SUMMARY

1. Baicalin was isolated and purified from the dry roots ofScutellaria baicalensis Georgi (Huangqin; a traditionally usedChinese medicinal herb) and its effect on the contractility of ratisolated mesenteric arteries was investigated and the role of theendothelium was examined.

2. The concentration-dependent contractile response toU46619 was enhanced by 10–5 mol/L baicalin in endothelium-intact rings, but this effect was abolished in the presence of 10–4 mol/L NG-nitro-L-arginine or in endothelium-denuded rings.

3. Pretreatment of endothelium-intact rings with baicalin (3 � 10–5 mol/L) markedly attenuated the relaxant response to A23187, thapsigargin and acetylcholine.

4. The present results indicate an important role for endo-thelial nitric oxide (NO) in the vascular response to baicalin.Baicalin appears to inhibit NO production and release in theendothelium and this mechanism is likely to be responsible forthe enhancement of the U46619-induced contraction and forinhibition of endothelial NO-mediated relaxation by baicalin inrat mesenteric artery.

Key words: baicalin, contraction, endothelium, rat mesentericartery, Scullaria baicalensis.

INTRODUCTION

The dry root of Scutellaria baicalensis Georgi (Huangqin) has beenused for centuries in Chinese herbal medicine. The antithrombotic,cholesterol-reducing and antiproliferaive effects of Huangqin orbaicalein and baicalin have been described.1–3 We have recently isolated and purified baicalein and baicalin, the two biologicallyactive flavonoids from Huangqin, and found that both compoundspossessed anti-oxidative activity4 and that baicalein induced endo-

thelium-dependent contraction and endothelium-independent relaxation in rat mesenteric arteries.5 In the present study, we further examined the role of endothelium-derived vasoactive factors in the vascular response to purified Huangqin baicalin.

METHODS

Experimental protocol

Male Sprague-Dawley rats (approximately 300 g) were killed by cervicaldislocation. The main branch of the superior mesenteric artery was dissectedand the surrounding connective tissue was trimmed off. The artery was cutinto three ring segments 3 mm in length. Each ring was mounted betweentwo stainless steel hooks in Krebs’ solution-filled baths. The Krebs’ solutioncontained (in mmol/L): NaCl 119; KCl 4.7; CaCl2 2.5; MgCl2 1; NaHCO3

25; KH2PO4 1.2; D-glucose 11.1. The bath solution was continuously bubbled with 95% O2 and 5% CO2 and kept at 37°C. During a 90 min equilibrium period, the basal tone was maintained at 5 mN. Isometric con-tractions were measured with a Grass FT03 force transducer (GrassInstrument Co., Quincy, MA, USA). In the first set of experiments, two concentration–effect curves to U46619 were established in control rings andin the presence of baicalin or baicalin plus NG-nitro-L-arginine (L-NNA) inendothelium-intact rings. The effect of baicalin was also examined onendothelium-dependent relaxation induced by A23187, thapsigargin oracetylcholine.

Drugs

Phenylephrine, acetylcholine, U46619, L-NNA, thapsigargin, A23187 wereused in this study. Baicalin was isolated and purified from the ground root of Huangqin4 and its chemical structure is shown in Fig. 1. Baicalin,thapsigargin, A23187 and U46619 were dissolved in dimethyl sulphoxide(DMSO) and further dilutions were made in fresh Krebs’ solution. At 0.2%v/v, DMSO did not affect U46619-induced tension.

Statistics

The effects of vasodilators on the U46619-induced sustained tone wereexpressed as percentages of the control and the IC50 is the drug concentrationthat caused half-maximal relaxation. Data are the mean�SEM from nexperiments. P < 0.05 obtained from two-tailed Student’s t-test was con-sidered significant.

RESULTS

U46619 induced a contractile response with an EC50 of 2.4 �

0.02 � 10–8 mol/L in endothelium-intact rings and 1.0 � 0.03 �

10–8 mol/L in endothelium-denuded rings (n = 4). The traces in

Proceedings of the Third Annual Scientific Meeting of the Institute ofCardiovascular Science and Medicine: Bridging Cardiovascular

Science and Medicine

BAICALIN-INDUCED VASCULAR RESPONSE IN RAT MESENTERICARTERY: ROLE OF ENDOTHELIAL NITRIC OXIDE

Yu Huang,* Suk Ying Tsang,* Xiaoqiang Yao,* Chi Wai Lau,* Ya Lun Su† and Zhen Yu Chen†

Departments of *Physiology and †Biochemistry, Chinese University of Hong Kong, Hong Kong, China

Correspondence: Dr Yu Huang, Department of Physiology, Faculty ofMedicine, Chinese University of Hong Kong, Shatin, NT, Hong Kong. Email:yu-huang@cuhk.edu.hk

Presented at the Third Annual Scientific Meeting of the Institute ofCardiovascular Science and Medicine, Hong Kong, October 1999. The papersin these proceedings have been peer reviewed.

Received 20 December 2001; accepted 20 February 2002.

Clinical and Experimental Pharmacology and Physiology (2002) 29, 721–724

722 Y Huang et al.

Fig. 2 show that baicalin (10–5 mol/L) enhanced U46619-inducedcontraction (EC50 1.6 � 0.05 � 10–8 mol/L; n = 4; Fig. 2b), whereasthis effect was absent in endothelium-denuded rings (EC50

8.9 � 0.04 � 10–9 mol/L; n = 4; Fig. 2c). At 10–4 mol/L, L-NNA

also potentiated U46619-induced contractions (EC50 7.9 �

0.4 � 10–9 mol/L; n = 4), but baicalin failed to affect the response to U46619 in the presence of L-NNA (EC50 8.4 � 0.8 � 10–9 mol/L; n = 4; Fig. 2b).

Figure 3a shows that, in U46619-contacted endothelium-intactrings, A23187 induced a concentration-dependent relaxation.Pretreatment with 3 � 10–5 mol/L baicalin (Fig. 3b) significantlyreduced the A23187-induced relaxation (IC50 1.68 � 0.17 � 10–8

mol/L for control and 3.41 � 0.27 � 10–8 mol/L for baicalin; n = 4;P < 0.05; Fig. 3c). Similarly, both thapsigargin (Fig. 3d) and acetylcholine (Fig. 3e) caused concentration-dependent relaxationwith respective IC50 values of 1.51 � 0.05 � 10 and 1.48 � 0.05 �

10–9 mol/L, respectively (both n = 4). Pretreatment with 3 �

10–5 mol/L baicalin significantly reduced the endothelium-dependentrelaxation (IC50 3.66 � 0.26 � 10–9 mol/L for thapsigargin and 3.66 � 0.27 � 10–9 mol/L for acetylcholine; n = 4; P < 0.05 comparedwith respective controls; Fig. 3d,e).

Fig. 2 (a) The enhancing effect of 10–5 mol/L baicalin on the contractile responses to U46619 (arrows) in an endothelium-intact ring. (b) Concentration–effectcurves for U46619 in the absence (�; n = 4) and presence of 10–5 mol/L baicalin (�; n = 4) or 10–4 mol/L NG-nitro-L-arginine (L-NNA; �; n = 4) or baicalinplus L-NNA (�; n = 4) in endothelium-intact rings. (c) Concentration–effect curves for U46619 in the absence (�; n = 4) and presence of 10–5 mol/L baicalin(�; n = 4) in endothelium-denuded rings.

Fig. 1 The chemical structure of baicalin purified from Huangqin.

Baicalin and endothelium 723

DISCUSSION

The present results show that purified baicalin from S. baicalensisGeorgi (Huangqin) enhanced the contractile response to U46619 inrat endothelium-intact mesenteric arteries and that this effect waseliminated in endothelium-denuded rings. Pretreament of endothe-lium-intact rings with L-NNA, an inhibitor of nitric oxide synthase(NOS), abolished the enhancing effect of baicalin on muscle ten-sion, indicating that baicalin may suppress endothelial nitric oxide(NO)-mediated vascular responses. Failure of baicalin to constrictthe rings suggests that baicalin did not influence the basal libera-tion of endothelial NO. Further experiments showed that baicalincould impair endothelium/NO-dependent relaxation induced by threewell-established endothelium-dependent vasodilators, namelyA23187, thapsigargin and acetylcholine. We have demonstrated pre-viously the inhibitory effect of baicalein, another flavonoid purifiedfrom Huangqin, on endothelium-dependent, but not -independent,

relaxation.5 At present, it is unknown how baicalin and baicaleininhibit NO formation and release in the endothelium. EndothelialNO production requires an increase of intracellular [Ca2+] andbaicalein has been reported to reduce endothelial [Ca2+]i in culturedhuman umbilical vein endothelial cells.6 However, this possiblemechanism could not explain the inhibitory effect of baicalin onrelaxation induced by A23187, thapsigargin and acetylcholinebecause these three dilators act at different sites to raise endothe-lial [Ca2+]i. It is possible that lipid-soluble baicalin may have a directeffect on endothelial NOS. It is yet to be determined whether baicalincan reduce endothelial NO-mediated vasodilatation in vivo.

ACKNOWLEDGEMENT

The authors thank the Hong Kong Research Grants Council for sup-port of this work.

Fig. 3 Traces showing the endothelium-dependent relaxation induced by A23187 (arrows) in the absence (a) and presence (b) of 3 � 10–5 mol/L baicalinin endothelium-intact rings. Concentration–response curves for (c) A23187, (d) thapsigargin and (e) acetylcholine (�, for control; �, 3 � 10–5 mol/L baicalin;n = 4 for both).

724 Y Huang et al.

REFERENCES

1. Kubo M, Matsuda H, Tani T, Arichi S, Kimura Y, Okuda H. Studies on Scutellariae radix XII. Anti-thrombic actions of various flavonoids from Scutellarieae radix. Chem. Pharm. Bull. 1985; 33:2411–15.

2. Huang HC, Wang HR, Hsieh LM. Antiproliferative effect of baicalein,a flavonoid from a Chinese herb, on vascular smooth muscle cell. Eur.J. Pharmacol. 1994; 251: 91–3.

3. Ying J, Guo LQ. Scutellaria baicalensis Georgi. In: Ying J, Gu LQ (eds).Research of Chinese Herbal Medicine and Clinical Application I.Beijing Xue–yue Press, Beijing. 1994; 559–69.

4. Chen ZY, Su YL, Bi YR, Tsang SY, Huang Y. Effect of baicalein andacetone extract of Scutellaria baicalensis on canola oil oxidation. J. Am.Oil Chem. Soc. 2000; 77: 73–8.

5. Chen ZY, Su YL, Lau CW, Law WI, Huang Y. Endothelium-dependentcontraction and direct relaxation induced by baicalein in rat mesentericartery. Eur. J. Pharmacol. 1999; 374: 41–7.

6. Kimura Y, Yokoi K, Matsushita N, Okuda H. Effects of flavonoids iso-lated from Scutellariae radix on the production of tissue-type plas-minogen activator and plasminogen activator inhibitor-1 induced bythrombin and thrombin receptor agonist peptide in cultured humanumbilical vein endothelial cells. J. Pharm. Pharmacol. 1997; 49:816–22.