Research Article Effects of an Aqueous Extract of Dangguijagyagsan on Serum Lipid Levels and Blood Flow Improvement in Ovariectomized Rats

Transcription

1 Evidence-Bsed Complementry nd Alterntive Medicine, Article ID , 8 pges Reserch Article Effects of n Aqueous Extrct of Dngguijgygsn on Serum Lipid Levels nd Blood Flow Improvement in vriectomized Rts In Sil Prk, Hye Won Lee, Jin Ah Ryuk, nd Byoung Seob Ko Koren Medicine Bsed Herbl Drug Development Group, Herbl Medicine Reserch Division, Kore Institute of rientl Medicine, 1672, Yuseong-dero, Dejeon , Republic of Kore Correspondence should be ddressed to Byoung Seob Ko; bsko@kiom.re.kr Received 26 July 214; Revised 12 August 214; Accepted 12 August 214; Published 7 September 214 Acdemic Editor: Myeong Soo Lee Copyright 214 In Sil Prk et l. This is n open ccess rticle distributed under the Cretive Commons Attribution License, which permits unrestricted use, distribution, nd reproduction in ny medium, provided the originl work is properly cited. Dngguijgygsn (DJS), trditionl herbl prescription, hs long been used to tret menopuse-relted symptoms. We identified the crdioprotective effects of n queous extrct of DJS using n ovriectomized (VX) nd ferric chloride- (FeCl-) induced crotid thrombosis rt model. Femle Sprgue-Dwley (SD) rts were ovriectomized or Shm-operted (Shm-control). The ovriectomized rts were divided into three groups: VX with sline (VX-control), spirin 3 mg/kg/dy (VX-ASA), nd DJS 1 mg/kg/dy (VX-DJS). The tretments were dministered for 5 weeks. Then, blood smples were collected to nlyze the serum lipid levels nd pltelet ggregtion. The topicl ppliction of 4% FeCl 3 induced intrvsculr thrombosis, which ws used to test thrombotic occlusion nd for histologicl exmintion. Body weight nd the levels of totl cholesterol (TC), triglyceride (TG), nd LDL-cholesterol (LDL-C) incresed in the VX rts. These effects were reduced by ASA nd DJS tretment. In ddition, ASA nd DJS tretment significntly inhibited pltelet ggregtion. These tretments lso incresed time to occlusion nd decresed both thrombus size nd the presence of collgen fibers in surrounding vessel wlls compred with the Shm-control nd VX-control groups. These results suggest tht DJS hs beneficil effects in terms of preventing crdiovsculr disese in menopusl womn becuse it cn reduce the serum lipid levels nd improve blood flow by inhibiting pltelet ggregtion nd thrombus formtion. 1. Introduction The incidence of crdiovsculr disese (CVD) remins the leding cuse of deth, with high morbidity nd mortlity fter menopuse. Estrogen deficiency my cuse n increse in crdiovsculr risks by ffecting lipoprotein metbolism, pltelet ggregtion bility, nd vessel resistnce. It is well estblished tht the incidence of CVD is due to hyperlipidemi, condition chrcterized by significnt increses in totl cholesterol (TC), triglycerides (TG), nd low-density lipoprotein cholesterol (LDL-C) nd reduction in highdensity cholesterol (HDL-C) [1, 2]. Pltelets from hypercholesterolemic ptients were more rective to ggregting regents such s epinephrine nd denosine diphosphte (ADP) thn were pltelets from norml individuls, nd pltelets from hypercholesterolemic ptients showed hyperctivity nd shortened pltelet survivl ex vivo [3]. Estrogen deficiency in VX nimls nd menopusl womn cn cuse ctivted pltelet ggregtion nd thrombosis by ffecting pltelet gonist hydrolysis nd nitric oxide production in blood vessels. An therosclerotic plque rupture releses collgen, lipids, nd smooth muscle cells nd leds to pltelet ctivtion nd occlusive thrombosis, thereby stimulting the cogultion cscde system. Thus, bnorml pltelet ctivtion plys key role in interrupting blood flow, for which ntipltelet drugs re vilble to prevent intrvsculr thrombosis [4]. Hormone replcement therpy (HRT) is the combintion tretment with estrogen or progestin. As primry therpy, HRT hs been observed to exert protective effect ginst menopusl crdiovsculr disese bymodultingtheserumlipidprofilesndugmentingthe response to therosclerosis [5]. However, HRT studies in menopusl women hve reported side effects such s brest cncer, thromboembolic disese, nd ischemic stroke [6].

2 2 Evidence-Bsed Complementry nd Alterntive Medicine Tble 1: Composition of DJS queous extrct nd ingredients. Ingredients Contents (g) Peoni lctiflor Plls Cnidium officinle Mkino Alism orientle Juzepzuk Angelic gigs Nki Pori cocos Wolf Atrctylodes jponic Koidzumi Totl 29. Considering the negtive side effects, herbl medicines hve been given further ttention s cndidtes for lterntive therpy for sfe nd effective HRT [7]. Dngguijgygsn (DngQui-Shoyo-Sn in Chinese; Tokishkuykusn in Jpnese) is trditionl medicinl prescription tht hs long been used for the tretment of menopuse-relted symptoms in Est Asi. Previous gynecologic reports relted to DJS hve documented its improvement of ovrin hormones [8], estrogenic ction [9], nd neurotoxicity effects of DJS under postmenopusl conditions [1]. However, no studies hve investigted the effectiveness of DJS in restoring blood flow. In this study, spirin ws used s positive control. Aspirin is widely used nti-thrombosis drug, nd low-dose spirin hs been shown to hve preventive effect on thrombus formtioninnimltesting[11]. Thus, we imed to investigte the orl dministrtion of DJS (1 mg/kg/dy) for 5 weeks to determine the beneficil effects on serum lipid levels nd blood flow improvement in VX nd thrombosis-induced rt models in comprison with Shm-control group. 2. Mterils nd Methods 2.1. Chemicls nd Regents. Acetonitrile (1%), formic cid (99.9%), nd distilled wter were purchsed from J.T. Bker (HPLC grde, USA). Reference compounds of lbiflorin nd peoniflorin were purchsed from Wko (Jpn). z- Ligustilide ws purchsed from ChromDex (USA). Nodkenin nd decursin were divided from Ministry of Food nd Drug Sfety (Kore) Plnt Extrcts. The formul of DJS consists of 6 herbs, including Peoni lctiflor (93.75 g), Cnidium officinle (56.25 g), Alism orientle (56.25 g), Angelic gigs (28.13 g), Pori cocos (28.13 g), nd Atrctylodis rhizom lb (28.13 g). Briefly, 29 g of the 6-herb mixture ws boiled in 6 L of distilled wter for 4 h t 1 C nd filtered using filter pper (Whtmn, Midstone, UK). After filtrtion, the extrct ws evported nd lyophilized. The DJS product (KIM PH 135)wsstoredtKoreInstituteofrientlMedicine (KIM, Dejeon, Kore) until used in this experiment. The composition of DJS extrct nd contents of ingredients re shown in Tble Chromtogrphic Conditions of HPLC-DAD. The contents lbiflorin, peoniflorin, z-ligustilide, decursin, nd nodkenin in the DJS wter extrct were nlyzed using n HPLC instrument (Agilent Technologies, USA) with Atlntis dc 18 column ( mm, 5 μm; Wters, USA). The mobile phse consisted of the solvents, distilled wter (A) nd cetonitrile with.1% formic cid (B). The following grdient ws used: min, A : B 9 : 1 (v/v); 2 min, A : B 75 : 25; 25 min, A : B 75 : 25; 3 min, A : B 5 : 5; 45 min, A:B2:8;nd6min,A:B:1.Themobilephseflow rte ws 1. ml/min, the column temperture ws 3 C, the injection volume ws 1 μl, nd UV detection ws t 23 nd 33 nm Animls. Seven-week-old femle Sprgue-Dwley rts (18 2 g) were purchsed from rient Bio (Seongnm, Gyeonggi, Kore). Animls were mintined t regulr 12 h light/drk cycle, t 21 ± 2 C, with reltive humidity of 5 ± 5%, nd were fed commercil diet (Rlston-Purin, St. Louis, M, USA). After 1 week of cclimtion, rts were Shm-operted (sline; Shm-control group), or bilterl ovriectomies were performed under generl nesthesi. The ovriectomized rts were rndomly divided into 3 groups: (A) vehicle control (sline; VX-control group) (B) spirin 3 mg/kg/dy (VX-ASA group); (C) DJS 1 mg/kg/dy (VX-DJS group). The rts were orlly dministered the drugs for 5 weeks. Food intke ws recorded dily, nd the body weight ws recorded weekly. The nimls used in this study were treted in ccordnce with the Guide for Cre nd Use of Lbortory Animls by the Institutionl Animl Cre nd Use Committee (IACUC) of Institute of rientl Medicine (pprovl number: 14-3, 14-21) Serum Lipid Levels. At the end of the experiment, rts were fsted for 12 h, nesthetized with Zoletil (1 ml/kg intrperitonelly), nd scrificed. Blood smples were collected from the inferior ven cv nd centrifuged t 3rpmfor1mintocollecttheserum.Mesurementsof TC, TG, nd HDL-C levels were performed enzymticlly using Roche Modulr P Autonlyzer (Roche Dignostics, Indinpolis, IN). The concentrtion of LDL-C ws clcultedusingthefriedewldeqution[12] Mesurement of Pltelet Aggregtion. Pltelet-rich plsm (PRP) ws seprted from the blood using n nticogulnt citrte dextrose (ACD) solution tht contined.8% citric cid, 2.2% sodium citrte, nd 2% dextrose t concentrtion of cells/ml. Pltelet ggregtion in response to collgen (2.5 μg/ml) ws performed in PRP using n ggregometer (Chrono-Log Co., Hrbortown, CA, USA). The extent of ggregtion ws expressed s percentge of the Shm-control stimulted with collgen FeCl 3 -InducedCrotidArteryThrombosisModels. Sline or test compounds were dministered 4 h before the initition of thrombotic occlusion. During opertive procedures, body temperture ws mintined t 37. ±.2 C using heting pd. After isoflurne nesthesi, one of the crotid rteries ws exposed nd Doppler flow probe (Powerlb/8SP, ADInstruments Pty Ltd, Cstle Hill, NSW, Austrli) ws plced on it.afterstbiliztionfor3min,vsculrinjurieswereinduced

3 Evidence-Bsed Complementry nd Alterntive Medicine 3 Tble 2: Effect of DJS dministrtion on body weight gin nd food intke in VX rts. Group Finl body weight (g) Body weight gin (g/dy) Food intke (g/dy) Shm ± ± ± 2.12 VX-control ± b 5.72 ± 2.43 b ± 2.84 VX-ASA ± b 5.39 ± 2.43 b ± 3.71 b VX-DJS ± b 5.11 ± 2.18 b ± 3.22 b Results re men ± S.D. (n =15). Mens of letters recorded s nd b within column indicted the sme level of body weight nd food intke within the vlues determined by one-wy ANVA. VX: ovriectomized; ASA: spirin; DJS: Dngguijgygsn. by topicl ppliction of FeCl 3 -sturted filter pper (2 2mm).Thefilterpperwsremovedndthecommoncrotid rtery ws wshed with sline. A Doppler flow probe ws plced on the exposed rtery nd blood flow ws mesured continuously with Lser Doppler Flowmetry (LDF; BFL21, Trnsonic Instrument, USA) for 3 min. To determine the time to occlusion (TT), rteril occlusion ws determined by decresed blood flow, nd complete occlusion ws defined s cesstion of the crotid rtery blood flow for 1 minutes Histology. The injured rtery of rts ws fixed in 4% prformldehyde nd embedded in prffin for histologicl nlysis. These were sectioned longitudinlly into 4 μm slices nd stined with hemtoxylin-eosin (H&E) nd Msson s trichrome. The thrombus for ech niml ws imged using light microscope (Axio Imger D2; Crl Zeiss Microimging, berkochen, Germny) nd the Zeiss AxioVision softwre (AxioVs4V4.8.1.; Crl Zeiss imging Solution) to obtin n imge slice tht represented the lrgest prt of the thrombus Sttisticl Anlysis. The results re presented s men ± stndrd devition (SD). Sttisticl significnce ws nlyzed using SPSS (version 2., SPSS Inc., Chicgo, IL, USA). Differences mong groups were compred with one-wy ANVA test, followed by Tukey s post hoc test. P vlues less thn.5 were considered significnt. 3. Results 3.1. HPLC Anlysis of Reference Compounds in DJS. The stndrd curves for the five components contining lbiflorin, peoniflorin,z-ligustilide, decursin, nd nodkenin were y= 13.26x+7.72 (R 2 1.), y = x (R 2.999), y = x (R 2 1.), y = x (R 2.999), nd y = 19.43x (R 2 1.), respectively. HPLC nlysis of DJS nd stndrd mixtures ws crried out t 23 nd 33 nm. The retention time of ech compound ws 15.6 min (lbiflorin), 17.1 min (peoniflorin), 22.3 min (nodkenin), 42.9 min (z-ligustilide), nd 43.8 min (decursin). The contents of ech component in DJS queous extrct were lbiflorin 2.97 ±.65 mg/g nd peoniflorin 16.17±.12 mg/g t 23 nm, nodkenin 3.63±.5 mg/g, zligustilide.11 ±.4 mg/g, nd decursin.2 ±.1 mg/g t 33 nm, respectively (Figure 1) Effects of DJS on Body Weight Gin nd Food Intke. The finl body weights fter 5-week tretments incresed significntly in the VX-control compred with tht in the Shm-control group (P <.5). VX-ASA nd VX- DJS groups showed similr body weight with VX-control group. In the VX-control group, tretment with sline demonstrted significnt increse in body weight gin compred to the Shm-control group (5.7 ± 2.4 g/dy versus 3.7 ± 2.7 g/dy, P <.1). The body weight gin ws slightly decresed in the VX-ASA (5.4 ± 2.4 g/dy) nd VX-DJS groups (5.1 ± 2.2 g/dy) compred with VX-control group, lthough not significntly compred with the Shm-control group (P >.5). When nimls were llowed free ccess to food following n experimentl period, the food intke ws the sme in the Shm-control (17.2 ± 2.12 g/dy) nd VX-control groups (17.72 ± 2.84 g/dy). Food intke ws incresed in the VX-ASA (19.14 ± 3.71 g/dy) nd VX- DJS groups (19.39 ± 3.22 g/dy) compred with the Shmcontrol (P <.1) nd VX-control groups (P <.1) (Tble 2) Effects of DJS on Serum Lipid Levels. vriectomy cused substntil increse in the TC, TG, nd LDL-C levels compred with the Shm-control group (P <.1, P <.5, nd P <.1). In VX rts, tretment with 3 mg/kg/dy of ASA (VX-ASA group) nd 1 mg/kg/dy of DJS (VX- DJS group) significntly ttenuted the increse in the TC, TG nd LDL-C levels but did not ffect HDL-C levels compred with the VX-control group (P <.5, resp.). Additionlly, the VX-ASA nd VX-DJS groups exhibited no significnt chnges in the HDL-C levels compred with the Shmcontrol nd VX-control groups (P >.5)(Figure 2) Effects of DJS on Pltelet Aggregtion. There ws no difference in the inhibition of pltelet ggregtion between the VX-control nd Shm-control groups (79.5% ± 7.4% versus 74.5% ± 9.%, P >.5). Tretment with 3 mg/kg/dy of ASA mrkedly suppressed pltelet ggregtion (32.3% ± 4.7%, P <.1), s did tretment with 1 mg/kg/dy of DJS (47.3% ± 2.6%, P <.1) compredwiththevx-control group (74.5% ± 9.%) (Figure 3) Effects of DJS in VX Rts with FeCl 3 -Induced Thrombosis. The men time to occlusion in the VX-control group ws significntly decresed (7.8 ±.84 min) compred with tht of the Shm-control group (3. ±. min). Therefore, the ppliction of 4% FeCl 3 to the externl surfce of the crotid rtery for 3 min induced rpid decrese in blood flow nd ws considered optiml for mesuring blood flow. In comprison, tretment with either 3 mg/kg of ASA

4 4 Evidence-Bsed Complementry nd Alterntive Medicine (mau) H H H H H Albiflorin (15.6 min) Peoniflorin (17.1 min) H H H H H H H (min) () 7 33 (mau) H H H H Nodkenin (22.3 min) CH 3 z-ligustilide (42.9 min) Decursin (43.8 min) (min) (b) Figure 1: HPLC-DAD chromtogrm of five reference compounds in DJS detected t 23 nm (lbiflorin, peoniflorin) nd 33 nm (nodkenin,z-ligustilide, nd decursin). DJS: Dngguijgygsn queous extrct (29.28 mg/ml). Lipid concentrtions (mg/dl) c b bc b b b TC TG HDL-C LDL-C c bc b Figure 2: Effects of ASA (3 mg/kg/dy) nd DJS (1 mg/kg/dy) on serum lipid profiles in SD rts compred with their corresponding controls: serum totl cholesterol (TC), triglyceride (TG), highdensity lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) levels in Shm (white br) nd VX SD rts fter 5 weeks of orl demonstrtion with sline (grey br), ASA (drk grey br, 3 mg/kg/dy), or DJS (blck br, 1 mg/kg/dy). Vlues of the sme mesured prmeter tht re not followed by the sme lphbeticl letter re significntly different (n =5 7 in ech group). Pltelet ggregtion (%) Shm-control VX-control VX-ASA VX-DJS Figure 3: Inhibition by ASA (3 mg/kg/dy) nd DJS (1 mg/ kg/dy)ofplteletggregtioninducedbycollgen(2.5μg/ml). Vlues of the sme mesured prmeter tht re not followed by the sme lphbeticl letter re significntly different (n =5in ech group). (VX-ASA group) (16.8 ± 1.3 min) nd 1 mg/kg of DJS (VX-DJS group) (13.2 ±.84 min) lso prolonged time to occlusion by 2.49 ±.33 rtio nd 1.94 ±.8 rtio, respectively (Figure4). c b

5 Evidence-Bsed Complementry nd Alterntive Medicine 5 Time to occlusion (min) Shm-control VX-vehicle VX-ASA d b c VX-DJS Figure 4: Time to occlusion of DJS in crotid rteril thrombosis model. The horizontl lines represent the men of the displyed vlues for time to occlusion in ech group. Vlues of the sme mesured prmeter tht re not followed by the sme lphbeticl letter re significntly different (n =5in ech group). 3.6.HistologyoftheCrotidArterywithThromboticcclusion fter FeCl 3 Tretment. Complete occlusion ws defined s the bsence of blood flow for 1 minutes fter the ppliction of FeCl 3. We collected those rteries nd processed them with H&E nd Msson s Trichrome for collgen fiber stining. In this study, histologicl chnges were observed in the crotid rteries fter FeCl 3 -induced thrombosis. In these imges, the type I collgen fiber stined blue, the nuclei stined blck, nd the bckground stined red. Thrombotic occlusion nd collgen fiber dmge were observed in the crotid rtery of the VX-control group fter FeCl 3 tretment. However, tretment with ASA nd DJS provided n excellent recovery of collgen fiber in the thrombotic vessels induced by FeCl 3 (Figure 5). 4. Discussion Postmenopusl women frequently experience weight gin nd re thus vulnerble to obesity-relted diseses, which led to n incresed risk of crdiovsculr diseses (CVD) such s coronry hert disese, hypertension, nd noninsulin-dependent dibetes mellitus [13, 14]. In the present study, body weight gin ws significntly incresed in VXcontrol group compred to Shm-control group for fiveweek tretment nd there were no significnt weight gin differences in VX-ASA nd VX-DJS groups compred to VX-control group. However, food intke in VX-ASA nd VX-DJS group hd much consumption compred to VXcontrol group nd these results suggest tht dministrtion of DJS could hve beneficil effect on obesity in menopuse. Lrge number of phrmcologicl studies were crried out on the influence of the menopuse on serum lipids nd lipoproteins profiles. Menopusl sttement increses the incidence of CVD due to hyperlipidemi, which is chrcterized by increses in totl cholesterol (TC), triglycerides (TG), nd low-density lipoprotein cholesterol (LDL-C) nd reduction in high-density lipoprotein cholesterol (HDL-C) [15 18]. Mny ttempts hve been mde to use herbl extrcts to improve serum lipid levels in postmenopusl women suffering from hyperlipidemi ssocited with CVD [19 21]. In the present study, DJS tretment significntly reduced the TC, TG, nd LDL-C levels (P <.5). Contrry to the conventionl studies, HDL-C level of VX-control did not pper to be significntly incresed (P >.5). These results indicte tht the rodent model for studying lipid metbolism physiology my hve limittions, but it still provides good model for studying the effects on serum lipid levels. Therefore, it is suggested tht the effects of the DJS on serum lipid levels re very complex nd should be investigted further. It hs been reported tht ovriectomy cused n elevtion of TC nd LDL-C in serum, leding to the development of therosclerosis nd coronry hert disese [22]. Furthermore, we further investigted the ntithrombosis ctivity bsed on theserumlipidlevelchngesofdjstretment. We lso determined the effect of DJS on thrombus formtion. Endothelil dysfunction is considered the first step in the therosclerotic process, nd our experimentl pproch ws bsed on endothelil injury nd pltelet ggregtion being key events in the pthogenesis of thrombus formtion [23, 24]. Intrvsculr thrombosis is involved series of complex events including pltelet dhesion, ctivtion, ggregtion, grnule relese, nd cogultion cscde ctivtion, is likely to pltelets contribute to thrombosis in severl wys [25]. Pltelets provide the membrne surfce required for the genertion of thrombin nd express surfce receptors tht ffect pltelet-pltelet nd pltelet-vessel wll interctions [26]. As positive control, we used spirin, one of the most widely used blood thinning gents, is used long-term in low doses to prevent hert ttcks, strokes nd blood clot formtioninpeoplethighriskfortheseevents[27, 28]. In this study, lthough the crdiovsculr protective effects of DJS tretment were not s strong s ASA, DJS tretment ws successful in inhibiting pltelet ggregtion nd thrombus formtion. Previous studies hve reported tht biochnin A, phytoestrogen tht cts s wek estrogen receptor gonist, restored endothelil function in n niml model of vsculr injury [29]. This study showed the possibility tht DJS tretment cn restore endothelil function in n niml model of crotid rtery injury, n effect similr to the vsculr effects of estrogen. Mesurement of pltelet ggregtion hs been useful method to exmine inhibition of thrombus formtion. Furthermore, FeCl 3 -induced thrombosis model method in rts is the optiml technique s it exhibits simplicity nd reproducibility. In previous test, we demonstrted the rteril response to vrious concentrtions (1 5%) of FeCl 3 nd found tht filter pper sturted with 4% concentrtion of FeCl 3 resulted in fst, effective recnliztion. FeCl 3 triggers n oxidtive vsculr endothelil mtrix. Then, pltelets interct with collgen nd vwf in the mtrix vi their respective pltelet surfce receptors, leding to pltelet dhesion [3]. According to the in vitro results, the in vivo ntithrombotic efficcy of DJS ws nticipted. Indeed, the orl dministrtion of DJS delyed the occlusion time in FeCl 3 -induced rtery thrombosis model. Notbly, DJS doubled the occlusion time t 1 mg/kg compred with the VX-control group. In this study, we did not design the Shm-vehicle nd VX-control groups to mesure time to

6 6 Evidence-Bsed Complementry nd Alterntive Medicine H&E 1x H&E 4x Msson s Trichrome 1x Msson s Trichrome 4x Shm-control VX-vehicle VX-ASA VX-DJS Figure 5: Histologicl exmintion of FeCl 3 -induced thrombus from the Shm-control group, VX-vehicle group, VX-ASA group, nd VX-DJS group. All sections were stined with hemtoxylin nd eosin (H&E, two left columns, nd mgnifiction 1, 4) nd Msson s trichrome (MT, two right columns, nd mgnifiction 1, 4). occlusion fter the FeCl 3 topicl ppliction. ur previous study investigted the effects of sline dministrtion on rts following Shm opertion nd ovriectomy, which is similr to the Shm-control nd VX-control groups. In support of our hypothesis, the histologicl stining methods lso demonstrted tht DJS inhibited rteril thrombus formtion nd dmge of collgen fiber in rteril vessel wll. Vrious clinicl studies hve shown tht herbl extrction components hve significnt ssocition ntioxidnt effects; it leds to ntithrombotic properties by increse of pltelet N relese nd rdicl scvenging ctivity [31, 32]. DJS consists of 6 herbs nd ech herb lso hs ntioxidnt effects [33 37]. We hypothesize tht ntioxidnt effects re responsible for the results from bove ntithrombotic studies. Femle sex ppers to be ssocited with crdioprotection nd menopuses re mjor risk fctors for crdiovsculr disese. In fct, the idel postmenopusl crdiovsculr protective therpy is expected to reproduce the beneficil effects of blood flow without producing ny dverse effects. Although the exct mechnism of ction remins to be clrified, we hve demonstrted the beneficil effects of DJS ontheimprovementofplsmlipidprofilesndrestortion of blood flow. These beneficil effects my be cused by (1) decresed TC, TG, nd LDL-C; (2) inhibition of pltelet ggregtion; or (3) inhibition of thrombus formtion. In conclusion, DJS could be cndidte for inhibiting the development of crdiovsculr risk fctors. Conflict of Interests The uthors declre tht there is no conflict of interests regrding the publiction of this pper. Authors Contributions InSilPrkndHyeWonLeecontributedeqully.InSilPrk nd Byoung Seob Ko conceived nd designed the experiments. In Sil Prk, Hye Won Lee, nd Jin Ah Ryuk performed the experiments nd wrote the pper. Byoung Seob Ko monitored the experiments nd discussed the results. Hye WonLeendByoungSeobKonlyzedthedtndreviewed the mnuscript. All uthors hve red nd pproved the pper. Acknowledgment This work ws supported by grnt from the Kore Institute of rientl Medicine (Grnt no. K148).

7 Evidence-Bsed Complementry nd Alterntive Medicine 7 References [1] L. Wroblewski Lissin nd J. P. Cooke, Phytoestrogens nd crdiovsculr helth, Journl of the Americn College of Crdiology,vol.35,no.6,pp ,2. [2] R. Rossi, T. Grimldi, G. riglini, G. Fntini, F. Coppi, nd M. G. Moden, Menopuse nd crdiovsculr risk, Pthophysiology of Hemostsis nd Thrombosis, vol.32,no.5-6,pp , 22. [3] T. M. Quinton, S. Kim, J. Jin, nd S. P. Kunpuli, Lipid rfts re required in Gα i signling downstrem of the P2Y12 receptor during ADP-medited pltelet ctivtion, Journl of Thrombosis nd Hemostsis,vol.3,no.5,pp ,25. [4] Y. Nkno, T. shim, H. Mtsuur, G. Kjiym, nd M. Kmbe, Effect of 17β-estrdiol on inhibition of pltelet ggregtion in vitro is medited by n increse in N synthesis, Arteriosclerosis, Thrombosis, nd Vsculr Biology, vol.18,no. 6, pp , [5]F.Grodstein,M.J.Stmpfer,J.E.Mnsonetl., Postmenopusl estrogen nd progestin use nd the risk of crdiovsculr disese, The New Englnd Journl of Medicine,vol.335,no.7,pp , [6] E. Brrett-Connor, L. Mosc, P. Collins et l., Effects of rloxifene on crdiovsculr events nd brest cncer in postmenopusl women, The New Englnd Journl of Medicine,vol. 355, no. 2, pp , 26. [7] H.J.Kng,R.Ansbcher,ndM.M.Hmmoud, Useoflterntive nd complementry medicine in menopuse, Interntionl JournlofGynecologyndbstetrics,vol.79,no.3,pp , 22. [8] S.Usuki,T.Nkuchi,S.Hig,ndK.Somey, Theimprovement of lutel insufficiency in fecund women by Tokishkuykusn tretment, Americn Journl of Chinese Medicine,vol. 3, no. 2-3, pp , 22. [9] M.H.Chung,S.Suzuki,T.Nishihr,ndM.Httori, Estrogenic effects of Kmpo formul, Tokishkuykusn, in prous ovriectomized rts, Biologicl nd Phrmceuticl Bulletin, vol.31,no.6,pp ,28. [1] J. Lee, D. Hwng, H. G. Kim, C. Lee, nd M. S. h, Dngguijkyk-sn protects dopmine neurons ginst 1-methyl- 4-phenyl-1,2, 3,6-tetrhydropyridine-induced neurotoxicity under postmenopusl conditions, Journl of Ethnophrmcology, vol. 139, no. 3, pp , 212. [11] F. W. Peter, R. J. P. M. Frnken, W. Z. Wng et l., Effect of low dose spirin on thrombus formtion t rteril nd venous micronstomoses nd on the tissue microcircultion, Plstic nd Reconstructive Surgery, vol. 99, no. 4, pp , [12] W. T. Friedewld, R. I. Levy, nd D. S. Fredrickson, Estimtion of the concentrtion of low-density lipoprotein cholesterol in plsm, without use of the preprtive ultrcentrifuge., Clinicl Chemistry,vol.18,no.6,pp ,1972. [13]M.E.MendelsohnndR.H.Krs, Theprotectiveeffects of estrogen on the crdiovsculr system, The New Englnd Journl of Medicine,vol.34,no.23,pp ,1999. [14]R.W.Robinson,N.Higno,ndW.D.Cohen, Incresed incidence of coronry hert disese in women cstrted prior to the menopuse, AMA Archives of Internl Medicine,vol.14, no. 6, pp , [15]G.A.Colditz,W.C.Willett,M.J.Stmpfer,B.Rosner,F.E. Speizer, nd C. H. Hennekens, Menopuse nd the risk of coronry hert disese in women, The New Englnd Journl of Medicine, vol. 316, no. 18, pp , [16] J.Jensen,L.Nils,ndC.Christinsen, Influenceofmenopuse on serum lipids nd lipoproteins, Mturits, vol. 12, no. 4, pp , 199. [17] M. G. Shlipk, J. A. Simon, E. Vittinghoff et l., Estrogen nd progestin, lipoprotein() nd the risk of recurrent coronry hert disese events fter menopuse, The Journl of the Americn Medicl Assocition, vol.283,no.14,pp , 2. [18] C. Bonithon-Kopp, P.-Y. Scrbin, B. Drne, A. Mlmejc, nd L. Guize, Menopuse-relted chnges in lipoproteins nd some other crdiovsculr risk fctors, Interntionl Journl of Epidemiology,vol.19,no.1,pp.42 48,199. [19] E. A. Lucs, D. A. Khli, B. P. Dggy, nd B. H. Arjmndi, Ethnol-extrcted soy protein isolte does not modulte serum cholesterolingoldensyrinhmsters:modelofpostmenopusl hypercholesterolemi, The Journl of Nutrition,vol.131, no. 2, pp , 21. [2] S. Hidk, Y. kmoto, nd M. Arit, A hot wter extrct of Chlorell pyrenoidos reduces body weight nd serum lipids in ovriectomized rts, Phytotherpy Reserch, vol.18,no.2,pp , 24. [21] Y. Prk, H. Kwon, M. K. Shimi, M. Rhyu, nd Y. Lee, Improved lipid profile in ovriectomized rts by red ginseng extrct, Phrmzie,vol.66,no.6,pp ,211. [22] M.Liu,X.Xu,W.Rng,Y.Li,ndH.Song, Influenceofovriectomy nd 17β-estrdiol tretment on insulin sensitivity, lipid metbolism nd post-ischemic crdic function, Interntionl Journl of Crdiology,vol.97,no.3,pp ,24. [23] P. M. Vnhoutte, Endothelil dysfunction nd therosclerosis, Europen Hert Journl, vol. 18, no. 5, pp. E19 E29, [24] U. Ruch, J. I. sende, V. Fuster, J. J. Bdimon, Z. Fyd, nd J. H. Chesebro, Thrombus formtion on therosclerotic plques: pthogenesis nd clinicl consequences, Annls of Internl Medicine,vol.134,no.3,pp ,21. [25] B. Furie nd B. C. Furie, Mechnisms of thrombus formtion, New Englnd Journl of Medicine, vol. 359, no. 9, pp , 28. [26] G. Dvì nd C. Ptrono, Pltelet ctivtion nd therothrombosis, The New Englnd Journl of Medicine, vol. 357, no. 24, pp , 27. [27] D. L. Bhtt, K. A. A. Fox, W. Hcke et l., Clopidogrel nd spirin versus spirin lone for the prevention of therothrombotic events, New Englnd Journl of Medicine,vol.354,no.16, pp , 26. [28] A.. Mree nd D. J. Fitzgerld, Vrible pltelet response to spirin nd clopidogrel in therothrombotic disese, Circultion, vol. 115, no. 16, pp , 27. [29] S. Schrepfer, T. Deuse, T. Münzel, H. Schäfer, W. Brendle, nd H. Reichenspurner, The selective estrogen receptor-β gonist biochnin A shows vsculoprotective effects without uterotrophic ctivity, Menopuse, vol.13,no.3,pp , 26. [3] A. Eckly, B. Hechler, M. Freund et l., Mechnisms underlying FeCl 3 -induced rteril thrombosis, JournlofThrombosisnd Hemostsis,vol.9,no.4,pp ,211. [31].Vitsev,S.Vrghese,S.Chkrbrti,J.D.Folts,ndJ. E. Freedmn, Grpe seed nd skin extrcts inhibit pltelet function nd relese of rective oxygen intermedites, Journl of Crdiovsculr Phrmcology, vol.46,no.4,pp , 25.

8 8 Evidence-Bsed Complementry nd Alterntive Medicine [32] J.E.Tnus-Sntos,M.Desi,L.R.Deketl., Effectsofendothelil nitric oxide synthse gene polymorphisms on pltelet function, nitric oxide relese, nd interctions with estrdiol, Phrmcogenetics nd Genomics, vol.12,no.5,pp , 22. [33] S. Wu, L. Ng, nd C. Lin, Antioxidnt ctivities of some common ingredients of trditionl chinese medicine, Angelic sinensis, Lycium brbrumnd Pori cocos, Phytotherpy Reserch, vol. 18, no. 12, pp , 24. [34]J.B.Jeong,S.Y.Ju,J.H.Prketl., Antioxidntctivityin essentil oils of Cnidium officinle mkino nd Ligusticum chunxiong hort nd their inhibitory effects on DNA dmge nd poptosis induced by ultrviolet B in mmmlin cell, Cncer Epidemiology,vol.33,no.1,pp.41 46,29. [35] H. J. Kim, E. J. Chng, S. H. Cho, S. K. Chung, H. D. Prk, nd S. W. Choi, Antioxidtive ctivity of resvertrol nd its derivtives isolted from seeds of Peoni lctiflor, Bioscience, Biotechnology nd Biochemistry, vol. 66, no. 9, pp , 22. [36] C.W.Hn,E.S.Kng,S.A.Hm,H.J.Woo,J.H.Lee,ndH.G. Seo, Antioxidtive effects of Alism orientle extrct in plmitte-induced cellulr injury, Phrmceuticl Biology,vol. 5,no.1,pp ,212. [37]C.Wng,K.Wng,L.Chen,D.Chou,ndW.Ling, Antioxidtive bilities of essentil oils from trctylodes ovt rhizome, Evidence-Bsed Complementry nd Alterntive Medicine, vol. 211, Article ID 24892, 8 pges, 211.

9 MEDIATRS of INFLAMMATIN The Scientific World Journl Gstroenterology Reserch nd Prctice Journl of Dibetes Reserch Interntionl Journl of Journl of Endocrinology Immunology Reserch Disese Mrkers Submit your mnuscripts t BioMed Reserch Interntionl PPAR Reserch Journl of besity Journl of phthlmology Evidence-Bsed Complementry nd Alterntive Medicine Stem Cells Interntionl Journl of ncology Prkinson s Disese Computtionl nd Mthemticl Methods in Medicine AIDS Behviourl Neurology Reserch nd Tretment xidtive Medicine nd Cellulr Longevity