【作者】 简洁；

【导师】 黄仁彬；

【作者基本信息】 广西医科大学 ， 药理学， 2009， 博士

【摘要】 玉郎伞(Yulangsan,YLS)为广西壮族习用药材。目前,在国内外只有本课题组对YLS进行了初步提取分离及相关药理、药效研究,结果表明:黄酮、皂苷和多糖为YLS的主要活性部位;YLS提取物具有降血压、抗自由基、减轻心肌缺血再灌注损伤、增加冠脉流量、护肝、抗炎、抗肿瘤等作用。上述研究均立足于YLS的大类活性成分,随着课题的深入,非常有必要明确YLS有效的活性单体成分及其作用机制。本课题正是在前期工作的基础上,按照中药现代化的思维模式,以药理活性为指导进行追踪筛选,运用先进的植化分离、纯化、鉴定技术,对中药玉郎伞黄酮进行深度研究,得到玉郎伞黄酮的活性单体;通过建立相应的体内外模型,应用直接加药和血清药理学两种给药方式,从体内、细胞、蛋白水平探讨玉郎伞活性黄酮单体对氧自由基、凝血、缺氧、心肌缺氧/复氧损伤、H2O2诱导心肌细胞凋亡的作用及可能机制,为进一步的新药研发提供理论和实验依据。目的:1.考察玉郎伞总黄酮的提取工艺:在前期研究的基础上,对玉郎伞总黄酮提取工艺进行优化。2.分离、纯化玉郎伞黄酮的单体成分并进行结构鉴定:运用先进的植化分离、纯化、鉴定技术,得到玉郎伞黄酮的活性单体并鉴定结构。3.玉郎伞黄酮单体的抗氧化、抗凝血、耐缺氧作用研究。4.研究玉郎伞黄酮单体及含药血清对心肌细胞缺氧/复氧损伤(H/R)的作用。5.研究玉郎伞黄酮单体及含药血清对H2O2诱发心肌细胞凋亡的作用及机制。方法:1. YLS总黄酮的提取工艺考察1.1 YLS总黄酮的鉴别:以浓氨水反应、三氯化铝反应、乙酸镁反应、盐酸-镁粉反应进行定性。1.2 YLS总黄酮的含量测定:以芦丁为对照品,在波长275nm处测定加入AlCl3试剂后的YLS提取物甲醇溶液吸光度,代入芦丁标准曲线方程得到供试品溶液中总黄酮的浓度,按下公式计算YLS与芦丁对照品对照所得的总黄酮含量:总黄酮含量%=待测液中总黄酮的浓度/待测液的浓度X 1000%总黄酮提取率%=总黄酮含量X提取物重量(g)/生药量(g) X 1000% 1.3 YLS总黄酮的提取方法优化:在前期研究的基础上,以提取率为主要考察指标,辅以提取时间、提取次数、试剂用量、提取成本、人员及环境保护等因素,对传统热回流提取、微波辅助萃取、冷浸提取等三种方式进行比较研究,以优化YLS总黄酮提取方法。2.玉郎伞黄酮成分的单体分离、纯化及结构鉴定2.1玉郎伞黄酮成分的单体分离2.1.1玉郎伞黄酮成分的初步分离:以YLS提取物为原料,加入石油醚萃取除色素、酯类,再经乙酸乙酯萃取,萃取液减压浓缩、冷冻真空干燥,得到乙酸乙酯萃物;取10cm×120cm玻璃层析柱(带辅助加压装置),以薄层层析硅胶H为分离固定相(样品:硅胶H=1:30比例),按PE→EtOAc的极性顺序采用PE/EtOAc混合溶剂由低到高梯度洗脱;洗脱液减压浓缩,经TLC检查,采用紫外荧光、碘熏等显色,合并相同流份后,减压浓缩、冷冻真空干燥。2.1.2玉郎伞黄酮成分的多级分离按本室方法建立体外·O2-和·OH发生体系,以抗氧化活性为测定指标,对初步分离所得的各部分物质进行体外抗氧化活性筛选,选择抗氧化活性最强的部分,以不同比例PE/EtOAc混合溶剂,经反复硅胶柱层析进行后续多级分离。2.2玉郎伞黄酮单体的纯化:经多次CHCl3/MeOH重结晶、制备薄层、制备液相(AKTA explore100快速纯化工艺开拓系统)等方法进行玉郎伞黄酮单体的纯化。2.3玉郎伞黄酮单体的纯度及结构鉴定2.3.1以TLC法分析单体纯度:采用三种不同配比的混合展开剂进行TLC分析。2.3.2 RP-HPLC法分析单体纯度色谱条件:色谱柱:Symmetry C18(4.6×150mm);检测波长:λ(?)=238nm、λ(П)=260nm;柱温:30℃;WATERS 2487 UV检测器;进样量20μg(达到柱超载);洗脱时间为主峰保留时间的2.5倍,以两种不同配比流动相进行检测。2.3.3测定YLS黄酮单体的甲醇溶液紫外吸收光谱2.3.4红外光谱分析官能团:常规KBr压片,测定红外光谱。2.3.5电喷雾离子质谱(ESI-MS)测定分子量2.3.6氢谱、碳谱鉴定分子结构:分别以DMSO-d6、CDCl3为溶剂,测定一维及二维核磁光谱。2.3.7 X-射线单晶衍射技术分析化合物的空间构型3.玉郎伞黄酮单体的抗氧化、抗凝血、耐缺氧作用研究3.1玉郎伞黄酮单体的抗氧化作用研究YLS A～B分别以1%DMSO溶解配成0.5、1.0、2.0 mg.ml-1浓度,按本室方法建立体外·O2-、·OH发生体系,设立正常对照、阳性对照(Vit.C)、YLS A～B的低、中、高剂量共8组,测定·O2-清除率、·O2-生成抑制率、·OH清除率等指标。3.2玉郎伞黄酮单体的抗凝血作用研究将90只小鼠随机分为正常对照、阳性对照、溶媒对照、YLS A～B的低、中、高剂量共9组,每组10只,均通过腹腔注射给药(0.2ml/20g),以毛细管法进行体内抗凝血作用研究。3.3玉郎伞黄酮单体的耐缺氧作用研究建立小鼠常压密闭缺氧模型,考察YLS两个黄酮单体预处理对缺氧存活时间的影响。4.研究玉郎伞黄酮单体及含药血清对心肌细胞H/R的作用利用原代培养3～4d的大鼠乳鼠心肌细胞,随机分为正常对照、H/R模型、阳性药维拉帕米、空白血清、溶媒(1%DMSO)、YLS A～B单体及含药血清低、中、高剂量共17组,除正常对照组外,均建立H/R模型;除正常对照、模型组外,于造模前加入相应药物或血清预孵2 h。倒置相差显微镜下观察H/R前后心肌细胞的形态学改变;以MTT法测定细胞存活力;按试剂盒说明测定H/R后各组心肌细胞培养上清液中MDA、T-SOD、GSH、LDH、NOS、TNF-а含量(活性)及心肌细胞Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶活性。5.玉郎伞黄酮单体及含药血清对H2O2诱发心肌细胞凋亡的作用及机制5.1玉郎伞黄酮单体及含药血清对H2O2诱发心肌细胞凋亡的作用在原代培养的心肌细胞培养液中加入终浓度为100μmol.L-1的H2O2,常规培养16h,建立心肌细胞凋亡模型,分组及给药方法同上(阳性药改用Vit.C),以Annex V-FITC和PI双染法检测心肌细胞凋亡。5.2玉郎伞黄酮单体及含药血清对NF-κBp65、Bcl-2、Bax蛋白表达的影响心肌细胞作爬片培养,造模、分组及给药方法同上。造模结束后,各组爬片细胞以4%多聚甲醛4℃过夜固定后,采用免疫组化方法检测NF-κBp65、Bcl-2、Bax蛋白的表达。结果:1.YLS总黄酮的提取方法优化确定以微波辅助萃取的方式进行玉郎伞总黄酮的提取。具体工艺为:将YLS饮片粉碎,以60%乙醇为提取溶剂,料液比1:8,提取温度< 60°C,微波功率240 W,提取时间20 min ,提取1次。按此方法操作,总黄酮提取率为:3.16%(以生药计),总黄酮含量为22.5% (以芦丁为对照)。2.玉郎伞黄酮成分的单体分离、纯化及结构鉴定YLS A:C17H16O3,淡黄色针晶,mp. 120.0-121.0℃;UV(MeOH)λmax (nm): 260,300;ESI-MS m/z:291.17 [M+Na]+,307.13 [M+K]+;IR(KBr)(cm-1):3501,3123,2929,2852,1789,1623,1604,1568,1528,1462,1372,1285,1228,1165,1133;1HNMR(DMSO-d6,500MHz)δ:3.86(6H,s,H-OCH3 X2,H-7,8),7.60(1H,brd,J=6.9 Hz,H-4),7.60(5H,m,H-2′、3′、4′、5′、6′),7.72(1H,J=8.8Hz,H-α),8.00(1H,d,J=8.8Hz,H-?),8.13(2H,brd,J=6.5Hz,H-3,5);13CNMR(DMSO-d6,125 MHz)δ:174.2(C-7′),110.6(C-α),121.5(C- ?),119.6(C-1),147.8(C-2,6),128.6(C-3,5),130.9(C-4),130.9(C-1′),129.2(C-2′、3′、4′、5′、6′),60.2(C-7),60.2(C-8)。结合HSQC、HMBC、NOSEY谱,确定其结构式为:Cis(顺)-2,6-二甲氧基查尔酮,首次从YLS植物中分离,经查阅SciFinder数据库表明:此化合物亦为首次分离及报道核磁数据。YLS B:C18H14O4,桔色方晶,mp. 135.0-136.0℃;UV(MeOH)λmax (nm): 238,350;ESI-MS m/z:295.00[M+H]+,610.99 [2M+ Na]+,263.35 [M?OMe]+;IR(KBr)(cm-1):3501,3133,2929, 2831,1739,1600,1562,1474,1380,1262,1219,1137,1102,1062,1025; 1HNMR(CDCl3,500MHz),δ:3.95(3H,s,-OCH3),7.2(1H,d,H-5’),8.2(1H,d,H-4’), 8.17(2H,s,H-2’,6’),7.77(1H,s,H-α),7.57(5H,m,H-2,3,4,5,6); 13CNMR(CDCl3,125MHz),δ:60.2(C-9’),104.2(C-α),110.0(C-5’),117.0(C-3’),119.8(C-8’),122.0(C-1’),128.4(C-2’),128.6(C-2,4,6),130.6(C-3,5),141.8(C-1),145.7(C-4’),150.0(C-6’),154.8(C-7’),158.2(C-?),175.0(C=O);Crystal data:Monocli- nic,P2(1)/c,a = 10.8813(16)nm,b = 9.7954(14) nm,c = 13.3473(19) nm,α= 90°,?= 103.697(2)°,γ= 90°,V=1382.2(3) nm3 ,Z=4,Dc=1.414 mg/m3, F(000)=616,μ=0.100 mm-1。确定其结构式为:Cis(顺)-6’-甲氧基- ?-羟基-苯骈呋喃查耳酮,系首次从YLS植物中分离。经查SciFinder数据库未见相同结构报道,为一新化合物。3.玉郎伞黄酮单体的抗氧化、抗凝血、耐缺氧作用3.1玉郎伞黄酮单体的体外抗氧化作用与空白对照组相比,YLS A～B单体的低、中、高浓度(终浓度分别为0.5、1.0、2.0 mg.ml-1)均能清除·O2-、·OH,抑制·O2-的生成(P<0.01)并呈浓度依赖性。其中,高浓度组效果最佳。3.2玉郎伞黄酮单体的抗凝血作用与正常对照组相比,YLS A～B低剂量组均无明显抗凝血作用(P> 0.05);YLS A～B中、高剂量组则可使凝血时间明显延长(P<0.01)。其中,高剂量组效果最好,抗凝作用与肝素钠相似(P>0.05 vs Heparin)。3.3玉郎伞黄酮单体的耐缺氧作用与正常对照组相比,YLS A～B低剂量组的存活时间无差异(P> 0.05),YLS A～B中、高剂量组则可显著延长缺氧小鼠的存活时间(P<0.01),其中,高剂量组效果最好,耐缺氧作用与Vit.C相似(P>0.05)。4.玉郎伞黄酮单体及含药血清对心肌细胞H/R的作用4.1心肌细胞形态学变化:光镜下可见原代培养3～4d后,心肌细胞融合成片,呈放射状排列,细胞之间伸出多个伪足交织成网状,细胞搏动同步化;H/R模型组心肌细胞胞浆空泡形成,细胞伪足收缩或消失,细胞折光率下降,异形心肌细胞数目增加并有大量细胞脱离、悬浮、溶解坏死,体内搏动幅度及频率减弱,节律不齐;YLS A～B单体及含药血清组可见少量的心肌细胞伪足回缩,细胞脱落悬浮现象,但损伤程度较模型组显著减轻且死亡细胞明显减少。4.2与模型组相比,YLS A～B单体及含药血清中、高剂量可显著增加心肌细胞活力及Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶活性、降低心肌细胞培养上清夜中MDA、LDH、iNOS、TNF-а含量(活性)、增加T-SOD、GSH、cNOS、tNOS活性(P< 0.05)。5.玉郎伞黄酮单体及含药血清对H2O2诱发心肌细胞凋亡的作用及机制5.1与模型组相比,中、高剂量的YLS A～B单体及含药血清能明显降低H2O2诱发所致心肌细胞的凋亡(P<0.01)并呈剂量依赖性,以高剂量组效果最明显。5.2与模型组相比,中、高剂量的YLS A～B单体及含药血清均能下调NF-κBp65、Bax蛋白表达,上调Bcl-2蛋白表达、增加Bcl-2/Bax比值(P<0.01)。结论:1.首次从YLS植物中分离出两个黄酮类活性单体,其中一个为新化合物,另一个系首次从植物中分离并报道核磁数据。2.两个YLS黄酮活性单体均具有抗氧化、抗凝血、耐缺氧、保护心肌细胞缺氧/复氧损伤、减轻心肌细胞凋亡的作用,并呈一定的剂量依赖性;其机制可能与抗氧化应激损伤、提高GSH、cNOS、Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶活性、降低LDH、iNOS、TNF-а活性、下调NF-κBp65和Bax蛋白表达、上调Bcl-2蛋白表达、增加Bcl-2/Bax比值而抑制心肌细胞凋亡有关。更多还原

【Abstract】 Yulangsan (YLS) is a folk herb widely used in Guangxi province. Its major active components of YLS include flavones, saponins and polycoses. Early researches showed that the extractives from YLS had the positive effects on hypertension, scavenging free radical, relieving myocardial ischemical reperfusion injury, increasing coronary artery flow rate , antiinflammatory and anticancer. Unfortunately, the prior researches were mainly focused on above- mentioned investigations, the active monomers and their mechanisms of action of YLS are not fully known.This study was designed to separate and purify the YLS flavonoid monom- ers and identify their molecular structure. Anti-oxidation in vitro, toleration to hypoxia and anticoagulation in vivo of YLS flavonoid monomers were investi- gated. At the same time, the hypoxia/ reoxygenation(H/R) injury and the H2O2- inducing cadiocyte apoptotic model of cultured neonatal rat cardiomyocytes were produced to inspect the possible mechanisms of YLS flavonoid monomers in vivo and in vitro.Objective:1.To study the technology for extracting total flavonoids from YLS.2.To separate and purify the YLS flavonoid monomers and identify their molecular structures. 3.To evaluate the effects of YLS flavonoid monomers on anti-oxidation in vitro, toleration to hypoxia and anticoagulation in vivo.4.To observe the effects of two flavone morphons on H/R injury in myo- cardial cells and explore its mechanisms.5.To investigate the effects of two flavone morphons on H2O2-induced cadiocyte apoptosis and its mechanisms.Methods:1.The technology for extracting total flavonoids from YLS1.1 Strong aqua reaction , AlCl3 reaction , MgAC reaction and HCl-Mg reaction were used to identify the total flavonoids of YLS.1.2 Rutin was used as control article, the OD275nm of YLS was determined and it was substed to the standard curvilinear equation.The contents of total fla- vonoids were calculated by the following formulas: Content %=C total flavonoids/CYLS×100% Extraction rate %= Content×WT extrac (g)/Crude drug (g)×100%1.3 To optimize the extraction of the total flavonoids from YLS A comparison of extract methods was conducted among the extractions of traditional heat refluxing, microwave and cold diffusion with the extraction yield of total flavonoids from YLS as major targets, meanwhile, the extraction time, extraction frequence, reagent dosage, extraction cost, and personnel and environmental protections were investigated .2.To separate and purify the YLS flavonoid monomers and identify their molecular structures.2.1 Separation:Using petroleum for getting rid of pigment and esters , acetoacetate as a abstracted solvent, the EtoAC extractant from YLS was extracted by the process of condense and cryodesiccation.Reciped a glass chromatographic column(10cm×120cm) aidding pressurize igniter,and gel silica H for TLC was used as separating fixed phase(sample:gel silica H equal 1:30 ). PE / EtOAc mixed solven was used for gradient elution according PE→EtOAc polarity order . The identical portions were merged by TLC.Antioxidative activity of the prefractionational fraction was sieved by using the system of proceeding oxyradical (·O2-) and hydroxy radical(·OH) were established in vitro. The optimal fraction was segregated in multilevel by gel silica column chromatography repeatedly, while different ratio PE/EtOAc was used as an eluant.2.2 Purification:Recryst,PTLC and PHPLC were used to purify the YLS flavonoid mono- mers.2.3 Identification:TLC and HPLC were used to determine the purity of two monomers.UV and IR were applyed to analysis the mother nucleus, substituent and functional groups. ESI-MS was taken to evaluate the molecular mass. NMR was adopted to characterize the molecular structure, while the steric configuration was assay- ed by x-ray monocrystal diffraction technology.3. To study the anti-oxidation of YLS flavonoid monomers in vitro, tolera- tion to hypoxia and anticoagulation in vivo.3.1 The anti-oxidation of YLS flavonoid monomers in vitroYLS A～B were dissolved in 1%DMSO as 0.5, 1.0, 2.0 mg.ml-1 respecti- vely. The cleaning and depressant effects of two flavone monomers from YLS on free radicals were studied by using the system of proceeding·O2- and·OH in vitro while 8 groups were established randomly as follows: normal, positive drug, and the low, middle, high-dose of YLS A～B group.3.2 The effects on anticoagulation in vivo90 mice were randomly divided into 9 groups with 10 mice in each group: normal, solvent,positive drug, and the low, middle, high-dose of YLS A～B group. Capillary tube method of thrombotest was used to investigate the anti- coagulated blood effects.3.3 The effects on toleration to hypoxiaHypoxia model of mice at normal pressure were used to evaluate the anti- hypoxia effects of two flavone monomers.4.The effects of two flavone morphons and the drug-containing sera on H/R injury in myocardial cellsThe hypoxia/reoxygenation injury model of cultured neonatal rat cardio- myocytes was developed in vitro. Active cardiocytes were picked out and divid- ed into following 17 groups : normal, H/R model, positive drug, blank serum, solvent, the low, middle, high-dose of YLS A～B morphons and the drug-con- taining serum.The cell morphous, the survival rate, the contents of MDA , T- SOD, GSH , LDH , NOS, TNF-аand the ctivities of Na+-K+-ATP, Ca2+-Mg2+- ATP enzymes were determined in the cultured neonatal rat cardiomyocytes inju- ried by H/R.5.The effects of two flavone morphons on cadiocyte apoptosis induced by H2O2 and its mechanisms.The cadiocyte apoptosis model was established by adding 100μmol·L-1 H2O2 as final concentration into cell culture fluid for 16h. The grouping and medication were same as above, while the apoptosis rate of the cadiocytes was detected by AnnexV-FITC/PI double staining.The immunohistochemical method was used to detect the protein expressions of NF-κBp65, Bcl-2 and Bax.Results:1.The technology on extracting total flavonoids from YLSThe microwave extraction was superior to the traditional heat refluxing extraction and cold diffusion. The specific technology was just as follows: an ethanol concentration of 60%; an extraction temperature below 60°C; a material to solvent ratio of 1:8; a microwave power of 240W; an extraction time of 20 min with the extraction carried out for one times. According to this technology, the extraction rate of total flavonoids was 3.16 % (vs crude drug), while the pur- ity was 22.5 % (vs rutin).2.The purification and identification of the YLS flavonoid monomersTwo flavonoid monomers were separated from YLS first time. YLS A: C17H16O3 , jasmineneedle crystal ,mp. 120.0-121.0℃;UV(MeOH) ?λmax(nm): 260 ,300; ESI-MS m/z: 291.17 [M+Na]+, 307.13 [M+K]+; IR(KBr)(cm-1):3501 , 3123 , 2929 , 2852 , 1789, 1623, 1604, 1568, 1528, 1462, 1372, 1285, 1228, 1165, 1133; 1HNMR(DMSO-d6 , 500MHz)δ:3.86(6H , s, H-OCH3 X2, H-7, 8), 7.60(1H, brd, J=6.9 Hz, H-4),7.60(5H, m, H-2′、3′、4′、5′、6′), 7.72(1H, J=8.8Hz, H-α),8.00(1H, d, J=8.8Hz, H-?),8.13(2H, brd, J=6.5Hz, H-3, 5); 13CNMR(DMSO-d6 , 125 MHz)δ: 174.2(C-7′),110.6(C-α),121.5(C- ?),119.6(C-1),147.8(C-2, 6),128.6(C-3, 5),130.9(C-4),130.9(C-1′),129.2(C-2′、3′、4′、5′、6′), 60.2(C-7), 60.2(C-8)。Chemical name: Cis-2,6- di-methoxyl chalcone. According to the results retrieving by SciFinder data base, it was separated and reported NMR data primary.YLS B: C18H14O4 , orange formula-crystal, mp. 135.0-136.0℃;UV(MeOH)λmax(nm): 238, 350; ESI-MS m/z: 295.00[M+H]+, 610.99 [2M+ Na]+, 263.35 [M?OMe]+; IR(KBr)(cm-1):3501, 3133, 2929, 2831, 1739, 1600, 1562, 1474, 1380, 1262, 1219, 1137, 1102, 1062, 1025; 1HNMR(CDCl3 , 500MHz)δ3.95(3H, s, -OCH3), 7.2(1H, d, H-5’), 8.2(1H, d, H-4’), 8.17(2H, s, H-2’, 6’), 7.77(1H, s, H-α),7.57(5H, m, H-2, 3, 4, 5, 6); 13CNMR(CDCl3 , 125MHz)δ60.2(C-9’), 104.2(C-α),110.0(C-5’), 117.0(C-3’), 119.8(C-8’), 122.0(C-1’), 128.4(C-2’), 128.6(C-2, 4, 6),130.6(C-3, 5),141.8(C-1),145.7(C-4’), 150.0(C-6’), 154.8(C-7’), 158.2(C-?),175.0(C=O);Crystal data: Monoclinic, P2(1)/c, a=10.8813(16)nm, b= 9.7954(14) nm, c= 13.3473(19) nm,α= 90°, ?=103.697(2)°,γ= 90°, V=1382.2(3)nm3 ,Z=4, Dc=1.414 mg/m3, F(000)=616 ,μ=0.100 mm-1.Chemical name : Cis-6’-methoxyl- ?- hydroxy- benzene-furan chalcone. It was a new compound because no identical structure was reported in SciFinder data base up to now. 3.The effects of YLS flavonoid monomers on anti-oxidation in vitro, toleration to hypoxia and anticoagulation in vivo.Two flavone monomers of low, middle and high concentrations (final concentrations were 0.5, 1.0, 2.0mg/ml respectively) could scavenge·O2- and·OH (P<0.05 vs control). Meanwhile, they inhibited the production of·O2- significantly (P<0.05 vs control). The effects were concentration-dependent. Two flavone monomers of middle and high dose could significantly prolong the bleeding time in mice and increased the survival time of hypoxia mice( P<0.05 vs control ). 4.The effects of two flavone morphons on H/R injury in myocardial cells Cadiocyte fused to flakiness and aligned radiatly under optical microscope, in which cell parapodium interlaced reticulation, cell beat synchronization could be observed . The endochylema vacuolization , cell parapodium contracted or disappeared, refractive index decreased, obform cadiocyte increased as well as cell detachment , float , dissolve, necrosis, cell beat extentand frequency inhibi- tion, and rhythm irregularity were displayed in H/R model group.These changes were attenuated significantly in two flavone morphons and their drug-containing sera of middle and high dosage groups(P<0.05 vs model). Compared with the model group, preconditioning by two flavone morphons and drug-containing sera of middle and high dosage could enhance the survival rate of myocardial cells after H/R. Meanwhile, it increased the activities of GSH, cNOS, tNOS, T-SOD, Na+-K+-ATP and Ca2+- Mg2+-ATP enzymes and decreased the releases of MDA , LDH , iNOS , and TNF-а(P<0.05) with dosage depend- ent.5.The effects of two flavone morphons on cadiocyte apoptosis induced by H2O2 and its mechanismsFCM tests showed that the apoptosis rate of cadiocytes in low , middle, high dose of two flavone morphons and their drug-containing sera groups decr- eased significantly compared with those of model group(P<0.01).In addition, compared with the model group, two flavone morphons and drug-containing sera groups at middle , high dose could inhibit significantly the protein expressions of NF-κBp65 and Bax, and it could increase the protein expressions of Bcl-2 and the ratio of Bcl-2/Bax(P<0.01).Conclusions:1.Two flavonoids monomers from YLS are separated initially.YLS A is separated and reported NMR data primary , while YLS B is a new compound according to the data base in SciFinder.2.Two flavone monomers have potent effects on scavenging free radicals, anti-hypoxia, anticoagulated blood, protecting H/R injury in myocardial cells and relieving cadiocytes apoptosis, meanwhile, it shows certain dose-effect relationship. Its protective mechanisms may be related to its anti-oxidative stress injury, increasing the activities of GSH, cNOS, tNOS, T-SOD, Na+-K+-ATP , Ca2+- Mg2+-ATP enzymes , decreasing the releases of MDA, LDH, iNOS, TNF-а, and relieving cadiocyte apoptosis by down-regulating the protein expressions of NF-κBp65 and Bax, up-regulating the protein expressions of Bcl-2 and the ratio of Bcl-2/Bax.更多还原