Effect of total flavonoid fraction of Astragalus complantus R.Brown on angiotensin II-induced portal-vein contraction in hypertensive rats.
The objective of the present study was to examine further the underlying mechanism of the antihypertensive effect of the total flavonoid (TF), extracted from the seed of Astragalus complanatus R. Brown.
Renovascular hypertension rats (RHR) were established by the two-kidney one clip (2K1C) method. The effect of TF on the contraction of portal vein was studied in an isolated preparation. The response of portal vein to angiotensin II (Ang II) was expressed as a percentage of the 100 mmol/l KC1 induced maximum contraction. We took the dose-response curve of portal vein to Ang II (from [10.sup.-9] to [10.sup.-6] mmol/1) as the control and then observed the change of curve after TF and Valsartan (Ang II receptor blocker) administration.
Ang II induced a concentration-dependent increase of the contraction amplitude (maximal increase, 46.53 [+ or -] 5.15% of 100mmol/l KCl induced contraction at Ang II [10.sup.-6]mmol/1 in RHR). The Ang II-induced portal vein contraction was prevented by TF with a concentration related manner (maximal inhibition amplitude from 46.53 [+ or -] 5.15% to 22.525 + 4.67% of 100 mmol/I KCl contraction at [10.sup.-6] mmol/l Ang II and 3.12 x [10.sup.-1] mg/I TF in RHR). The effect of TF on Ang II-induced portal vein contraction was similar to Valsartan.
These results showed that the antihypertensive action of TF was attributed to the dilation of vessels and is related to the blockade of the Ang II receptor.
[C] 2008 Published by Elsevier GmbH.
Keywords: Astragalus complanatus; Total flavonoid fraction; Portal vein; Angiotensin II receptor blocker; Contraction
Astragalus complanatus R.Brown is a widely used herbal material in traditional Chinese medicine. In China pharmacopein (PRC Pharmacopeia, 1990) it is indicated that the seed increases Yang, acts primarily on kidney and liver channels and improve function of sexual performance. Scientists had made a great deal of research on its chemical constituents and pharmacological effects, obtained 22 extracts, 43 fractions from seeds of Astragalus complanatus R.Brown, and found that it had anti-inflammation, lipid-modifying and liver-protective effects and improved the immune function and promoted blood circulation (Mulrow, 1998). The total flavonoid (TF) is an active fraction extracted from seeds of Astragalus complanatus R.Brown. During the past two decades, over 16 flavonoids have been isolated, including complanatoside, neocomplanoside, myricomplanoside, rhamnocitrin 3-O-[beta]-D glucoside, astragalin, kaempferol 3-O-[alpha]-L-arabinoside, daucosterol, myricetin, kaempferol, calycosin 7-O-glucoside, ononin, rhamnocitrin, formononetin, myricetin 5'-O-[beta]-D-glucopyranoside, myricetin 3-O-[beta]-D-glucopyranoside, quercetin 3-O-[beta]-D-glucopyranoside (isoquercitrin) (Chen and Liu, 1998; Cui et al., 1989, Gu et al., 1997).
Previous studies performed in animals have demonstrated the antihypertensive efficacy of the TF in anesthetized dogs (Tang and Xie, 1987). The antihypertensive efficacy of TF has also been demonstrated in anesthetized, normotensive rats (Yin et al., 1988). Our previous researches have proved that TF can decrease the blood pressure in renovascular hypertensive rats (RHR) and the spontaneously hypertensive rat (SHR) (Xue et al., 2002). This antihypertensive efficacy due to the decrease of total peripheral resistance was associated with renin-angiotensin system (Li et al., 2005). The aim of the present study was to find the underlying mechanism of the antihypertensive action in isolated portal vein in vitro method.
Materials and methods
Adult male Wistar rats (290 [+ or -] 30 g) were provided by the animal breeding center of Shan Dong University. Adult male spontaneously hypertensive rats (SHR) (230 [+ or -] 50g) were purchased from Cardiovascular Hospital of Beijing, China. All animals were housed in a temperature-controlled room (22 [+ or -] 1 [degrees]C) and in group of four per cage (polypropylene with a bedding of wood shavings) with water and food available ad libitum and were allowed to acclimate for at least 2 weeks. The experiments were approved by the Shan Dong University Animal Care and Use Committee.
A two-kidney one-clip (2K1C) model of renovascular hypertension (RHR) was induced as previously described (Mai et al. 1993). In brief, male Wistar rats weighting 150-180 g were anesthetized with hexobarbital (40 mg/kg i.p.). A U-shape sliver clip (0.2-0.25 mm ID) was placed on the left renal artery to cause partial renal occlusion. The right kidney remained untouched. The shame control group received similar surgical intervention except that the sliver clip was not inserted. Systolic blood pressure was measured weekly by tail-cuff plethysmography with rats under consciousness. Only those rats that developed systolic blood pressure above 150mmHg within 3 weeks were considered to 2K1C hypertensive rats and enrolled in this experiment (Thurston et al., 1980).
The portal vein was removed quickly in RHR (n = 12) and SHR (n = 12) under hexobarbital anesthesia (40 mg/kg i.p.) according to previously described methods (Sutter and Ljung, 1977). The Vein was dissected into helical strips (1.5 mm* 10 mm) under the microscope. One end of the portal vein was firmly fixed to the bottom of the organ bath containing Tyrode solution (NaCl 130, KC1 4.7, [KH.sub.2][PO.sub.4] 1.18, Mg[SO.sub.4]. 7[H.sub.2]O 1.17, Ca[Cl.sub.2].2[H.sub.2]O 1.6, Na[HCO.sub.3] 14.9, dextrose 5.5, CaNa-EDTA 0.01 mmol/l, pH = 7.4), which was maintained at 37[degrees]C and aerated with 95% [O.sub.2] and 5% [CO.sub.2]. The other side was mounted on a force-displacement transducer (LWA-20). The resting tension was set to 150-250 mg based on adjusting the blood vessel to a point at which maximum peak-to peak oscillations were observed. The experiment began after the vascular strip was equilibrated for about 2 h.
The response of portal vein to Ang II was presented as a percentage of the 100mmol/l KCl induced maximum contraction amplitude. The dose response curve of portal vein to Ang II served as the control group (Mikkelsen et al., 1984). The vascular preparations were exposed to increasing concentrations of Ang II (starting at [10.sup.-9] mmol/1) until the maximal contractile effect was achieved (ending at [10.sup.-6]mmol/l). The dose-effective curve of portal vein to Ang II was tested after TF (3.12 x [10.sup.-2]mg/1 and 3.12 x [10.sup.-1] mg/l) and Valsartan ([10.sup.-7]mmol/1) administration.
All data were expressed as mean values [+ or -] SD. The portal vein's reactions to increasing concentration of Ang II after TF administration were statistically compared with the control group and the group after Valsartan administration. The different groups were compared by one-way ANOVA analysis. A value of p > 0.05 was considered to be statistically significant.
Drugs and reagents
TF (purity 98.2%, quality control standard 90 [+ or -] 10%, No 010308) was supplied by the Center of Physiology and Pharmacology of the Medical Sciences Academy of Shan Dong Province, PR China. Ang II and Valsartan (N-(l-oxopentyl)-N-[[2'-( l H-tetrazol-5-yl)[1,1'-biphenyl]-4-yl]methyl]-L-Valine) were purchased from the Sigma Chemical Company and TianJin Nuohua pharmaceutial factory.
Ang II in concentrations from [10.sup.-9] to [10.sup.-4] mmol/l produced a concentration-dependent increase in portal vein contraction in both of SHR and RHR (maximal amplitude 35.44 [+ or -] 4.42% and 46.53 [+ or -] 5.15% of 100 mmol/1 KCI-induced contraction at [10.sup.-6] mmol/1 Ang II in SHR and RHR separately, Table 1). TF inhibited the Ang II-induced portal vein contraction significantly in both SHR (maximal amplitude from 35.44 [+ or -]4.42% to 22.567 [+ or -]3.29% of 100 mmol/1 KCI-induced contraction at [10.sup.-6] mmol/1 Ang II and 3.12 X [10.sup.-1] mg/l TF, p < 0.01, compared with control) and RHR (maximal amplitude from 46.53 [+ or -] 5.15% to 22.525 [+ or -]4.67% of 100mmol/l KC1 contraction at [10.sup.-6]mmol/1 Ang II and 3.12 X [10.sup.-1] mg/1 TF, p < 0.01, compared with control) (Table 1). The tendency of the dose-response curve of TF on Ang II-induced portal vein contraction was similar to Valsartan in both SHR and RHR (Table 1 and Fig. 1).
[FIGURE 1 OMITTED]
Table 1. Effect of TF and Valsartan on Ang II-induced portal contraction on SHR and RHR Ang II concentration (mmol/l) [10.sup.-9] [10.sup.-8] [10.sup.-7] [10.sup.-6] SHR Control 26.844 29.78 32.34 35.44 [+ or -] [+ or -] [+ or -] [+ or -] 4.5 3.79 % 4.28 % 4.41 % % TF (3.12 x 15.32 17.391 20.062 22.567 [10.sup.-1] [+ or -] [+ or -] [+ or -] [+ or -] mg/l) 2.2% **(#) 2.83% **# 3.05% **# 3.29% **# TF (3.12 x 19.912 21.86 25.889 27.703 [10.sup-2] [+ or -] [+ or -] [+ or -] [+ or -] mg/l) 2.59% * 3.03% * 3.78% ** 4.5% ** Valsartan 18.985 21.664 25.538 28.074 ([10.sup.-7] [+ or -] [+ or -] [+ or -] [+ or -] mmol/l) 2.37% ** 2.92% ** 3.62% ** 3.68% ** RHR Control 37.193 41.589 43.973 46.53 [+ or -] [+ or -] [+ or -] [+ or -] 4.34% 4.96% 5.4% 5.15% TF (3.12 x 13.28 16.514 20.078 22.525 [10.sup.-1] [+ or -] [+ or -] [+ or -] [+ or -] mg/l) 2.52% **# 3.28% **# 4.54% **# 4.67% **# TF (3.12 x 20.987 24.264 27.101 30.437 [10.sup-2] [+ or -] [+ or -] [+ or -] [+ or -] mg/l) 2.89% ** 3.31% ** 4.27% ** 4.68% ** Valsartan 21.509 23.114 26.039 29.223 ([10.sup.-7] [+ or -] [+ or -] [+ or -] [+ or -] mmol/l) 3.65% ** 3.73% 4.03% ** 4.1% ** The contraction amplitude was expressed as a percentage of the 100 mmol/l KCI induced maximum contraction amplitude. Values are expressed as means [+ or -] SD, n = 12, * p < 0.05, ** p < 0.01, versus control, # p < 0.05, versus Valsartan.
Flavonoids are nearly ubiquitous in plants. Plants and spices containing flavonoids have been used for thousands of years in traditional Eastern medicine. Over 4000 structurally unique flavonoids have been identified in plant sources (Harborne, 1986). Flavonoids are rich in seeds, citrus fruits, olive oil, tea, and red wine. They are low molecular weight compounds composed of a three-ring structure with various substitutions. This basic structure is comprised of two benzene rings (A and B) linked through a heterocyclic pyran or pyrone (with a double bond) ring (c) in the middle. Flavonoids can be subdivided according to the presence of an oxy group at position 4, a double bond between carbon atoms 2 and 3, or a hydroxyl group in position 3 of the C (middle) ring. In the flavonoid structure, a phenyl group is usually substituted at the 2-position of the pyrone ring. In isoflavonoids, the substitution is at the 3-position.
It has been demonstrated that the flavonoids possess anti-inflammatory, antioxidant, antiallergic, hepatoprotective, antithrombotic, antiviral, and anticarcinogenic activities (Middleton et al., 2000). In recent years it has been a major rekindling of interest in the studies of plant flavonoids on the cardiovascular system. The effects of flavone on myocardial postischemic -reperfusion recovery was studied by Ning et al. (1993). They found that flavone treatment caused better recovery of left ventricular developed pressure. Moreover end-diastolic pressures were significantly lower in the flavone-treated group compared with control (Ning et al., 1993).
In the present study we extracted the total flavonoid fraction (TF) from seeds of Astragalus complanatus R.Brown to investigate its hypotensive effect and the underlying mechanism in RHR and SHR. Our previous research demonstrated that TF can reduce the blood pressure in hypertensive animal models effectively but it had no significant effect on cardiac output, heart rate and dp/dtmax (Xue et al., 2002). Its depressive effect is probably due to the decrease of total peripheral resistance (TPR) (Li et al., 2005).The portal veins from hypertensive rats are functionally different from those of normotensive rats. The altered functional properties of portal vein in several respects resemble those of arterial resistance vessels (Sutter and Ljung, 1977) and can be used to investigate the underlying mechanism that TF decrease the TPR in hypertensive rats.
We found that TF inhibits Ang II-induced portal vein contraction in a dose-dependent manner in RHR and SHR through a blood vessel dilation mechanism. Ang II, the main effector peptide of the RAS, can induce the vasoconstriction through binding to the ATI receptor (Kaschina and Unger, 2003). In vitro experiments showed no other neurohumor factor affected the Ane II-induced portal vein contraction except the binding of AngII in AT1 receptor. The dose-response curve of Ang II-induced portal contraction after TF administration was similar to the curve after administration of the Ang II receptor blockers Valsartan. From these findings we conclude that TF attaches Ang II ATI receptor in the fashion of Ang II receptor blocker.
Hypertension is a major risk factor for stroke, coronary heart disease, heart failure and renal disease. Central to many of these pathophysiological processes is the renin-angiotensin system. Ang II receptor blockers are widely used in the treatment of hypertension and hypertension-related end organ damage, which are very effective antihypertensive drugs (Unger, 2002).
During the past three decades, traditional Chinese medicine, based primarily on plant materials, has been adopted throughout much of the Western world and became one of the fastest-growing healthcare choices in the United States. We demonstrated that total flavonoid has the ability to decrease the blood pressure in hypertensive animals and this potent vasodilatation agent acts probably on the Ang II receptor. In the other way as a Chinese traditional medicine it is more economical and exhibits less side effect than synthetic drugs.
This study was supported by a grant from Ninth Five-year Key Foundation of Health System in Shandong Province (9715), China. We thank the assistance provided by Professor Chai-Xiang Shu and Zhao-Ai Ping from Medical Sciences Academy of Shan Dong Province, People's Republic of China. We thank Dr. M.E. Kreis and Dr. M.'H. Mueller from Ludwig Maxillians University, Munich, Germany for revising the language.
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B. Xue (a), Jx. Li (b), Q. Chai (c), Zx Liu (c), Lb. Chen (b), *
(a) Department of Pathophysiology, School of Medicine, Shandong University, Jinan 250012, PR China
(b) Institute of Physiology, School of Medicine. Shandong University, Jinan 250012, PR China
(c) Shandong Academy of Medical Sciences, Jinan 250061, PR China
* Corresponding author. Tel.: + 86 531 88381910; fax: + 86 531 88382502.
E-mail address: firstname.lastname@example.org (L. Chen).
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|Author:||Xue, B.; Li, Jx.; Chai, Q.; Liu, Zx; Chen, Lb.|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Sep 1, 2008|
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