Diphasic Effects of Astragalus membranaceus BUNGE (Leguminosae) on Vascular Tone in Rat Thoracic Aorta

1450 Biol. Pharm. Bull. 28(8) 1450 1454 (2005) Vol. 28, No. 8 Diphasic Effects of Astragalus membranaceus BUNGE (Leguminosae) on Vascular Tone in Rat Thoracic Aorta Bi-Qi ZHANG, a Shen-Jiang HU,*,a,d Li-Hong QIU, a Qi-Xian SHAN, b Jian SUN, a Qiang XIA, b and Ka BIAN c,d a Department of Cardiology, The First Affiliated Hospital, College of Medicine, Zhejiang University; No. 79, Qingchun St, Hangzhou 310003, Zhejiang, P.R. China: b Department of Physiology, College of Medicine, Zhejiang University; No. 353, Yan an St, Hangzhou 310031, Zhejiang, P.R. China: c Department of Integrative Biology and Pharmacology, The University of Texas-Houston Medical School; 6431 Fannin, Houston, Texas, 77030, U.S.A.: and d E-Institute of Shanghai Universities, Division of Nitric Oxide and Inflammatory Medicine; No. 1200, Chalun St, Shanghai 201203, P.R. China. Received March 22, 2005; accepted April 28, 2005 This study was designed to investigate the effects of the aqueous ethanol extract of Astragalus membranaceus BUNGE (Leguminosae) on rat thoracic aorta. Isometric tension was recorded in response to drugs in organ bath. In endothelium-intact aortic rings, A. membranaceus extract induced a significant dose-dependent relaxation of the rings precontracted by phenylephrine, which could be inhibited by preincubation with L-N(w)-nitro-arginine methyl ester or methylthioninium chloride. In endothelium-denuded ones, the extract could dose-dependently relax the rings contracted by phenylephrine, not by KCl; and it could also attenuate contractile response to phenylephrine, not to caffeine or phorbol-12,13-diacetate in Ca 2 -free medium; but it failed to affect the CaCl 2 - induced enhancement of contractile response to phenylephrine in Ca 2 -free medium. These results indicate that nitric oxide signaling and Ca 2 -handling pathway are involved in the A. membranaceus extract-induced vasodilatation. Key words Astragalus membranaceus; nitric oxide; calcium ion; thoracic aorta; vasomotion The roots of Astragalus membranaceus BUNGE (Leguminosae) are amongst the most popular and important Qi (pronounce chee) tonifying adaptogenic herbs in China, their use dates back more than 2000 years, and are recorded in Shen Nong s Materia Medica. According to ethnobotancical data collected in China, A. membranaceus has been prescribed for centuries for diarrhea, frequent colds, spontaneous sweats, fatigue, and edema. Furthermore, A. membranaceus is alleged to possess the effects of antioxidative damage, 1) immune-stimulation, 2) and antiviral infection. 3) Recently, it has received more attention that the fact recorded in ancient Chinese texts that A. membranaceus possessed an antihypertensive effect by raising the yang qi of the spleen and kidney, since deficiency qi formed by the disorder of spleen and kidney is considered to be the basic pathogeneses of hypertension. 4) A great deal of studies demonstrated that A. membranaceus was beneficial on the process of hypertension in animals and humans, 5 7) and a cooperative mechanism of renin angiotensin aldosterone system, kallikrein bradykinin system, and central neuropeptide 8) was used to explain this effect of A. membranaceus. We also found in the past experiments that the administration of A. membranaceus could attenuate the progression of blood pressure in spontaneously hypertensive rats concomitant with restoration of baroreflex sensitivity and reduction of Ca 2 concentration in lymphocyte and vascular smooth muscle cells (VSMC). 9 11) Additionally, in vivo experiment had demonstrated that A. membranaceus could induce the vasodilatation. 12) However, there is no experiment evidence available to show the exact mechanism of vasorelaxation produced by A. membranaceus. The present study is conducted to elucidate various possible mechanisms in vasomotor response of A. membranaceus. MATERIALS AND METHODS Plant Material and Preparation of the Extracts A. membranaceus roots were collected in the northern region of Gansu Province, China, in the autumn (2001) and identified by the Quality Assessment of Di ao Group, Sichuang, China. And a voucher specimen was deposited in the Academic Department of Di ao Group (STQ-C-00203). A. membranaceus roots were air dried. A crude extract was prepared by decoction of 2.0 10 2 g in 500 ml water for 3 times (90 min per time). The obtained extract was combined, filtered and concentrated. Then all the aqueous extract was extracted with 400 ml ethanol (75% and 85%) twice. After the ethanol was retrieved, the active fractions were recovered, filtered and lyophilized (yield 0.28%). Consequently, the final extract was used in the study after dissolution in distilled water at 37 C and stirred for 60 min. Chemicals Phenylephrine (PE), acetylcholine (ACh), L- N(w)-nitro-arginine methyl ester (L-NAME), methylthioninium chloride (MC), phosphoramidon, ethylene glycol-bis [b-aminoethyl ether]-n,n,n,n -tetraacetic acid (EGTA), caffeine and phorbol-12,13-diacetate (PD) were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). They were dissolved in distilled water and diluted with Krebs Henseleit (K H) solution before use. All chemicals were of the highest purity available. Experiment Animals The investigation conformed to the Chinese Regulation for Administration of Laboratory Animal (SSTC 1988/2). Health male Sprague Dawley rats weighing 260 280 g (Laboratory Animal Center of Zhejiang College of Traditional Chinese Medicine, Hangzhou, China) were housed in conventional cages with free access to water and rodent chow at the controlled temperature and humidity with a 12-h light/dark cycle. Preparation of Thoracic Arterial Rings from Sprague To whom correspondence should be addressed. e-mail: s0hu0001@hotmail.com 2005 Pharmaceutical Society of Japan

August 2005 1451 Dawley Rats Rats were euthanized by decapitation using small animal guillotine. The thoracic aorta was immediately isolated and immersed in oxygenated K H solution 4 C of the following composition (in mm): NaCl 118.3; KCl 4.7; MgSO 4 1.2; KH 2 PO 4 1.2; NaHCO 3 25.0; CaCl 2 2.5; Glucose 11.0. Then the adherent connective tissue was cleaned and blood vessel was cut into 3 4 mm rings, with the special care of avoiding damage of endothelium. In some preparations, endothelium was mechanically removed by gently rubbing the lumen. The rings were mounted horizontally between two stirrups in organ bath filled with 10 ml K H solution at 37 C, ventilated continuously with 95% O 2 and 5% CO 2. The isometric tension was recorded with a force transducer (JZ101, metrical range: 0 5.0 g) and MedLab 5.0v recording system (Nanjing Medease Science and Technology Co. Ltd). Experimental Procedure of Isometric Tension Rings were equilibrated for 60 min at 2.0 g resting tension, and then challenged with KCl (6.0 10 2 M) at least 3 times until a reproducible maximal contractile response was obtained. After a further equilibration period of 30 min, the integrity of the endothelium was assessed in all preparations by determining the ability of ACh (1.0 10 5 M) to induce more than 80% relaxation of rings pre-contracted with PE (1.0 10 6 M). The endothelium was considered to be removed when there was less than 10% relaxation response to ACh. Protocol 1: Effects of A. membranaceus on Vascular Tone: The first series of the experiments were conducted to assess endothelium-dependent or independent effects of A. membranaceus on isolated aortic rings. When the tension was at resting state or reached a plateau induced by PE (3.0 10 7 M), A. membranaceus (2.0 10 5 2.0 10 1 g/l) was cumulatively added into the organ bath with 10 min interval. The rings with intact or denuded endothelium were always tested in parallel. Protocol 2: Endothelium-Independent Vasomotor Response by A. membranaceus: The second series of the experiments were designed to determine the underlying mechanisms of A. membranaceus-induced vasomotor response in aortic rings without endothelium. In contrast to the response of A. membranaceus (2.0 10 5 2.0 10 1 g/l) to PE, KCl (3.0 10 2 M) was also employed to constrict the denuded aortic rings. A plateau contractile response to KCl or PE was also obtained as a time control. Additionally, after the preparations were serially washed and equilibrated for 60 min by Ca 2 -free medium (mm: NaCl 118.3, KCl 4.7, KH 2 PO 4 1.2, MgSO 4 1.2, NaHCO 3 25, Glucose 11.0, EGTA 0.05), PE (3.0 10 7 M), PD (1.0 10 6 M) (an activator of protein kinase C, PKC), 13) or caffeine (2.5 10 2 M) (an activator of ryanodine receptor) 14) was used to contract the denuded rings. In order to explore the probable effect of extracellular Ca 2 inflow, 2.5 mm CaCl 2 was added after PE stimulated the rings. Then the effect of A. membranaceus (6.0 10 3 g/l) was tested. A time control (same protocol as above except for the omission of A. membranaceus to the second application of PE, PD or caffeine) was always run in parallel. Following the initial control response in Ca 2 -free medium and before the second test contraction (to PE or caffeine), a 6.0 10 2 M KCl-induced contraction in K H solution was obtained to refill the Ca 2 stores in the vascular tissues. Protocol 3: Endothelium-Dependent Vasomotor Response by A. membranaceus: In the third series of experiments, the potential influence of A. membranaceus on PE-induced vasomotor function in the intact rings was examined. For pretreatment, endothelium-intact rings were incubated with L- NAME (1.0 10 4 M) (a specific inhibitor of nitric oxide synthase), MC (1.0 10 5 M) (an inhibitor of guanylate cyclase) for 15 min 15) and phosphoramidon (5.0 10 6 M) (an inhibitor of endothelin converting enzyme) for 20 min 16) before administration of PE. Then the response curves of A. membranaceus were recorded. The rings with or without the treatment of inhibitors were always tested in parallel. Statistical Analysis Relaxant responses were expressed as the percentage decreases of the magnitude of the contraction induced by PE or KCl ( 100%) before the application of vasodilators, and the contractile responses as the ratio of the responses following and before the application of A. membranaceus (second response/first response). All results are expressed as mean S.E.M. Statistical analysis was performed with t-test or ANOVA followed by Newman Keuls test. Differences were accepted as statistically significant at p values 0.05 (GraphPad Prism). RESULTS PE (1.0 10 6 M) induced a similar sustained contraction of aortic rings in each group with a peak tension of about 3.11 0.27 g in intact aortic rings and 3.02 0.19 g in denuded ones. Effects of A. membranaceus on the Vasotension All doses (2.0 10 5 2.0 10 1 g/l) of A. membranaceus exhibited negligible vasomotor actions on aortic rings with or without endothelium at resting tension (Fig. 1). A. membranaceus (2.0 10 5 2.0 10 1 g/l) produced dose-dependent relaxation in endothelium-denuded rings (Fig. 2), after the plateau tension induced by PE. However, in PE-precontracted endothelium-intact rings, low doses (2.0 10 4 6.0 10 3 g/l) of A. membranaceus showed a more significant relaxation than that in denuded ones (Fig. 2), while high dose (2.0 10 2 2.0 10 1 g/l) of the extract induced a transient contraction (Figs. 2, 3). Endothelium-Independent Vasodilatation by A. membranaceus In contrast to the effect on PE-contracted endothelium-denuded rings in K H solution, A. membranaceus (2.0 10 4 2.0 10 1 g/l) had no dilatation-effect on rings stimulated by KCl (3.0 10 2 M) (Fig. 4). And in the experiment with Ca 2 -free medium, aortic contraction elicited by PE, caffeine or PD was decreased substantially, as these vascular contractions in Ca 2 -free medium are mediated only via the release of Ca 2 from the intracellular stores or phosphorylation of myosin light chain. A. membranaceus (6.0 10 3 g/l) could inhibit the contraction induced by PE (3.0 10 7 M), but not block the extracellular Ca 2 inflow, because 2.5 mm CaCl 2 induced a similar enhancement of contraction by PE, compared with the control (95.84 2.63% vs. 96.96 1.87%, p 0.05). Furthermore, A. membranaceus (6.0 10 3 g/l) failed to induce an attenuation of contractile response by PD (1.0 10 6 M) and caffeine (2.5 10 2 M) (Fig. 5). Endothelium-Dependent Vasomotor Effects by A. membranaceus Pre-incubation of the intact rings with L-

1452 Vol. 28, No. 8 Fig. 1. Effects of A. membranaceus on the Vasomotion of Aorta Rings in the Resting Tension A. membranaceus (2.0 10 5 2.0 10 1 g/l) was cumulatively added into the bathing solution in 10 min interval during the resting phase (2.0 g) in (A) endothelium intact or (B) removed rings. Values of each group are expressed as mean S.E.M. of 8 experiments. Fig. 2. Vasorelaxant Effects of A. membranaceus (2.0 10 5 2.0 10 1 g/l) on Phenylephrine (3.0 10 7 M)-Induced Contractions in Aortic Rings (A) with or (B) without Endothelium Values of both groups are expressed as mean S.E.M. of 8 experiments. Contractile responses are expressed as the percentage from maximal contraction elicited by phenylephrine (3.0 10 7 M). p 0.05, p 0.01, compared with the control group. Fig. 3. Transient Vasocontractile Response of A. membranaceus (Closed Circle) against Phenylephrine-Induced Endothelium-Intact Aortic Rings With (A) or (B) without the incubation of phosphoramidon (5.0 10 6 M) for 20 min before phenylephrine (3.0 10 7 M) was added, the effect of A. membranaceus (2.0 10 2, 6.0 10 2 and 2.0 10 1 g/l, showed by a, b, c point) was obtained. For it was similar with Fig. 2, the effect of A. membranaceus (2.0 10 5 6.0 10 3 g/l) was cut. Values of both groups are expressed as mean S.E.M. of 8 experiments. Contractile responses are expressed as the percentage from maximal contraction elicited by phenylephrine (3.0 10 7 M). p 0.05, p 0.01, compared with corresponding values before administration. NAME (1.0 10 4 M) markedly, incompletely inhibited A. membranaceus (6.0 10 3 g/l)-induced relaxation from 36.0 to 7.3% (Fig. 6). Similarly, MC (1.0 10 5 M) also showed an inhibition of endothelium-dependent vasodilatation from 36.0 to 8.0% (Fig. 6). Thus, NO signaling might play a major, but not full role in the vasodilatation effect of A. membranaceus. When the dose of A. membranaceus in the organ bath was stepwise raised to 2.0 10 2 2.0 10 1 g/l, a transient contraction could be observed in endothelium-intact rings. Pretreatment of endothelium-intact rings with phosphoramidon (5.0 10 6 M) significantly attenuated the transient contraction response (Fig. 3). DISCUSSION The present study first and detailedly explored the in vitro vascular effects of A. membranaceus in aortic rings isolated from rats. In the course of the study, a number of novel observations had been made, which contribute to better scien-

August 2005 1453 Fig. 4. Vasorelaxant Effects of A. membranaceus (2.0 10 5 2.0 10 1 g/l) on KCl (3.0 10 2 M)-Induced Contractions in Aortic Rings without Endothelium Values of both groups are expressed as mean S.E.M. of 8 experiments. Contractile responses are expressed as the percentage from maximal contraction elicited by KCl (3.0 10 2 M). Fig. 5. Inhibitory Effect of A. membranaceus (6.0 10 3 g/l) on Phenylephrine (3.0 10 7 M)-, Phorbol-12,13-diacetate (1.0 10 6 M) and Caffeine (2.5 10 2 M)-Induced Contractions in Ca 2 -Free Medium in Endothelium- Denuded Rings Each column with a bar represents mean S.E.M. of 8 experiments. Data are expressed as the percentage of the test response ratio (after/before A. membranaceus) with respect to the time control response ratios. p 0.01, compared with value in the corresponding vehicle group. Fig. 6. Vasorelaxant Effects of A. membranaceus (6.0 10 3 g/l) on Phenylephrine (3.0 10 7 M)-Induced Contraction in Presence of L-N(w)- Nitro-arginine Methyl Ester (L-NAME) (1.0 10 4 M) and Methylthioninium Chloride (MC) (1.0 10 5 M) in K H Solution in Endothelium-Intact Rings In the untreatment group, A. membranaceus or vehicle group was not pretreated with L-NAME or MC. Each column with a bar represents mean S.E.M. of 8 experiments. Contractile responses are expressed as the percentage from maximal contraction elicited by phenylephrine (3.0 10 7 M). p 0.05, p 0.01, compared with value of the corresponding vehicle group. p 0.01, compared with value of the experiment subgroup in the untreatment group. tific understanding of their therapeutic use for antihypertension associated with Qi deficiency caused by the disorder of spleen and kidney in China. First, although neurohumoral factors may contribute to antihypertension for the crude extract of A. membranaceus observed previously, 8) it is apparent that direct endothelium-dependent and independent vasorelaxation should be prominently taken into account according to this study. Second, A. membranaceus dose-dependently dilated endothelium-denuded rings contracted with PE. However, the agent had no effect on the vessel contracted by KCl. The cellular mechanism of contraction involved in the response to KCl and PE is different. KCl induce Ca 2 influx via voltagedependent Ca 2 channel, which further activate Ca 2 -induced Ca 2 release through ryanodine-receptor. PE increase intracellular Ca 2 concentration with two mechanisms, 1) through receptor-gated Ca 2 channels; 2) mobilizes Ca 2 from intracellular stores via the inositol-1,4,5-trisphosphate (IP 3 ) receptor or induces secondly myosin light chain phosphorylation via activating PKC. 17,18) That is, the vasorelaxant action of A. membranaceus seems to occur in a receptor-dependent manner in SMVC of rat aortas. However, the result that A. membranaceus failed to inhibit the contraction caused by addition of CaCl 2 in Ca 2 free solution was probably only due to its effects on the intracellular pathways. This hypothesis was confirmed in the further experiments that the contractions induced by PD and caffeine in Ca 2 -free medium were not affected by A. membranaceus equally. Thus, it appears that the IP 3 -induced Ca 2 release channels might be the site of action for A. membranaceus on endothelium-removed aortas. Third, NO is a potent vasodilator synthesized in the endothelium 19) by NO synthase, and causes VSMC relaxation through the activation of soluble guanylate cyclase. 20) The present study demonstrated that the extract of A. membranaceus dose-dependently inhibited the contraction induced by PE in intact aorta isolated from rats. This vasorelaxant action was mostly inhibited by treatment with L- NAME or MC. Endothelium-dependent relaxation of A. membranaceus seemed to be associated with NO signaling via guanylate cyclase activation since both L-NAME and MC could attenuate this response. This result was supported by other studies which had shown that A. membranaceus could increase the plasma level of NO 21) and induce the release of NO from VSMC in vitro by NO-soluble guanylate cyclasecyclic guanosine monophosphate pathway. 22) Finally, it was also observed that high dose (2.0 10 2 2.0 10 1 g/l) A. membranaceus induced a transient contraction in arterial rings pre-constricted by PE. This result was identical with that obtained in another report where treatment with high dose of A. membranaceus reversibly increased blood pressure of patients without hypertension previously. 23) The mechanism underlying is under investigation. Blockage of the contraction by endothelin-converting enzyme phosphoramidon may suggest the involvement of an endothelin receptor mediated signaling. In summary, the findings indicated that A. membranaceus have diphasic effects of relaxation and contraction on the aorta rings. The mechanisms of relaxation may include inhibition of intracellular Ca 2 release in vascular smooth muscle cells, and endothelium dependent relaxation mediated by

1454 Vol. 28, No. 8 the NO-GC pathway, whilst the effect of transient contraction of A. membranaceus at a high dose was related with endothelin release. Acknowledgements This work was supported by grants from Province Administration of Traditional Chinese Medicine, Zhejiang, P.R. China (Project Number: 2000C54 and 2003C078); Foundation for Returnee from Personal Department of Zhejiang Province, P.R. China (Project Number: ZRZ-2001275); and by E-Institutes of Shanghai Municipal Education Commission, P.R. China (Project Number: E- 04010). The authors are grateful to Prof. Wenquan Liang of the Department of Pharmacology, Zhejiang University, for his technical help. REFERENCES 1) Toda S., Shirataki Y., J. Ethnopharmacol., 68, 331 333 (1999). 2) Jiao Y., Wen J., Yu X., Chin. J. Integrated Tradit. Chin. West. Med., 19, 356 358 (1999). 3) Huang Z. Q., Qin N. P., Ye W., Chin. J. Integrated Tradit. Chin. West. Med., 15, 328 330 (1995). 4) Gu Y., Deng D. M., Chin. Arch. Tradit. Chin. Med., 19, 324 326 (2001). 5) Jia S. Q., Tianjin Pharm., 6, 24 26 (1994). 6) Zhao C. L., Tradit. Chin. Med. Res., 14, 25 26 (1998). 7) Zha Y. Z., J. Tradit. Chin. Med., 41, 329 (2000). 8) Song D. J., Gu D. G., Mao S. Y., Chin. Tradit. Herb. Drugs, 20, 25 28 (1989). 9) Chen Z. K., Hu S. J., Sun J., Xia Q., Shen Y. L., Zheng X., Chin. J. Lab. Diagn., 7, 403 405 (2003). 10) Chen Z. K., Hu S. J., Sun J., Xia Q., Shen Y. L., Tradit. Chin. Drug Res. & Clin. Pharmacol., 14, 372 374 (2003). 11) Chen Z. K., Hu S. J., Zheng X., Wang G. B., Sun J., Xia Q., Shen Y. L., Chin. J. Chin. Mater. Med., 28, 155 158 (2003). 12) Guo Z. G., Xue S. W., J. Tradit. Chin. Med., 21, 73 75 (1980). 13) Ko W. H., Yao X. Q., Lau C. W., Law W. I., Chen Z. Y., Kwok W., Ho K., Huang Y., Eur. J. Pharmacol., 399, 187 196 (2000). 14) Chew D. K., Orshal J. M., Khalil R. A., Hypertension, 42, 818 824 (2003). 15) Huang Y., Life Sci., 60, 1749 1756 (1997). 16) Zimmermann M., Jung C. S., Vatter H., Raabe A., Seifert V., Acta Neurochir. (Wien), 144, 1213 1219 (2002). 17) Karaki H., Ozaki H., Hori M., Mitsui-Saito M., Amano K., Harada K., Miyamoto S., Nakazawa H., Won C. J., Sato K., Pharmacol. Rev., 49, 157 230 (1997). 18) Kobayashi S., Gong M. C., Somlyo A. V., Somlyo A. P., Am. J. Physiol., 260, C364 370 (1991). 19) Moncada S., Palmer R. M., Higgs E. A., Pharmacol. Rev., 43, 109 142 (1991). 20) Martin E., Davis K., Bian K., Lee Y. C., Murad F., Semin. Perinatol., 24, 2 6 (2000). 21) He L., Wang R. G., You Z. L., Yang Z. W., Chen X. D., Li J., Zhan X. L., Wang J., Acad. J. Hunan Tradit. Chin. Med., 23, 4 6 (2003). 22) Wang K. H., Foreign Med. Sci.: TCM Section, 18, 38 39 (1996). 23) Wang C. H., Zhang J. J., Shandong J. Tradit. Chin. Med., 15, 351 (1996).