Absorbed Bioactive Compounds Replicate GuanxinⅡ-Induced Endothelium-Associated in/ex vivo Vasodilation

Objective:To develop an interference-free and rapid method to elucidate GuanxinⅡ(GXⅡ)'s representative vasodilator absorbed bioactive compounds(ABCs)among enormous phytochemicals.Methods:The contents of ferulic acid,tanshinol,and hydroxysafflor yellow A(FTA)in GXⅡ/rat serum after the oral administration of GXⅡ(30 g/kg)were detected using ultra-performance liquid chromatography-mass spectrometry.Totally 18 rats were randomly assigned to the control group(0.9%normal saline),GXⅡ(30 g/kg)and FTA(5,28 and 77 mg/kg)by random number table method.Diastolic coronary flow velocity-time integral(VTI),i.e.,coronary flow or coronary flow-mediated dilation(CFMD),and endothelium-intact vascular tension of isolated aortic rings were measured.After 12 h of exposure to blank medium or 0.5 mmol/L H_(2)O_(2),endothelial cells(ECs)were treated with post-dose GXⅡof supernatant from deproteinized serum(PGSDS,300μL PGSDS per 1 m L of culture medium)or FTA(237,1539,and 1510 mg/m L)for 10 min as control,H_(2)O_(2),PGSDS and FTA groups.Nitric oxide(NO),vascular endothelial growth factor(VEGF),endothelin-1(ET-1),superoxide dismutase(SOD),malondialdehyde(MDA)and phosphorylated phosphoinositide 3 kinase(p-PI3K),phosphorylated protein kinase B(p-AKT),phosphorylated endothelial nitric oxide synthase(p-e NOS)were analyzed.PGSDS was developed as a GXⅡproxy of ex vivo herbal crude extracts.Results:PGSDS effectively eliminates false responses caused by crude GXⅡpreparations.When doses equaled the contents in GXⅡ/its post-dose serum,FTA accounted for 98.17%of GXⅡ-added CFMD and 92.99%of PGSDS-reduced vascular tension.In ECs,FTA/PGSDS was found to have significant antioxidant(lower MDA and higher SOD,P<0.01)and endothelial function-protective(lower VEGF,ET-1,P<0.01)effects.The increases in aortic relaxation,endothelial NO levels and phosphorylated PI3K/Akt/e NOS protein induced by FTA/PGSDS were markedly abolished by NG-nitro-L-arginine methyl ester(L-NA,e NOS inhibitor)and wortmannin(PI3K/AKT inhibitor),respectively,indicating an endothelium-dependent vasodilation via the PI3K/AKT-e NOS pathway(P<0.01).Conclusion:This study provides a strategy for rapidly and precisely elucidating GXⅡ's representative in/ex vivo cardioprotective absorbed bioactive compounds(ABCs)-FTA,suggesting its potential in advancing precision ethnomedicine.

冠心Ⅱ号煎剂的质量研究

目的:考察同一批冠心Ⅱ号组方药材在同一煎煮工艺条件下煎剂中有效成分含量的变异程度。方法:选择同一批组方药材,尽可能地控制煎煮过程的各个细节,测定5批次冠心Ⅱ号煎剂中有效成分的含量并计算其平均值。结果:以生药量计,丹参素为(0.773±0.0 656)mg/g,原儿茶醛为(36.591±3.0 590)μg/g,芍药苷为(2.655±0.2 454)mg/g,阿魏酸为(85.052±7.5 469)μg/g。结论:如果以批次间成分含量变异系数小于10%为标准,各有效成分含量均达到要求;如果以成分含量批次间无统计学差异的标准来判断,则需要将更多影响煎剂有效成分含量的因素纳入质量控制范畴。

冠心Ⅱ号对急性心肌梗塞大鼠心肌损伤和心肌酶的影响

目的观察冠心Ⅱ号对急性心肌梗塞大鼠心肌病理损伤和血清心肌酶的作用。方法连续给大鼠灌胃冠心Ⅱ号标准汤剂5.6ml/kg或等容积生理盐水7d,采用结扎左冠状动脉前降支(LAD)法建立大鼠急性心肌梗塞模型,24h后取血用比色法测定血清中谷-草转氨酶(AST)、肌酸激酶(CK)、肌酸激酶同工酶(CK-MB)、乳酸脱氢酶(LDH)水平;取心肌,固定、常规石蜡包埋、切片、HE染色,光镜观察心肌组织病理损伤程度。结果冠心Ⅱ号组大鼠血清中AST,CK,CK-MB及LDH的水平较模型对照组下降(P<0.05);冠心Ⅱ号组大鼠心肌病理损伤等级低于模型对照组(P<0.05)。结论冠心Ⅱ号对急性心肌梗塞有保护作用和改善心肌酶作用。

Network Pharmacology-Based Exploration of Synergistic Mechanism of Guanxin Ⅱ Formula (冠心Ⅱ号方) for Coronary Heart Disease

Objective:To study the pharmacological mechanism of Guanxin Ⅱ formula(冠心Ⅱ号方,GXⅡ)for treatment of coronary heart disease(CHD).Methods:A network pharmacology-based method was utilized.First candidate compounds,targets of GXⅡ were collected using Pharm Mapper,BATMAN-TCM,Drug Bank and Swiss Target Prediction,and targets on CHD were mined from Gene Cards,Dis Genet,Drug Bank and GEO.Afterwards,the big hub compounds and targets were chosen in the candidate compounds-direct therapeutic targets on the CHD(C-T)network and the direct therapeutic targets on the CHD(T-D)network.Furthermore,the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis were performed to identify the enriched terms.Finally,a molecular docking simulation strategy was adopted to verify the binding capacity between the big hub compounds and big hub targets on CHD.Results:First,114 candidate compounds were selected with the following criteria:OB 30%,DL 0.18,and HL 4 h.Then,1,035 targets of GXⅡ were gathered,while 928 targets on CHD were collected.Afterwards,196 common targets of compound targets and therapeutic targets on CHD were defined as direct therapeutic targets acting on CHD.In addition,the contribution index(CI)in the C-T network was calculated,and 4 centrality properties,including degree,betweenness,closeness and coreness,in the T-D network,4 big hub compounds,and 6 big hub targets were eventually chosen.Furthermore,the GO and KEGG analysis indicated that GXⅡ acted on CHD by regulating the reactive oxygen species metabolism,steroid metabolism,lipid metabolism,inflammatory response,proliferation,differentiation and apoptosis.The docking results manifested excellent binding capacity between the 4 big hub compounds and 6 big hub targets on CHD.Conclusion:This network pharmacology-based exploration revealed that GXⅡ might prevent and inhibit the primary pathological processes of CHD.

冠心Ⅱ号提取工艺改进及其对急性心肌梗死大鼠心脏功能的影响

目的对冠心Ⅱ号进行提取工艺改进并观察其对急性心肌梗死(AMI)大鼠心脏功能的影响。方法将冠心Ⅱ号提取工艺进行改进和优化得到精制过滤液和纯化洗脱液,并采用高效液相色谱仪进行主要成分分析。采用左冠状动脉前降支下结扎法制备AMI模型,造模成功的50只大鼠按随机数字表法分为模型组、尼可地尔片组(2.7 mg/kg体重)、复方川丹颗粒组(川丹颗粒,4.60 g生药/kg体重)、冠心Ⅱ号精制组(4.60 g生药/kg体重)、冠心Ⅱ号纯化组(4.60 g生药/kg体重)组,每组10只。另设10只穿线不结扎为假手术组。术后第2天开始灌胃给药,连续4周,假手术组、模型组灌胃等容积纯净水。采用高频超声心动图检测各项心脏形态学和功能指标。ELSIA法检测心肌钙蛋白T(cTnT)、内皮素-1(ET-1)、降钙素基因相关肽(CGRP)、肌酸激酶同工酶MB(CK-MB)、血栓素B2(TXB2)和6-酮-前列腺素F1α(6-Keto-PGF1α)水平。TTC法染色测量心肌梗死面积,HE染色观察心肌病理形态和炎症细胞浸润状态。结果丹酚酸B和芍药苷外标含量分别为川丹颗粒0.767%、0.418%,冠心Ⅱ号精制组4.247%、1.546%,冠心Ⅱ号纯化组32.758%、12.150%。与假手术组比较,模型组ET-1、TXB2升高(P<0.05,P<0.01),心脏超声学检测指标改善(P<0.05,P<0.01)。与模型组比较,冠心Ⅱ号方两组和尼可地尔片组心脏超声学检测指标改善(P<0.05,P<0.01),大鼠心重指数、梗死面积比值、cTnT、ET-1和TXB2水平亦降低(P<0.05,P<0.01);川丹颗粒组部分心脏超声学检测指标改善(P<0.05,P<0.01),大鼠心重指数、cTnT、ET-1和TXB2降低(P<0.05,P<0.01);各给药组大鼠心肌缺血区的心肌细胞炎症浸润和细胞形态不同程度改善。结论冠心Ⅱ号提取工艺改进的主要有效成分丹酚酸B和芍药苷含量有较大提高,可提高AMI后大鼠心脏的射血分数、抑制心室重构及改善心功能,部分指标优于川丹颗粒。

基于高效液相色谱技术对冠心Ⅱ号抗抑郁作用物质基础的研究

目的:在前期冠心Ⅱ号具有抗抑郁作用的研究基础上,采用高效液相色谱(HPLC)技术,通过分析冠心Ⅱ号主要活性成分在大鼠体内血清及脑组织的含量测定,揭示冠心Ⅱ号抗抑郁作用的物质基础。方法:SD大鼠灌胃冠心Ⅱ号(2.5 g/kg),30 min后处死取血清及脑组织,采用HPLC检测血清及脑组织中羟基红花黄色素A、阿魏酸、丹酚酸B的含量,并计算各单体成分的血脑屏障(BBB)透过率。色谱柱:Agilent Zorbax SB-C18(250 mm×4.6 mm,5μm);流动相:乙腈(A)-0.1%磷酸(B);柱温:35℃;流速:1.0 mL/min。结果:血清中羟基红花黄色素A、阿魏酸、丹酚酸B的线性回归方程分别为Y=3.9765X+0.4310(r=0.9999),Y=70.262X+69.1410(r=0.9999),Y=34.729X-2.4044(r=0.9996)。脑组织中羟基红花黄色素A、阿魏酸、丹酚酸B的线性回归方程分别为Y=4.5869X-0.0963(r=0.9998),Y=70.859X+100.0400(r=0.9981),Y=42.176X+18.2420(r=0.9995)。3种活性单体成分在血清及脑组织质量浓度比较,差异无统计学意义(P>0.05)。羟基红花黄色素A、阿魏酸、丹酚酸B在大鼠正常状态的BBB透过率分别为1.22%、1.27%及0.87%,三者均能透过BBB。结论:冠心Ⅱ号灌胃大鼠后血清及脑组织中均能检测到羟基红花黄色素A、阿魏酸、丹酚酸B这3种活性单体成分,这三者可能是冠心Ⅱ号抗抑郁作用的潜在物质基础,并且均能透过BBB。