* CorrespCollege oDongdaemE-mail: hy

PHYTOTHERAPY RESEARCHPhytother. Res. 27: 1308–1312 (2013)Published online 30 October 2012 in Wiley Online Library(wileyonlinelibrary.com) DOI: 10.1002/ptr.4874

Copyright

Endothelium-Independent Vasorelaxation Effectsof Sigesbeckia glabrescens (Makino) Makino onIsolated Rat Thoracic Aorta

Kyungjin Lee, Jinho Jung, Gabsik Yang, Inhye Ham, Youngmin Bu, Hocheol Kim andHo-Young Choi*Department of Herbology, College of Korean Medicine, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701,Republic of Korea

The present study aimed to investigate the vasorelaxant effect of the methanol extract of Sigesbeckia glabrescens(Makino) Makino (MESG) on rat aortic rings and mechanism of action. MESG inhibited both noradrenalinebitartrate (NA)- and potassium chloride (KCl)-induced contraction of endothelium-intact aortic rings in aconcentration-dependent manner. Removal of the endothelium did not influence the effect of MESG on NA-precontracted aortic rings. Pretreatment withMESG (250mg/mL) inhibited calcium chloride-induced vasocontractionof NA- or KCl-precontracted endothelium-denuded aortic rings. It also relaxed phorbol-12-myristate-13-acetate-induced contraction of aortic rings in a concentration-dependent manner. In addition, Bay K8644 (an L-typecalcium channel opener) vasocontracted in MESG pretreated aortic rings. On the other hand, the inositol 1,4,5-triphosphate receptor, the ryanodine receptor, the Rho-kinase inhibitor (Y-27632), a soluble guanylyl cyclase blocker(1-H-[1,2,4]-oxadiazolo-[4,3a]-quinoxalin-1-one), and K+ channel blockers (glybenclamide, tetraethylammonium,and 4-aminopyridine) did not affect the effect of MESG. These results suggested that the mechanism underlyingthe vasorelaxant effect of MESG is mediated by endothelium-independent pathways. This specifically refers toblockade of the influx of extracellular Ca2+ via receptor-operative Ca2+ channels and voltage-dependent Ca2+

channels and inhibition of a protein kinase C-mediated cellular pathway. Copyright © 2012 JohnWiley& Sons, Ltd.

Keywords: Sigesbeckia glabrescens; vasorelaxation; rat aortic rings; protein kinase C; receptor-operative Ca2+ channel; voltage-dependent Ca2+ channel.

INTRODUCTION

Hypotensive effects are related to a variety of activitiessuch as regulation of blood volume, peripheral vascularresistance, vasoreactivity, and activities related to thenervous system and renin-angiotensin system (Perezand Musini, 2008). Vasoreactivity is a basic, importantphenomenon that directly influences arteries of thecirculatory system. Therefore, many researchers haveinvestigated the vasorelaxant effects of various plantextracts as hypotensive agents (Park et al., 2009; Xiaet al., 2008; Koh et al., 2007; Kang et al., 2005).The medicinal plant Sigesbeckia glabrescens (Makino)

Makino (Asteraceae) is an annual herb that is widelydistributed throughout East Asia. The aerial part of thisplant is commonly known as ‘Jindeukchal’ in Korea. Ithas been used in traditional medicine to treat arthritis,paralysis, and dermatological diseases (Kim et al.,1998) and reportedly exerts various pharmacologicaleffects such as antiallergic (Kim et al., 2001), anticancer(Jun et al., 2006), anti-inflammatory (Kim et al., 2008),antidiabetic (Kim et al., 2006), and hypotensive activities(Shin 2000). Several diterpenoids have been isolated(Kim et al., 2008; Liu and Röder 1991), and one such

ondence to: Dr Ho-Young Choi, Department of Herbology,f Korean Medicine, Kyung Hee University, 1 Hoegi-dong,un-gu, Seoul 130-701, Republic of Korea.choi@khu.ac.kr

© 2012 John Wiley & Sons, Ltd.

compound, kirenol, has been used to qualitatively identifyS. glabrescens (Cheng et al., 2005). Although, the vasore-laxant effect of S. glabrescens was reported previously,there was no data about the mechanism of action (Shin2000). Thus, we designed the present study to investigatethe exact mechanisms underlying the vasorelaxant effectof S. glabrescens. We used isolated rat aortic rings toassess the effects of the methanol extract of the aerial partof S. glabrescens (MESG) on various vasorelaxant orvasocontraction factors.

MATERIALS AND METHODS

Chemicals and drugs. Noradrenaline bitartrate (NA),acetylcholine (ACh), potassium chloride (KCl), calciumchloride (CaCl2), ethylene glycol-bis (b-aminoethylether)-N,N,N0,N0-tetraacetic acid (EGTA), phorbol-12-myristate-13-acetate (PMA), tetraethylammonium (TEA),glybenclamide, 4-aminopyridine (4-AP), caffeine, BayK8644, Y-27632, nifedipine, and 1-H-[1,2,4]-oxadiazolo-[4,3a]-quinoxalin-1-one (ODQ), and dimethyl sulfoxide(DMSO) were purchased from Sigma Aldrich (St. Louis,MO, USA). All other reagents were of analytical purity.

Sample preparation. S. glabrescens (Makino) Makinowas collected during September 2006 from Pocheon,Kyunggi-Province, Republic of Korea. ProfessorHocheol Kim of Kyung Hee University identified the

Received 22 July 2011Revised 18 September 2012Accepted 04 October 2012

1309VASORELAXATION EFFECTS OF SIGESBECKIA GLABRESCENS (MAKINO) MAKINO

plant. For the standardization, the content of kirenol inraw material was analyzed by high-performance liquidchromatography and found that it contained 0.67%(data not shown). A voucher specimen (SG001) ofS. glabrescens was deposited at the Department ofHerbology, College of Oriental Medicine, KyungHee University. Dried aerial parts of S. glabrescens(500.0 g) were extracted three times with 100% MeOH(5.0 L) for 2 h in a reflux apparatus. After reflux andfiltration, the extracts were evaporated using a rotaryevaporator at 60 �C and lyophilized to yield 68.0 g ofcrude extract. MESG (1.0 g) was dissolved in DMSO(10.0mL).

Preparation of rat aortic rings. Sprague–Dawley rats(weight, 240–260 g) were purchased from Narabio(Seoul, Korea). Animal care and all experimentalprocedures were approved and conducted accordingto Kyung Hee University Institutional Animal Careand Use Committee (KHUASP(SE)-09-040). Rats werehoused under controlled temperature (22� 2 �C), constanthumidity, a 12 h light/dark cycle (lights on from 07:00 to19:00), and food and water were available ad libitum.Rats were killed by cervical dislocation after etheranesthesia, the thoracic aorta was rapidly removed andimmersed inKrebs-Henseleit solution [KHS, composition(mM): NaCl, 118.0; KCl, 4.7; MgSO4, 1.2; KH2PO4, 1.2;CaCl2, 2.5; NaHCO3, 25.0; and glucose, 11.1; pH 7.4],maintained at 37 �C, and aerated with a mixture of 95%O2 and 5%CO2. Aortic rings were cut into approximately2mm and placed between two tungsten stirrups andconnected to an isometric force transducer. The vesselsegments were allowed to equilibrate for 1 h at a restingtension of 2.0 g. The KHS was replaced every 15minduring the equilibration period. The changes in isometrictension were recorded via transducers connected to aGrass Polygraph recording system (Grass Instrument Co.,Rhode Island, USA). When required, the endotheliumwas removed by gently rubbing the lumen of the vesselwith a thin cotton swab. The presence of functionalendothelium was verified by the ability of ACh (10mM)to induce more than 80% relaxation in rings that wereprecontracted by NA (1mM). In endothelium-denudedrings, ACh caused less than 10% relaxation. Ca2+ freeKHS was prepared by replacing CaCl2 with EGTA(1mM).

Effect of MESG on NA- and KCl-induced contractionin endothelium-intact or -denuded aortic rings.Endothelium-intact aortic rings were incubated withMESG (50, 100, or 250 mg/mL) for 10min, and differentconcentrations of NAwere added (1�10–10–5� 10–6M).In another experiment, rings were precontracted withKCl (60mM). After a plateau was attained, MESG wasadded (50, 100, or 250mg/mL). To investigate whetherthe effect was related to endothelium, the relaxant effectsof MESG (50, 100, or 250mg/mL) on endothelium-intactand -denuded aortic rings precontracted by NA (1mM)were tested.

Effect of MESG on Ca2+ channels. To investigate theeffect of MESG on extracellular Ca2+-induced contraction,endothelium-denuded aortic rings were preincubated withMESG (250mg/mL) and DMSO (2.5 mL/mL) for 15minbefore contraction with NA (1mM) or KCl (60mM) in

Copyright © 2012 John Wiley & Sons, Ltd.

Ca2+ free KHS and then the contractile responses inducedby CaCl2 (0.01–10mM) were investigated.

To investigate whether the effect of MESG wasrelated to L-type Ca2+ channel, the same procedurewere conducted with Bay K8644 (1 nM–10 mM), anL-type Ca2+ channel opener instead of CaCl2. Responsesto Bay K8644 were expressed as increased amount fromthe baseline which is precontracted by KCl (60mM).

To investigate the effect of MESG on the intracellularrelease of Ca2+ from the sarcoplasmic reticulum (SR)during contraction, endothelium-denuded aortic ringswere contracted (C1) by NA (1 mM) or caffeine(20mM) after a 15-min incubation in Ca2+ free KHS.After contraction, the rings were washed twice withKHS and incubated for 40min to refill the intracellularCa2+ stores. After incubation with Ca2+ free KHS for15min, the rings were contracted (C2) once more byapplying NA or caffeine without (control) and withpretreatment with MESG (250 mg/mL) 10min beforecontraction. Data were obtained by calculating the ratiobetween C1 and C2.

Effects of MESG on PMA-induced contraction.Endothelium-denuded aortic rings were precontractedwith PMA (1mM), a protein kinase C (PKC) activator,in Ca2+ free KHS. After a plateau was attained, MESGwas added (10–250 mg/mL).

Role of K+ channels, Rho-kinase pathway, and solubleguanylyl cyclase (sGC) in MESG-induced relaxation.Endothelium-denuded aortic rings were preincubatedwith various agents, a K+ channel blocker [TEA(5mM), glybenclamide (10 mM), or 4-AP (1mM)], aselective Rho-kinase inhibitor [Y-27632 (1mM)], or aspecific inhibitor of sGC [ODQ (1 mM)], for 15minbefore the addition of NA (1mM). Once a plateau wasattained, MESG (250 mg/mL) was added.

Data analysis. Data are expressed as mean� standarderror of mean (SEM). Statistical comparisons betweentwo groups were evaluated using Student’s t-test, andamong multiple groups were made by using a one-wayANOVA followed by Tukey’s post-hoc test. All statisticalanalyses were performed using SPSS v.13.0 statisticalanalysis software (SPSS Inc., Chicago, IL, USA).P valuesless than 0.05 were considered statistically significant.

RESULTS

Effect of MESG on NA- or KCl-induced contraction

MESG caused a concentration-dependent relaxationon NA- or KCl-induced contraction (Fig. 1A, 1B). Inthese experiments, treatment of DMSO (25 mL, finalconcentration of 2.5 mL/mL in KHS filled chamber) didnot alter the contractions induced by NA or KCl(Fig. 1A, 1B). Therefore, we did not consider the effectof DMSO in further experiments. In addition, MESGcaused concentration-dependent relaxation in both NA-precontracted endothelium-intact and -denuded aorticrings (Fig. 1C).

Phytother. Res. 27: 1308–1312 (2013)

Figure 1. (A) Inhibitory effect of MESG on NA-induced contractionof aortic rings. (B) Inhibitory effect of MESG on KCl-precontractedaortic rings. (C) Vasorelaxant effect of MESG on NA-precontractedaortic rings with (E+) or without (E-) endothelium. Valuesare expressed as mean�SEM (n=5–7). *P<0.05 vs. control.**P<0.01 vs. control.

Figure 2. (A) Inhibitory effect of MESG (250mg/mL) on thecontraction induced by extracellular Ca2+ in endothelium-denudedaortic rings precontracted by NA (1mM) or KCl (60mM). (B)Contraction responses induced by Bay K8644 (1 nM–10 mM) onaortic rings precontracted with KCl (60mM) in the presence orabsence of MESG. (C) Inhibitory responses of MESG (250mg/mL)on NA (1mM)- or caffeine (20mM)-induced transient contractionsof aortic rings. C2/C1 represents the ratio of the second contractionto the first contraction. Values are expressed as mean�SEM(n=4–6). **P<0.01 vs. control (NA), ##P<0.01 vs. control (KCl).

1310 K. LEE ET AL.

Effect of MESG on Ca2+ channels

Preincubation with MESG significantly inhibited the con-tractions induced by extracellular CaCl2 (1 and 10mM)adding both in NA- and KCl-precontracted aortic rings(Fig. 2A). In addition, BayK8644 (1 nM–10mM) contracted

Copyright © 2012 John Wiley & Sons, Ltd.

more in KCl-induced vasocontraction in a concentration-dependent manner. When MESG (250mg/mL) preincu-bated, the effects of Bay K8644 were higher than theabsence of MESG. However, higher concentration of

Phytother. Res. 27: 1308–1312 (2013)

1311VASORELAXATION EFFECTS OF SIGESBECKIA GLABRESCENS (MAKINO) MAKINO

BayK8644 (1 and 10mM) produced no further contractionor even relaxation (Fig. 2B). In addition, MESG did notinfluence the NA-induced transient contraction ratio orthe caffeine-induced transient contraction ratio (Fig. 2C).

Effect of MESG on PMA-induced contraction

The cumulative application of MESG resulted in aconcentration-dependent relaxation of the PMA-inducedcontraction, and it showed 51.3%of vasorelaxant effect in250mg/mL treatment (Fig. 3).

Figure 4. Relaxation responses induced by MESG (250mg/mL) onendothelium-denuded aortic rings precontracted with NA (1mM)in the presence of glybenclamide (1mM), TEA (5mM), 4-AP(1mM), ODQ (1mM), or Y-27632 (1 mM). Values are expressed asmean�SEM (n=4–8).

Role of K+ channels, Rho-kinase pathway and sGC inMESG-induced relaxation

The vasorelaxant effects of MESG on NA-precontractedaortic rings were not altered by incubation of the ringswith glybenclamide, TEA, 4-AP, ODQ, or Y-27632(Fig. 4).

DISCUSSION

In the present study, MESG had a relaxant effect onboth NA- and KCl-precontracted aortic rings. Theremoval of functional endothelium did not change theseresponses. MESG inhibited the vasocontraction inducedby Ca2+ supplementation in aortic rings precontractedwith NA or KCl in Ca2+ free KHS. MESG also had arelaxant effect on PMA-precontracted aortic rings. Onthe other hand,MESGdid not inhibit the vasocontractioninduced by NA or caffeine in Ca2+ free KHS. TEA,glybenclamide, 4-AP, ODQ, and Y-27632 did not affectthe vasorelaxant effect of MESG.The vascular endothelium and smooth muscle play

important roles in vasorelaxation (Ersoy et al., 2008). Inthe present study, MESG equally relaxed endothelium-intact and -denuded aortic rings precontracted by NAor KCl. These results suggest that the vasorelaxantmechanism underlying the effects of MESG are regulated

Figure 3. Vasorelaxant effect ofMESGonPMA (1mM)-precontractedendothelium-denuded aortic rings. Values are expressed as mean�SEM (n=4). *P<0.05 vs. control. **P<0.01 vs. control.

Copyright © 2012 John Wiley & Sons, Ltd.

through vascular smooth muscle-related factors and notthrough endothelial-related factors. In vascular smoothmuscle, vasorelaxation is regulated by the influx ofextracellular Ca2+ via transmembrane Ca2+ channels,Ca2+ release from intracellular stores, PKC activation,opening of K+ channels, and direct inhibition of sGC(Chen et al., 2009).

We first examined the effect of an influx of extracellularCa2+ on MESG-induced relaxation. The influx of extra-cellular Ca2+ is mainly regulated by receptor operatedcalcium channels (ROCCs) or voltage-dependent calciumchannels (VDCCs) (Karaki et al., 1997). MESG inhibitedthe vasoconstriction induced by Ca2+ supplementation inaortic rings that had been precontracted with NA. Inaddition, MESG inhibited the vasoconstriction inducedby Ca2+ supplementation in aortic rings that had beenprecontracted withKCl. These results suggest thatMESGcan inhibit the entry of extracellular Ca2+ via ROCCs and/or VDCCs. Activation of VDCCs involved mainly phos-phorylation of L-type calcium channels, Ca2+-dependentinactivation via calmodulin, and direct G-protein-mediated inhibition of the neuronal N and P/Q channels(Dolphin 2009). In the present study, Bay K8644, anL-type Ca2+ channel opener, contracted aortic rings morein MESG pretreatment than non-treatment control. Thissuggests that MESG inhibits the entry of extracellularCa2+ via L-type Ca2+ channels.

The tension of vascular smooth muscle is regulatedby intracellular Ca2+ release from the SR via specificinositol-1,4,5-triphosphate receptor channels (IP3R) orryanodine receptor channels (RyR) (Ehrlich andWatras1988). In the present study, MESG pretreatment did notinhibit the transient contraction of aortic rings inducedby NA or caffeine in Ca2+ free KHS. These resultssuggest that the intracellular Ca2+ release from the SRvia the IP3R or RyR pathway is not involved inMESG-induced relaxation.

MESG decreased the PMA-induced contraction ofaortic rings in Ca2+ free KHS in a concentration-dependent manner. PMA activate PKC and inducesustained contraction via increasedCa2+ sensitivity withouta measurable increase in the intracellular Ca2+ level(Jiang and Morgan 1987). This result indicates that the

Phytother. Res. 27: 1308–1312 (2013)

1312 K. LEE ET AL.

PKC-mediated pathway may be another mechanism bywhich MESG induces vasorelaxation.The vasorelaxant effects ofMESGwere not affected by

pretreatment with TEA, glybenclamide, 4-AP, Y-27632,or ODQ. The opening of K+ channels in vascular smoothmuscle cells is a major mechanism involved in the regula-tion of muscle contractility and vascular tone (Nelson andQuayle 1995). TEA, glybenclamide, and 4-AP are well-known K+ channel blockers used to investigate the K+

channel-related mechanisms of the vasorelaxant effectsof various agents. sGC is known to overload cGMP intosmooth muscle cells, inducing vasorelaxation (Rho et al.,2002). Y-27632 is a Rho-kinase inhibitor that blocksagonist-induced Ca2+ sensitization of smooth muscle(Shimokawa 2002). Our results indicate that the relaxanteffect of MESG on the rat aortic rings is not related tothe opening of K+ channels, Rho-kinase pathway, or thecGMP pathway.

Copyright © 2012 John Wiley & Sons, Ltd.

In conclusion, our results suggest that the vasorelaxantmechanism of MESG is an endothelium-independentpathway that include blockade of the influx of extracellu-lar Ca2+ through ROCCs and VDCCs involving mainlyL-type voltage-gated Ca2+ channels and inhibition of thePKC-mediated cellular pathway. However, the Rho-kinase pathway, IP3 pathway, RyR pathway, K+ channels,and sGC are not involved in the relaxant effect ofMESG.

Acknowledgement

This work was supported by the Stage of Brain Korea 21 project inKorean Medical Science Center.

Conflict of Interest

The authors have declared that there is no conflict of interest.

REFERENCES

Chen GP, Ye Y, Li L, Yang Y, Qian AB, Hu SJ. 2009. Endothelium-independent vasorelaxant effect of sodium ferulate on ratthoracic aorta. Life Sci 84: 81–88.

Cheng ZH, Chou GX, Wang ZT. 2005. A study on quality standardfor Herba Siegesbeckidae. Zhongguo Zhong Yao Za Zhi 30:257–259.

Dolphin AC. 2009. Calcium channel diversity: multiple roles ofcalcium channel subunits. Curr Opin Neurobiolo 19: 237–244.

Ehrlich BE, Watras J. 1988. Inositol 1,4,5-trisphosphate activatesa channel from smooth muscle sarcoplasmic reticulum. Nature336: 583–586.

Ersoy S, Orhan I, Turan NN, Sahan G, Ark M, Tosun F. 2008.Endothelium-dependent induction of vasorelaxation byMelissa officinalis L. ssp. officinalis in rat isolated thoracicaorta. Phytomedicine 15: 1087–1092.

Jiang MJ, Morgan KG. 1987. Intracellular calcium levels in phorbolester-induced contractions of vascular muscle. Am J Physiol253: H1365–1371.

Jun SY, Choi YH, Shin HM. 2006. Siegesbeckia glabrescensinduces apoptosis with different pathways in human MCF-7and MDA-MB-231 breast carcinoma cells. Oncol Rep 15:1461–1467.

Kang DG, Lee JK, Choi DH, Shon EJ, Moon MK, Lee HS. 2005.Vascular relaxation by the methanol extract of Sorbus cortexvia NO-cGMP pathway. Biol Pharm Bull 28: 860–864.

Karaki H, Ozaki H, Hori M, et al. 1997. Calcium movements,distribution, and functions in smooth muscle. Pharmacol Rev49: 157–230.

Kim HC, Ahn DK, Lee SI. 1998. Antihypertensive effects ofcrude and steamed with alcohol Siegesbeckiae Herba on thespontaneous hypertensive rats. Kor J Herbology 13: 53–58.

Kim HM, Lee JH, Won JH, et al. 2001. Inhibitory effect onimmunoglobulin E production in vivo and in vitrobySiegesbeckiaglabrescens. Phytother Res 15: 572–576.

Kim JY, Lim HJ, Ryu JH. 2008. In vitro anti-inflammatory activity of3-O-methyl-flavones isolated from Siegesbeckia glabrescens.Bioorg Med Chem Lett 18: 1511–1514.

Kim S, Na M, Oh H, et al. 2006. PTP1B inhibitory activity ofkaurane diterpenes isolated from Siegesbeckia glabrescens.J Enzyme Inhib Med Chem 21: 379–383.

Koh SB, Kang MH, Kim TS, et al. 2007. Endothelium-dependentvasodilatory and hypotensive effects of Crotalaria sessilifloraL. in rats. Biol Pharm Bull 30: 48–53.

Liu K, Röder E. 1991. Diterpenes from Siegesbeckia glabrescens.Planta Med 57: 395–396.

Nelson MT, Quayle JM. 1995. Physiological roles and properties ofpotassium channels in arterial smooth muscle. Am J Physiol268: C799–822.

Park JY, Shin HK, Lee YJ, Choi YW, Bae SS, Kim CD. 2009. Themechanism of vasorelaxation induced by Schisandra chinensisextract in rat thoracic aorta. J Ethnopharmacol 121: 69–73.

Perez MI, Musini VM. 2008. Pharmacological interventions forhypertensive emergencies: a Cochrane systematic review.J Hum Hypertens 22: 596–607.

Rho EH, Perkins WJ, Lorenz RR, Warner DO, Jones KA. 2002.Differential effects of soluble and particulate guanylyl cyclaseon Ca(2+) sensitivity in airway smooth muscle. J Appl Physiol92: 257–263.

Shimokawa, H. 2002. Rho-kinase as a novel therapeutic target intreatment of cardiovascular diseases. J Cardiovasc Pharmacol39: 319–327.

Shin H. 2000. Effects of Siegesbeckia glabrescens on the vascularrelaxation and antioxidative status. J Korean Oriental Med21: 77–83.

Xia M, Qian L, Zhou X, Gao Q, Bruce IC, Xia Q. 2008. Endothelium-independent relaxation and contraction of rat aorta inducedby ethyl acetate extract from leaves of Morus alba (L.).J Ethnopharmacol 120: 442–446.

Phytother. Res. 27: 1308–1312 (2013)