited a synergistic increase and a subtractive cardiac effect, respectively. As expected, rue decreased responses to green beans and kelp. All three extracts together produced an additive effect on AR. This effect was ascribed to a balance of the negative chronotropic effects of green beans and kelp and the positive chronotropic effect of rue. The positive inotropic effect of rue also explained the changes in A T when mixtures of rue and kelp or of rue and green beans were attempted. Although green beans or kelp alone did not have an effect on AT, when given together, surprisingly, they decreased A T so much that the positive effect of rue was nullified. That kelp or rue alone, but not green beans, relaxed KCI preconstricted tail artery strips of rats indicated a direct effect of these plant extracts on the vasculature. None of them had any effect on the basal tension of the strips (i.e., the unstimulated vasculature). Kelp and rue were likely to be antihypertensives. They produced a subtractive effect on the decrease in contractility when used together. This finding suggested that at some point, these two plant extracts have a common pathway of action in smooth muscle relaxation. Despite the fact that the mechanism of action of these plant extracts on cardiovascular tissue was not examined in the present study and therefore remains unknown, the present findings provide data on why these extracts should be used together therapeutically, as herbal medicines often are (Zhou, 1995). These extracts interacted when used together: synergism, summation or subtraction. Whenever an herb is chosen to treat a disease, a concoction preparation is made that is an "aqueous" extract of the herb for easy administration and perhaps absorption. The amount varies with the seriousness of the disease, because a dose-dependent relation usually exists. However, being a "crude" extract, it contains many active substances with diverse effects (including the one treating or relieving a particular symptom of the disease). Any effects other than those related to the symptom in question are considered "side" el-
fects. These side effects are of prime concern when the dose is high. For example, kelp used as an antihypertensive has been shown to be very effective in lowering BP through a number of mechanisms (Chiu and Fung, 1997). It slowed the heart rate in a dose-dependent manner, as observed in the present study. Used alone, kelp might not be desirable as an antihypertensive. If kelp is used together with rue, which also is hypotensive, their combined effect is an additive one (Chiu and Fung, 1997). Importantly, the decrease in heart activity due to kelp is attenuated, and indeed the contractility of the myocardium becomes stronger owing to rue. Kelp's effect on the vasculature is also compromised by rue. However, it is not good to use rue in the absence of kelp, because it disturbs the balance by increasing the cardiac activity. Trying to strike a balance and checking the physiological or pharmacological activity of different dosages as well as different plant combinations instead of a single plant, because plants in combination may share certain pharmacological properties with varying efficacy toward a symptom, provides a better way of achieving the same objective with less "side" or undesirable effect. The data obtained in this study supported this contention.
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