玉郎伞黄酮成分的单体分离与药效研究
|
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。
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 YLS
1.1 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 vitro
YLS 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 vivo
90 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 hypoxia
Hypoxia 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 cells
The 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 YLS
The 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 monomers
Two 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 mechanisms
FCM 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.
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。
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 YLS
1.1 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 vitro
YLS 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 vivo
90 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 hypoxia
Hypoxia 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 cells
The 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 YLS
The 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 monomers
Two 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 mechanisms
FCM 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.
引文
[1]广西壮族自治区卫生厅.广西中药材标准[S].南宁:广西科学技术出版社,1990
[2]黄仁彬,林兴,蒋伟哲,等.玉郎伞化学成分对自发性高血压大鼠血压的影响[J].中国医院药学杂志,2006;26(2):130-4
[3]张靖贤,孔晓龙,蒋伟哲.龙眼参中多糖的含量测定[J].中国现代应用药学杂志,2002;19(2):108
[4]黄忠仕,林兴,李江等.龙眼参多糖对D一半乳糖所致的痴呆小鼠脑组织NO、SOD、MDA的影响[J].中国老年学杂志,2005;25(2):176-7
[5]段小群,林兴,焦杨,等.龙眼参多糖对四氯化碳诱导大鼠原代肝细胞损伤的保护作用[J].中国药房,2006;17(15):1132-4
[6]黄仁彬,段小群,焦杨,等.玉郎伞提取物对小鼠急性化学性肝损伤的保护作用及其机制的研究[J].广西医科大学学报,2003;20(6):874-7
[7]孔晓龙,蒋伟哲,黄仁彬,等.龙眼参对自发性高血压大鼠和SD大鼠血压的影响[J].中草药,2001;32(8):727-30
[8]黄仁彬,焦杨,蒋伟哲,等.玉郎伞提取物对心脏血流动力学和冠脉流量的影响[J].中国医院药学杂志,2003;23(6):321-2
[9]焦杨,段小群,孔晓龙,等.玉郎伞提取物对大鼠心肌缺血再灌注损伤的保护作用[J].中国医院药学杂志,2004;24(12):326-7
[10]焦杨,段小群,黄仁彬,等.玉郎伞提取物对超氧阴离子自由基和羟自由基的抑制和清除作用[J].广西医科大学学报,2004;21(1):22-3
[11]孔晓龙,蒋伟哲,林自中.龙眼参提取物对耐药口腔上皮样癌细胞株KB/MIT及耐药乳腺癌细胞株MCF一7/ADR的逆转作用[J].广西医科大学学报2003;20(4):495-6
[1]黄仁彬,林兴,蒋伟哲,等.玉郎伞化学成分对自发性高血压大鼠血压的影响[J].中国医院药学杂志,2006;26(2):130-4
[2]苏东林,单杨,李高阳.紫外分光光度法测定柑桔皮中总黄酮的含量[J].食品研究与开发,2007;(8):312-4
[3]黄富远,高明,楼云雁.黄酮类化合物的研究概况[J].中华中医药学刊,2007;25(8):32-5
[4]王艳,张铁军.微波萃取技术在中药有效成分提取中的应用[J].中草药,2005;36(3):470-3
[1]焦杨,段小群,黄仁彬,等.玉郎伞提取物对超氧阴离子自由基和羟自由基的抑制和清除作用[J].广西医科大学学报,2004;21(1):22-3
[2] Ting Han, Huiliang Li, Qiaoyan Zhang, et al. New thiazinediones and other components from Xanthium strumarium[J].Chemistry of Natural Compo- unds, 2006; 42(5):567-71
[3]赖毅勤,周宏兵.近年来黄酮化合物提取和分离方法研究进展[J].食品与药品,2007;9(4):54-9
[4]张胜帮,赵玲玲.黄酮类化合物的提取纯化研究进展[J].温州大学学报(自然科学版),2007;28(5):25-30
[1]周新,李宏杰.黄酮类化合物的生物活性及临床应用进展[J].中国新药杂志,2007;16(5):350-5
[2]焦杨,段小群,黄仁彬,等.玉郎伞提取物对超氧阴离子自由基和羟自由基的抑制和清除作用[J] .广西医科大学学报,2004;21(1):22-3
[3]张卫华,张振林,黄显峰,等.茜草饮片炒炭前后止血机制的比较[J].中华中医药杂志,2006;21(3):160-2
[4]简洁,梁钢.心脑肾康的组方筛选、初步稳定性及耐缺氧研究[J].广西医科大学学报,2007;24(3):551-3
[5]陈健,黄媛恒,王乃平,等.玉郎伞多糖和皂苷对氧自由基清除作用的研究[J].中药药理与临床,2007;23(5):100-02
[6]张绪东,蒋伟哲,焦杨,等.玉郎伞黄酮对大鼠心肌缺血再灌注损伤的保护作用[J].中国新药与临床杂志,2008;27(6):412-5
[7] J.Chen,X.Lin,Y.Jiao. Protection by Yulangsan polysaccharide against focal cerebral ischemia-reperfusion injury in rats and the mechanism[J].中国新药杂志,2008;17(13):1118-20
[1]焦杨,段小群,孔晓龙,等.玉郎伞提取物对大鼠心肌缺血再灌注损伤的保护作用[J].中国医院药学杂志,2004;24(12):326-7
[2]杨瑞丰,潘伟.心肌细胞培养进展[J].中国心血管病研究杂志,2005;3(8): 634-6
[3] Kong CR, Bursac N, Tung L.Mechanoelectrical excitation by fluid jets in monolayers of cultured cardiac mycocytes[J].J Appl Physiol,2005;98(6):2328-36
[4]陈淳媛,孙跃女,杨作成.新生大鼠心肌细胞原代培养[J].实用预防医学,2007;14(4):983-5
[5]何蕾,张莉,黄靖香.乳鼠心肌细胞的体外培养及生物学特性研究.解放军医学杂志,2007;32(5):506-7
[6]邵华,金秀东,梁军.不同差速贴壁时间对心肌细胞培养的影响实验性研究[J].牡丹江医学院学报,2008;29(1):3-4
[7]张中海,包琳,史巧云. Balb/c乳鼠心肌细胞的分离和体外培养方法的优化[J].哈尔滨医科大学学报,2007;41(4):324-6
[8]张金艳,李贻奎,马堃，等.冠心苏合丸系列组方药物血清对心肌细胞缺氧复氧损伤的影响[J].中国新药杂志,2007;16(18):1481-4
[9]张绪东,蒋伟哲,焦杨,等.玉郎伞黄酮对大鼠心肌缺血再灌注损伤的保护作用[J].中国新药与临床杂志,2008;27(6):412-5
[10]杨兰,郝卫东,尚兰琴,等.四逆汤水提物对缺氧-复氧损伤心肌细胞的保护作用[J].中华中医药杂志,2005;20(6):338-41
[11]蔡诗琴,龚培力.二氢石蒜碱对大鼠乳鼠培养心肌细胞缺氧/复氧损失的保护作用[J].中国药理学通报,2005;21(8):999-1002
[12] Zingarelli B, Hake PW, Yang Z, et al. Absence of inducible nitric oxidesynthase modulates early reperfusion-induced NF-kappaB and AP-1 activation and enhances myocardial damage[J]. FASEB J, 2002;16(3):327-42
[13]王乐,曾秋棠,谢江.白藜芦醇预处理对体外大鼠心肌缺血再灌注损伤的保护作用[J].临床心血管病杂志,2006;22(1):47-9
[14]Gao F, Gao E, Yue TL, et al. Nitric oxide mediates the anti-apoptotic effect of insulin in myocardial ischemia-reperfusion: the roles of PI3-kinase, Akt, and endothelial nitric oxide synthase phosphorylation[J]. Circulation, 2002;105(12):1497-502
[15]Kawahara K ,Takase M ,Yamauchi Y. Increased vulnerability to ischemia/ reperfusion-induced ventricular tachyarrhythmias by pre-ischemic inhibition of nitric oxide synthase in isolated rat hearts[J]. Cardiovasc Pathol, 2003;12(1):49-56
[16]Mehlhorn U, Bloch W, Krahwinkel A, et al. Activation of myocardial constitutive nitric oxide synthase during coronary artery surgery[J]. Eur J Cardiothorac Surg,2000;17(3):305-11
[17]Wang W, Sawicki G,Schulz R. Peroxynitrite-induced myocardial injury is mediated through matrix metalloproteinase-2[J]. Cardiovasc Res, 2002;53(1):165-74
[18]Gok S, Vatansever S, Vural K, et al. The role of ATP sensitive K+ channels and of nitric oxide synthase on myocardial ischemia/reperfusion- induced apoptosis[J].Acta Histochem, 2006;108(2):95-104
[19]储岳峰,柯永胜,俞国华.茶皂苷减轻心肌缺血再灌注损伤的实验研究[J].中国心血管病研究杂志,2007;5(4):296-8
[20]曹文军,陈瑞芬,刘国贞.槲皮素对缺血再灌注大鼠心肌损伤的保护作用[J].首都医科大学学报,2004;25(3):311-3
[1]徐菲菲,刘秀华,祝筱梅.低氧预处理通过上调钙网蛋白表达减轻大鼠心肌细胞氧化应激损伤[J].生理学报,2008;60(1):29-37
[2]王丽伟,刘凤芝,陆景坤.黄酮醇类化合物对心肌细胞凋亡过程caspase- 3、Bcl-2、Bax蛋白表达的影响[J].中国药学杂志,2007;42(10):749-54
[3]王睿,徐长庆.丹参注射液对阿霉素所致心肌细胞损伤的保护作用[J].齐齐哈尔医学院学报,2007;28(1):17-9
[4]陆景坤,王丽伟,刘凤芝.不同类型黄酮对过氧化氢诱导的心肌细胞凋亡的影响[J].中国临床药理学与治疗学,2007;12(6):260-5
[5]潘志伟,王秋娟,柳仁民,等.淫羊霍苷预适应对乳鼠心肌细胞的保护作用[J].中国药科大学学报,2007;38(1):73-6
[6]周蓓,吴力军,田代真一.水飞蓟宾对异丙肾上腺素引起的大鼠心肌细胞损伤的保护作用及机制[J].药学学报,2007;42(3):263-8
[7]王娜,关鹏,常彦忠.大豆异黄酮对乳鼠心肌细胞氧化损伤的保护作用[J].河北中医药学报,2007;22(1):31-2
[8] Zhao ZQ ,Morris CD, Budde JM,etal.Inhibition of myocardial apoptosis reduces infaret size and improves regional contraetile disfunction during reperfusion[J].Cardiovasc Res, 2003;59 (11):132-42
[9]张彦清,刘宝江.异丙酚对乳鼠心肌细胞缺氧/复氧损伤后凋亡的影响[J].心脏杂志,2007;19(2):392-4
[10] Scarabelli TM ,StePhanou A ,Pasini E ,etal.Different signaling pathways induce apoptosis in endothelial cells and cadiocytes during ischemia/ re- perfusion injury[J].Circ Res,2004; 90(6):745-8
[11]Borutaite V, Brown GC, Mitochondria in apoptosis of ischemic heart[J].FEBS Lett, 2003;541(3):1-5
[12]吴青,陶宏凯,陶大昌,等.缺血再灌注诱导心肌细胞凋亡及凋亡相关基因表达的研究[J].中国心血管病研究杂志,2004;2(11):905-8
[13]谭兴琴.凋亡因子Bcl-2/Bax与心肌缺血再灌注损伤[J].重床医学,2007;36(18):1885-7
[14]裴兆辉,马虹,朱妙章.血管紧张素П诱导心肌细胞凋亡机制的研究进展[J].心脏杂志,2005;17(5):482-3
[15]杨晓红,崔炜,王雅依.阿托伐他丁对糖尿病大鼠心肌细胞NF–κB、TNF-α表达的影响[J].解放军医学杂志,2008;33(3):282-4
[16]庄梅,方颖,吴立荣.地塞米松对大鼠缺血再灌注心肌细胞凋亡及HSP -70、NF–κB蛋白表达的影响[J].中国老年学杂志,2008;28(15):1467-9
[17]Bergmann MV, Loser P, Dietz R, et al.Effects of NF-Kappa B inhibition on TNF-αinduced apoptosis and downstream pathways in cardiomyocytes[J]. J Mol Cell Cardiol,2001;33(6):1223-32
[18]胡波,张晓刚.糖基化终产物诱导心肌细胞炎症反应的研究[J].中国病理生理杂志,2007;23(12):2341-5
[19]Li C, Kao RL ,Ha T,et al.Early activation of IKK beta during in vivo myocardial isehemia[J].Am J Physiol Heart Circ Physiol,2001;280(3): 1264-71
[20]李乐,高小丽,丁宝兴.青藤碱抑制H2O2诱导乳鼠心肌细胞凋亡[J].中国中药杂志,2008;33(8):939-42
[21]杨平,王迪,敖杰男.心脉通抗氧化应激所致心肌细胞凋亡的机制探讨[J].中国新药与临床药理,2007;18(4):279-82
[22]朱汉华,李浪. TNF-α介导的心肌细胞凋亡信号转导通路在心肌损伤中的作用[J].心血管病学进展,2008;29(2):329-31
[23]Chandrasekar B, Colston JT,dela SD,et al. TNF-αand H2O2 induced L-18 and L-18R beta expression in cardiomyocytes via NF-kappaB [J]. Biochem Biophys Res Commun. 2003;303(4):1152-8
[1]龙春,高志强,陈凤鸣,等.黄酮类化合物的结构—抗氧化活性关系研究[J].重庆文理学院学报(自然科学版),2006;5(2):13-7
[2]高金燕,曾小花,王毅辉.从西芹中提取黄酮类物质的初步研究[J].中国食品添加剂,2004;(3):19-21
[3]郭雪峰,岳永德.黄酮类化合物的提取、分离纯化和含量测定方法的研究进展[J].安徽农业科学,2007;35(26):8083-6
[4]朱红梅,赵猛,王文晖.酶法提取沙棘叶中黄酮的研究[J].农业与技术.2008;(6):34-6
[5]许云峰,张臻,周建芹.内生镰刀菌漆酶辅助提取槐米中总黄酮的研究[J].中草药,2008;39(11):1639-42
[6]刘志刚,颜仁梁,徐昌瑞.正交试验法优选黄芩总黄酮苷元酶解后提取工艺[J].中药新药与临床药理,2008;19(2):143-5
[7]陈永刚,刘晓涵,林励.超声波提取法提取化橘红中黄酮类物质的工艺优选[J].中药材,2009;32(1):138-42
[8]梁惠花,刘晓河,王志宝.坝上油菜蜂花粉中总黄酮的超声提取及含量测定[J].张家口医学院学报,2003;20(4):8-9
[9]王艳,张铁军.微波萃取技术在中药有效成分提取中的应用[J].中草药,2005;36(3):470-3
[10]元英进,刘明言,董岸杰.中药现代化生产关键技术[M].北京:化学工业出版社,2002
[11]张英,俞卓裕,吴晓琴.中草药和天然植物有效成分提取新技术——微波协助萃取[J].中国中药杂志,2004;29(2):104-9
[12]Zhao EL , Gai QQ, Zhang HR. Investigation ofmicrowave assisted extrac- tion of seabuckthorn flavonoids[J]. Food FermentInd, 2004;30 (12):148-50
[13]Yue ME , JiangTF , ShiYP . Fast determination of flavonoids in Hippophae rhamnoides and itsmedicinalpreparation by capillary zone electrophores isusing dimethyl-β-cy-clodextrin asmodifier[J]. Talanta ,2004;62:695-9
[14]郭雪峰,岳永德.黄酮类化合物的提取、分离纯化和含量测定方法的研究进展[J].安徽农业科学,2007;35(26):8083-6
[15]钱亚琴,刘汉清,庄炜. SFE—CO2萃取技术在中药提取分离中的应用[J].南京中医药大学学报,2004;20(4):254-6
[16]姚开,贾冬英,何强,等.大豆异黄酮主要单体组分的分离方法[J].四川大学学报(工程科学版),2004;36(3):77
[17]Satu-Maaril H,Antti H,Krislilna W,eta1.Metabolism of the soy isofla- vonesdaidzein.genistein alld glycitein in human subjects:Identiifcation of new metabolitc shaving an intactisol favonoid skeleton[J].J Steroid Biochem Mol Biol,2005;89 (5):285-9
[18]曹群华,瞿伟菁,李家贵,等.大孔树脂吸附纯化沙棘籽渣总黄酮的研究[J].中国中药杂志,2005;30(3):225-7
[19]任荣军.大孔树脂吸附技术在黄酮类成分分离纯化中的应用[J].中国药业,2008;17(16):71-3
[20]闫磊,再安,焱文.大孔树脂分离纯化山楂总黄酮的研究[J].湖北中医学院学报,2007;9(2):33-5
[21]孙希云,张琦,孟宪军.大孔吸附树脂分离纯化马齿苋中黄酮类化合物的研究[J].食品与发酵工业,2006;32(2):124-7
[22]武夏明,刘逢芹,李宏建.黄酮类中药成分提取分离技术的应用[J].齐鲁药事,2008;27(7):419-21
[23]傅博强,刘士力,王小如.XDA-1大孔吸附树脂对甘草酸及甘草总黄酮的吸附分离[J].现代中药研究与实践,2004;18(增刊):45-9
[24]张纪兴,李坚,程国华.大孔树脂吸附法富集地锦草总黄酮的工艺研究[J].中药材,2006;29(2):123-5
[25]关海君,田娟娟,白志明.滤膜技术在大豆异黄酮生产中的应用[J].中国油脂,2006;31(3):35-6
[26]赵貤,王亚群,李琳.高效毛细管电泳法分离黄酮类化合物的进展[J].华西医学,2002;17(4):577-8
[27]刘宁宁,李爱峰,柳仁民.高速逆流色谱在分离纯化中草药黄酮类成分中的应用[J].化学试剂,2008;30(5):346-50
[28]WU Su-juan ,SUN Ai-ling ,LIU Ren-min. Separation and purification of baicalin andwogonoside fromthe Chinese medicinal plant Scutellaria baica- lensis Georgi by high-speed countercurrent chromatography [J]. J. Chroma- togr. A , 2005;1066(2):243-7
[29]LI Hua-bin , CHEN Feng. Isolation and purification of baicalein , wogonin and oroxylin A from the medicinal plant Scutellaria baicalensis by high- speed counter-current chromatography[J]. J.Chromatogr.A, 2005;1074(2):107-10
[30]ZHOU Ting-ting, ZHU Zhen-yu, WANG Chen, et al.On-line purity monitor- ing in high-speed countercurrent chromatography:application of HSCCC- HPLC-DAD for the preparation of 5-HMF,neomangiferin and mangiferin from Anemarrhena asphodeloides Bunge [J]. J.Pharm. Biomed.Anal, 2007;44(1):96-100
[31]王巧娥,王小如,曹建敏,等.高速逆流色谱法分离纯化甘草查尔酮甲和胀果香豆素甲[J].现代中药研究与实践,2004;18(增刊):53-6
[32]周瑞雪.苦荞总黄酮提取分离及质量控制研究.中国优秀硕士学位论文,2007
[33]付斌.藜蒿有效成分的提取、分离及抗乙肝病毒的实验研究.中国优秀硕士学位论文,2007
[34]王晓梅,曹稳根.黄酮类化合物药理作用的研究进展[J].宿州学院学报,2007;22(1):105-8
[35]周新,李宏杰.黄酮类化合物的生物活性及临床应用进展[J].中国新药杂志,2007;16(5):350-5
[36]BENT HH . The biochemistry andmedical significance of the flavonoids[J]. Pharmacol Ther , 2007;101(2):67-70
[37]李云峰,袁莉,杨明.棉籽总黄酮抗抑郁作用的研究[J].中国药理学通报,2006;22(1):48-50
[38]郝吉利,周兰兰,江秋虹.黄蜀葵总黄酮抗脑卒中后抑郁作用的研究[J].中国药房,2007;(12):652-5
[39]梁统,覃燕梅,丁航,等.侧柏总黄酮的抗炎作用[J].沈阳药科大学学报, 2005;22(4):301-3
[40]陈琳,程文明,胡成穆,等.豹皮樟总黄酮抗炎作用及部分机制研究[J].安徽医科大学学报, 2004;39(6):439-42
[41]李叶,唐浩国,刘建学.黄酮类化合物抑菌作用的研究进展[J].农产品加工·学刊,2008;(12):53-6
[42]谢鹏,张敏红.黄酮类化合物抑菌作用的研究进展[J].动物保健,2004;(12):35
[43]韩淑琴,杨洋,黄涛,等.仙人掌提取物的抑菌机理[J].食品科技,2007;(3):38-9
[44]Katarzyna Ulanowska, Aleksandra Tkaczyk, Grazyna Konopa, et al.Differ- ential antibacterial activity of genistein arising from global inhibition of DNA,RNA and protein synthesis in some bacterial strains[J].Archives of Microbiology,2006;184(5):271-8
[45]王超云.黄芩苷对化学性肝损伤的保护作用[J].中草药,2005;36(5):730- 2
[46]徐庆,宋芸娟,李丽亚,等.荔枝核总黄酮的抗鸭乙型肝炎病毒的作用[J].世界华人消化杂志,2005;13(17):2082-5
[47]蒋伟哲,孔晓龙,黄仁彬,等.龙眼参对小鼠化学性肝损伤的保护作用及其机制研究[J].中国药房,2004;15(7):398-401
[48]朱会平.黄酮类化合物促进癌细胞凋亡的分子机理研究进展[J].生物技术通讯,2006;17(3):455-6
[49]高昕,王喜军.黄酮类化合物抗肿瘤作用研究进展[J].中医药信息,2005;22(5):33-4
[50]农朝赞,黄华艺.黄酮类化合物抗肿瘤作用的研究应用[J].中国药房, 2004; 15(9): 568-9
[51]唐勇.鸡血藤黄酮类组分抗肿瘤活性研究[J].中国实验方剂学杂志,2007;13(2):51-4
[52]OFER M, WOLFFRAM S, KOGGEL A, et al. Modulation of drug transport by selected flavonoids: Involvement of P-gp and OCT[J]. EurJ Pharma Sci, 2005;25(3):263-71
[53]LI MTRAKULP , K HANTAMATO, PINTHA K. Inhibition of P- glycopro- tein function and expression by kaempferol and quercetin[J]. J Chemother, 2005;17(1):86-95
[54]林晓贞,梁钢,黎莉,等. EGCG逆转人耐药肝癌细胞株多药耐药的体内实验研究[J].广西医科大学学报,2006;(1):48-51
[55]兰真,曾凡骏,曾里,等.生物类黄酮对心血管系统的调节及作用机理[J].现代预防医学,2005;32(6):613-5
[56]韩兆轩,王维民.类黄酮在心血管疾病中的研究进展[J].农产品加工·学刊,2007;(12):31-4
[57]李乐,孙晓东,高晓利,等.绞股蓝总黄酮对麻醉犬心脏血流动力学及冠脉血流量的作用[J].浙江工业大学学报,2006;34(5):521-8
[58]杨青,郭彩清,油继辉,等.黄酮类物质的生理功能及应用发展动态[J].贵州农业科学,2007;35(2):266-8
[2]黄仁彬,林兴,蒋伟哲,等.玉郎伞化学成分对自发性高血压大鼠血压的影响[J].中国医院药学杂志,2006;26(2):130-4
[3]张靖贤,孔晓龙,蒋伟哲.龙眼参中多糖的含量测定[J].中国现代应用药学杂志,2002;19(2):108
[4]黄忠仕,林兴,李江等.龙眼参多糖对D一半乳糖所致的痴呆小鼠脑组织NO、SOD、MDA的影响[J].中国老年学杂志,2005;25(2):176-7
[5]段小群,林兴,焦杨,等.龙眼参多糖对四氯化碳诱导大鼠原代肝细胞损伤的保护作用[J].中国药房,2006;17(15):1132-4
[6]黄仁彬,段小群,焦杨,等.玉郎伞提取物对小鼠急性化学性肝损伤的保护作用及其机制的研究[J].广西医科大学学报,2003;20(6):874-7
[7]孔晓龙,蒋伟哲,黄仁彬,等.龙眼参对自发性高血压大鼠和SD大鼠血压的影响[J].中草药,2001;32(8):727-30
[8]黄仁彬,焦杨,蒋伟哲,等.玉郎伞提取物对心脏血流动力学和冠脉流量的影响[J].中国医院药学杂志,2003;23(6):321-2
[9]焦杨,段小群,孔晓龙,等.玉郎伞提取物对大鼠心肌缺血再灌注损伤的保护作用[J].中国医院药学杂志,2004;24(12):326-7
[10]焦杨,段小群,黄仁彬,等.玉郎伞提取物对超氧阴离子自由基和羟自由基的抑制和清除作用[J].广西医科大学学报,2004;21(1):22-3
[11]孔晓龙,蒋伟哲,林自中.龙眼参提取物对耐药口腔上皮样癌细胞株KB/MIT及耐药乳腺癌细胞株MCF一7/ADR的逆转作用[J].广西医科大学学报2003;20(4):495-6
[1]黄仁彬,林兴,蒋伟哲,等.玉郎伞化学成分对自发性高血压大鼠血压的影响[J].中国医院药学杂志,2006;26(2):130-4
[2]苏东林,单杨,李高阳.紫外分光光度法测定柑桔皮中总黄酮的含量[J].食品研究与开发,2007;(8):312-4
[3]黄富远,高明,楼云雁.黄酮类化合物的研究概况[J].中华中医药学刊,2007;25(8):32-5
[4]王艳,张铁军.微波萃取技术在中药有效成分提取中的应用[J].中草药,2005;36(3):470-3
[1]焦杨,段小群,黄仁彬,等.玉郎伞提取物对超氧阴离子自由基和羟自由基的抑制和清除作用[J].广西医科大学学报,2004;21(1):22-3
[2] Ting Han, Huiliang Li, Qiaoyan Zhang, et al. New thiazinediones and other components from Xanthium strumarium[J].Chemistry of Natural Compo- unds, 2006; 42(5):567-71
[3]赖毅勤,周宏兵.近年来黄酮化合物提取和分离方法研究进展[J].食品与药品,2007;9(4):54-9
[4]张胜帮,赵玲玲.黄酮类化合物的提取纯化研究进展[J].温州大学学报(自然科学版),2007;28(5):25-30
[1]周新,李宏杰.黄酮类化合物的生物活性及临床应用进展[J].中国新药杂志,2007;16(5):350-5
[2]焦杨,段小群,黄仁彬,等.玉郎伞提取物对超氧阴离子自由基和羟自由基的抑制和清除作用[J] .广西医科大学学报,2004;21(1):22-3
[3]张卫华,张振林,黄显峰,等.茜草饮片炒炭前后止血机制的比较[J].中华中医药杂志,2006;21(3):160-2
[4]简洁,梁钢.心脑肾康的组方筛选、初步稳定性及耐缺氧研究[J].广西医科大学学报,2007;24(3):551-3
[5]陈健,黄媛恒,王乃平,等.玉郎伞多糖和皂苷对氧自由基清除作用的研究[J].中药药理与临床,2007;23(5):100-02
[6]张绪东,蒋伟哲,焦杨,等.玉郎伞黄酮对大鼠心肌缺血再灌注损伤的保护作用[J].中国新药与临床杂志,2008;27(6):412-5
[7] J.Chen,X.Lin,Y.Jiao. Protection by Yulangsan polysaccharide against focal cerebral ischemia-reperfusion injury in rats and the mechanism[J].中国新药杂志,2008;17(13):1118-20
[1]焦杨,段小群,孔晓龙,等.玉郎伞提取物对大鼠心肌缺血再灌注损伤的保护作用[J].中国医院药学杂志,2004;24(12):326-7
[2]杨瑞丰,潘伟.心肌细胞培养进展[J].中国心血管病研究杂志,2005;3(8): 634-6
[3] Kong CR, Bursac N, Tung L.Mechanoelectrical excitation by fluid jets in monolayers of cultured cardiac mycocytes[J].J Appl Physiol,2005;98(6):2328-36
[4]陈淳媛,孙跃女,杨作成.新生大鼠心肌细胞原代培养[J].实用预防医学,2007;14(4):983-5
[5]何蕾,张莉,黄靖香.乳鼠心肌细胞的体外培养及生物学特性研究.解放军医学杂志,2007;32(5):506-7
[6]邵华,金秀东,梁军.不同差速贴壁时间对心肌细胞培养的影响实验性研究[J].牡丹江医学院学报,2008;29(1):3-4
[7]张中海,包琳,史巧云. Balb/c乳鼠心肌细胞的分离和体外培养方法的优化[J].哈尔滨医科大学学报,2007;41(4):324-6
[8]张金艳,李贻奎,马堃，等.冠心苏合丸系列组方药物血清对心肌细胞缺氧复氧损伤的影响[J].中国新药杂志,2007;16(18):1481-4
[9]张绪东,蒋伟哲,焦杨,等.玉郎伞黄酮对大鼠心肌缺血再灌注损伤的保护作用[J].中国新药与临床杂志,2008;27(6):412-5
[10]杨兰,郝卫东,尚兰琴,等.四逆汤水提物对缺氧-复氧损伤心肌细胞的保护作用[J].中华中医药杂志,2005;20(6):338-41
[11]蔡诗琴,龚培力.二氢石蒜碱对大鼠乳鼠培养心肌细胞缺氧/复氧损失的保护作用[J].中国药理学通报,2005;21(8):999-1002
[12] Zingarelli B, Hake PW, Yang Z, et al. Absence of inducible nitric oxidesynthase modulates early reperfusion-induced NF-kappaB and AP-1 activation and enhances myocardial damage[J]. FASEB J, 2002;16(3):327-42
[13]王乐,曾秋棠,谢江.白藜芦醇预处理对体外大鼠心肌缺血再灌注损伤的保护作用[J].临床心血管病杂志,2006;22(1):47-9
[14]Gao F, Gao E, Yue TL, et al. Nitric oxide mediates the anti-apoptotic effect of insulin in myocardial ischemia-reperfusion: the roles of PI3-kinase, Akt, and endothelial nitric oxide synthase phosphorylation[J]. Circulation, 2002;105(12):1497-502
[15]Kawahara K ,Takase M ,Yamauchi Y. Increased vulnerability to ischemia/ reperfusion-induced ventricular tachyarrhythmias by pre-ischemic inhibition of nitric oxide synthase in isolated rat hearts[J]. Cardiovasc Pathol, 2003;12(1):49-56
[16]Mehlhorn U, Bloch W, Krahwinkel A, et al. Activation of myocardial constitutive nitric oxide synthase during coronary artery surgery[J]. Eur J Cardiothorac Surg,2000;17(3):305-11
[17]Wang W, Sawicki G,Schulz R. Peroxynitrite-induced myocardial injury is mediated through matrix metalloproteinase-2[J]. Cardiovasc Res, 2002;53(1):165-74
[18]Gok S, Vatansever S, Vural K, et al. The role of ATP sensitive K+ channels and of nitric oxide synthase on myocardial ischemia/reperfusion- induced apoptosis[J].Acta Histochem, 2006;108(2):95-104
[19]储岳峰,柯永胜,俞国华.茶皂苷减轻心肌缺血再灌注损伤的实验研究[J].中国心血管病研究杂志,2007;5(4):296-8
[20]曹文军,陈瑞芬,刘国贞.槲皮素对缺血再灌注大鼠心肌损伤的保护作用[J].首都医科大学学报,2004;25(3):311-3
[1]徐菲菲,刘秀华,祝筱梅.低氧预处理通过上调钙网蛋白表达减轻大鼠心肌细胞氧化应激损伤[J].生理学报,2008;60(1):29-37
[2]王丽伟,刘凤芝,陆景坤.黄酮醇类化合物对心肌细胞凋亡过程caspase- 3、Bcl-2、Bax蛋白表达的影响[J].中国药学杂志,2007;42(10):749-54
[3]王睿,徐长庆.丹参注射液对阿霉素所致心肌细胞损伤的保护作用[J].齐齐哈尔医学院学报,2007;28(1):17-9
[4]陆景坤,王丽伟,刘凤芝.不同类型黄酮对过氧化氢诱导的心肌细胞凋亡的影响[J].中国临床药理学与治疗学,2007;12(6):260-5
[5]潘志伟,王秋娟,柳仁民,等.淫羊霍苷预适应对乳鼠心肌细胞的保护作用[J].中国药科大学学报,2007;38(1):73-6
[6]周蓓,吴力军,田代真一.水飞蓟宾对异丙肾上腺素引起的大鼠心肌细胞损伤的保护作用及机制[J].药学学报,2007;42(3):263-8
[7]王娜,关鹏,常彦忠.大豆异黄酮对乳鼠心肌细胞氧化损伤的保护作用[J].河北中医药学报,2007;22(1):31-2
[8] Zhao ZQ ,Morris CD, Budde JM,etal.Inhibition of myocardial apoptosis reduces infaret size and improves regional contraetile disfunction during reperfusion[J].Cardiovasc Res, 2003;59 (11):132-42
[9]张彦清,刘宝江.异丙酚对乳鼠心肌细胞缺氧/复氧损伤后凋亡的影响[J].心脏杂志,2007;19(2):392-4
[10] Scarabelli TM ,StePhanou A ,Pasini E ,etal.Different signaling pathways induce apoptosis in endothelial cells and cadiocytes during ischemia/ re- perfusion injury[J].Circ Res,2004; 90(6):745-8
[11]Borutaite V, Brown GC, Mitochondria in apoptosis of ischemic heart[J].FEBS Lett, 2003;541(3):1-5
[12]吴青,陶宏凯,陶大昌,等.缺血再灌注诱导心肌细胞凋亡及凋亡相关基因表达的研究[J].中国心血管病研究杂志,2004;2(11):905-8
[13]谭兴琴.凋亡因子Bcl-2/Bax与心肌缺血再灌注损伤[J].重床医学,2007;36(18):1885-7
[14]裴兆辉,马虹,朱妙章.血管紧张素П诱导心肌细胞凋亡机制的研究进展[J].心脏杂志,2005;17(5):482-3
[15]杨晓红,崔炜,王雅依.阿托伐他丁对糖尿病大鼠心肌细胞NF–κB、TNF-α表达的影响[J].解放军医学杂志,2008;33(3):282-4
[16]庄梅,方颖,吴立荣.地塞米松对大鼠缺血再灌注心肌细胞凋亡及HSP -70、NF–κB蛋白表达的影响[J].中国老年学杂志,2008;28(15):1467-9
[17]Bergmann MV, Loser P, Dietz R, et al.Effects of NF-Kappa B inhibition on TNF-αinduced apoptosis and downstream pathways in cardiomyocytes[J]. J Mol Cell Cardiol,2001;33(6):1223-32
[18]胡波,张晓刚.糖基化终产物诱导心肌细胞炎症反应的研究[J].中国病理生理杂志,2007;23(12):2341-5
[19]Li C, Kao RL ,Ha T,et al.Early activation of IKK beta during in vivo myocardial isehemia[J].Am J Physiol Heart Circ Physiol,2001;280(3): 1264-71
[20]李乐,高小丽,丁宝兴.青藤碱抑制H2O2诱导乳鼠心肌细胞凋亡[J].中国中药杂志,2008;33(8):939-42
[21]杨平,王迪,敖杰男.心脉通抗氧化应激所致心肌细胞凋亡的机制探讨[J].中国新药与临床药理,2007;18(4):279-82
[22]朱汉华,李浪. TNF-α介导的心肌细胞凋亡信号转导通路在心肌损伤中的作用[J].心血管病学进展,2008;29(2):329-31
[23]Chandrasekar B, Colston JT,dela SD,et al. TNF-αand H2O2 induced L-18 and L-18R beta expression in cardiomyocytes via NF-kappaB [J]. Biochem Biophys Res Commun. 2003;303(4):1152-8
[1]龙春,高志强,陈凤鸣,等.黄酮类化合物的结构—抗氧化活性关系研究[J].重庆文理学院学报(自然科学版),2006;5(2):13-7
[2]高金燕,曾小花,王毅辉.从西芹中提取黄酮类物质的初步研究[J].中国食品添加剂,2004;(3):19-21
[3]郭雪峰,岳永德.黄酮类化合物的提取、分离纯化和含量测定方法的研究进展[J].安徽农业科学,2007;35(26):8083-6
[4]朱红梅,赵猛,王文晖.酶法提取沙棘叶中黄酮的研究[J].农业与技术.2008;(6):34-6
[5]许云峰,张臻,周建芹.内生镰刀菌漆酶辅助提取槐米中总黄酮的研究[J].中草药,2008;39(11):1639-42
[6]刘志刚,颜仁梁,徐昌瑞.正交试验法优选黄芩总黄酮苷元酶解后提取工艺[J].中药新药与临床药理,2008;19(2):143-5
[7]陈永刚,刘晓涵,林励.超声波提取法提取化橘红中黄酮类物质的工艺优选[J].中药材,2009;32(1):138-42
[8]梁惠花,刘晓河,王志宝.坝上油菜蜂花粉中总黄酮的超声提取及含量测定[J].张家口医学院学报,2003;20(4):8-9
[9]王艳,张铁军.微波萃取技术在中药有效成分提取中的应用[J].中草药,2005;36(3):470-3
[10]元英进,刘明言,董岸杰.中药现代化生产关键技术[M].北京:化学工业出版社,2002
[11]张英,俞卓裕,吴晓琴.中草药和天然植物有效成分提取新技术——微波协助萃取[J].中国中药杂志,2004;29(2):104-9
[12]Zhao EL , Gai QQ, Zhang HR. Investigation ofmicrowave assisted extrac- tion of seabuckthorn flavonoids[J]. Food FermentInd, 2004;30 (12):148-50
[13]Yue ME , JiangTF , ShiYP . Fast determination of flavonoids in Hippophae rhamnoides and itsmedicinalpreparation by capillary zone electrophores isusing dimethyl-β-cy-clodextrin asmodifier[J]. Talanta ,2004;62:695-9
[14]郭雪峰,岳永德.黄酮类化合物的提取、分离纯化和含量测定方法的研究进展[J].安徽农业科学,2007;35(26):8083-6
[15]钱亚琴,刘汉清,庄炜. SFE—CO2萃取技术在中药提取分离中的应用[J].南京中医药大学学报,2004;20(4):254-6
[16]姚开,贾冬英,何强,等.大豆异黄酮主要单体组分的分离方法[J].四川大学学报(工程科学版),2004;36(3):77
[17]Satu-Maaril H,Antti H,Krislilna W,eta1.Metabolism of the soy isofla- vonesdaidzein.genistein alld glycitein in human subjects:Identiifcation of new metabolitc shaving an intactisol favonoid skeleton[J].J Steroid Biochem Mol Biol,2005;89 (5):285-9
[18]曹群华,瞿伟菁,李家贵,等.大孔树脂吸附纯化沙棘籽渣总黄酮的研究[J].中国中药杂志,2005;30(3):225-7
[19]任荣军.大孔树脂吸附技术在黄酮类成分分离纯化中的应用[J].中国药业,2008;17(16):71-3
[20]闫磊,再安,焱文.大孔树脂分离纯化山楂总黄酮的研究[J].湖北中医学院学报,2007;9(2):33-5
[21]孙希云,张琦,孟宪军.大孔吸附树脂分离纯化马齿苋中黄酮类化合物的研究[J].食品与发酵工业,2006;32(2):124-7
[22]武夏明,刘逢芹,李宏建.黄酮类中药成分提取分离技术的应用[J].齐鲁药事,2008;27(7):419-21
[23]傅博强,刘士力,王小如.XDA-1大孔吸附树脂对甘草酸及甘草总黄酮的吸附分离[J].现代中药研究与实践,2004;18(增刊):45-9
[24]张纪兴,李坚,程国华.大孔树脂吸附法富集地锦草总黄酮的工艺研究[J].中药材,2006;29(2):123-5
[25]关海君,田娟娟,白志明.滤膜技术在大豆异黄酮生产中的应用[J].中国油脂,2006;31(3):35-6
[26]赵貤,王亚群,李琳.高效毛细管电泳法分离黄酮类化合物的进展[J].华西医学,2002;17(4):577-8
[27]刘宁宁,李爱峰,柳仁民.高速逆流色谱在分离纯化中草药黄酮类成分中的应用[J].化学试剂,2008;30(5):346-50
[28]WU Su-juan ,SUN Ai-ling ,LIU Ren-min. Separation and purification of baicalin andwogonoside fromthe Chinese medicinal plant Scutellaria baica- lensis Georgi by high-speed countercurrent chromatography [J]. J. Chroma- togr. A , 2005;1066(2):243-7
[29]LI Hua-bin , CHEN Feng. Isolation and purification of baicalein , wogonin and oroxylin A from the medicinal plant Scutellaria baicalensis by high- speed counter-current chromatography[J]. J.Chromatogr.A, 2005;1074(2):107-10
[30]ZHOU Ting-ting, ZHU Zhen-yu, WANG Chen, et al.On-line purity monitor- ing in high-speed countercurrent chromatography:application of HSCCC- HPLC-DAD for the preparation of 5-HMF,neomangiferin and mangiferin from Anemarrhena asphodeloides Bunge [J]. J.Pharm. Biomed.Anal, 2007;44(1):96-100
[31]王巧娥,王小如,曹建敏,等.高速逆流色谱法分离纯化甘草查尔酮甲和胀果香豆素甲[J].现代中药研究与实践,2004;18(增刊):53-6
[32]周瑞雪.苦荞总黄酮提取分离及质量控制研究.中国优秀硕士学位论文,2007
[33]付斌.藜蒿有效成分的提取、分离及抗乙肝病毒的实验研究.中国优秀硕士学位论文,2007
[34]王晓梅,曹稳根.黄酮类化合物药理作用的研究进展[J].宿州学院学报,2007;22(1):105-8
[35]周新,李宏杰.黄酮类化合物的生物活性及临床应用进展[J].中国新药杂志,2007;16(5):350-5
[36]BENT HH . The biochemistry andmedical significance of the flavonoids[J]. Pharmacol Ther , 2007;101(2):67-70
[37]李云峰,袁莉,杨明.棉籽总黄酮抗抑郁作用的研究[J].中国药理学通报,2006;22(1):48-50
[38]郝吉利,周兰兰,江秋虹.黄蜀葵总黄酮抗脑卒中后抑郁作用的研究[J].中国药房,2007;(12):652-5
[39]梁统,覃燕梅,丁航,等.侧柏总黄酮的抗炎作用[J].沈阳药科大学学报, 2005;22(4):301-3
[40]陈琳,程文明,胡成穆,等.豹皮樟总黄酮抗炎作用及部分机制研究[J].安徽医科大学学报, 2004;39(6):439-42
[41]李叶,唐浩国,刘建学.黄酮类化合物抑菌作用的研究进展[J].农产品加工·学刊,2008;(12):53-6
[42]谢鹏,张敏红.黄酮类化合物抑菌作用的研究进展[J].动物保健,2004;(12):35
[43]韩淑琴,杨洋,黄涛,等.仙人掌提取物的抑菌机理[J].食品科技,2007;(3):38-9
[44]Katarzyna Ulanowska, Aleksandra Tkaczyk, Grazyna Konopa, et al.Differ- ential antibacterial activity of genistein arising from global inhibition of DNA,RNA and protein synthesis in some bacterial strains[J].Archives of Microbiology,2006;184(5):271-8
[45]王超云.黄芩苷对化学性肝损伤的保护作用[J].中草药,2005;36(5):730- 2
[46]徐庆,宋芸娟,李丽亚,等.荔枝核总黄酮的抗鸭乙型肝炎病毒的作用[J].世界华人消化杂志,2005;13(17):2082-5
[47]蒋伟哲,孔晓龙,黄仁彬,等.龙眼参对小鼠化学性肝损伤的保护作用及其机制研究[J].中国药房,2004;15(7):398-401
[48]朱会平.黄酮类化合物促进癌细胞凋亡的分子机理研究进展[J].生物技术通讯,2006;17(3):455-6
[49]高昕,王喜军.黄酮类化合物抗肿瘤作用研究进展[J].中医药信息,2005;22(5):33-4
[50]农朝赞,黄华艺.黄酮类化合物抗肿瘤作用的研究应用[J].中国药房, 2004; 15(9): 568-9
[51]唐勇.鸡血藤黄酮类组分抗肿瘤活性研究[J].中国实验方剂学杂志,2007;13(2):51-4
[52]OFER M, WOLFFRAM S, KOGGEL A, et al. Modulation of drug transport by selected flavonoids: Involvement of P-gp and OCT[J]. EurJ Pharma Sci, 2005;25(3):263-71
[53]LI MTRAKULP , K HANTAMATO, PINTHA K. Inhibition of P- glycopro- tein function and expression by kaempferol and quercetin[J]. J Chemother, 2005;17(1):86-95
[54]林晓贞,梁钢,黎莉,等. EGCG逆转人耐药肝癌细胞株多药耐药的体内实验研究[J].广西医科大学学报,2006;(1):48-51
[55]兰真,曾凡骏,曾里,等.生物类黄酮对心血管系统的调节及作用机理[J].现代预防医学,2005;32(6):613-5
[56]韩兆轩,王维民.类黄酮在心血管疾病中的研究进展[J].农产品加工·学刊,2007;(12):31-4
[57]李乐,孙晓东,高晓利,等.绞股蓝总黄酮对麻醉犬心脏血流动力学及冠脉血流量的作用[J].浙江工业大学学报,2006;34(5):521-8
[58]杨青,郭彩清,油继辉,等.黄酮类物质的生理功能及应用发展动态[J].贵州农业科学,2007;35(2):266-8