枳实降血脂药效组分生物效应评价方法研究
|
摘要
1.研究目的
研究枳实(Fructus Aurantii Immaturus)降血脂药效组分及其生物效应,并与韩国枳实(Fructus Ponciri)比较;建立枳实降血脂药效组分的生物效应评价方法,探讨枳实药效组分生物质量标准,为建立中药药效组分质量标准评价新体系奠定基础。
2.研究方法
采用高效液相色谱法(HPLC)测定枳实降血脂的药效组分,并进行与降血脂相关的生物效应指标分析。
利用高脂饲料和Triton WR-1339诱导SD大鼠造高血脂模型,验证枳实降血脂生物效应。利用HPLC-药效组分生物活性测定结合法,确定枳实降血脂药效组分。选取和高血脂相关的体外生物效应指标研究枳实及其药效组分的降血脂效应,即采用氧化低密度脂蛋白(Ox-LDL)损伤人脐静脉内皮细胞株(HUVEC)模型,研究枳实及其药效组分对HUVEC细胞间黏附分子-1(ICAM-1)表达和细胞培养液中一氧化氮(NO)含量的影响;采用过氧化氢(H2O2)和叔丁基过氧化氢(t-BOOH)诱导的人肝癌细胞株(HepG2)氧化损伤模型,研究枳实及其药效组分对乳酸脱氢酶(LDH)漏出、丙二醛(MDA)含量、总抗氧化能力(T-AOC)、Cu-Zn-超氧化物歧化酶(Cu-Zn-SOD)活力、谷胱甘肽过氧化物酶(GSH-Px)活力、过氧化氢酶(CAT)活力影响;利用流式细胞技术,研究枳实及其药效组分对线粒体膜电位(MMP)的影响;利用单细胞凝胶电泳技术,研究枳实对H2O2和t-BOOH诱导的HepG2细胞脱氧核糖核酸(DNA)氧化损伤的保护作用。
3.研究结果
3.1枳实降血脂的药效组分
(1)枳实的药效组分
以药典规定的70%乙醇提取物为标准中药的研究样品,其药效组分为:柚皮苷-橙皮苷-枸橘苷-新橙皮苷-柚皮素-橙皮素(223.66:18.50:254.49:321.00:4.46:1);其中水层部分的药效组分:柚皮苷-橙皮苷-新橙皮苷-柚皮素(178.51:15.18:257.67:1);氯仿层部分的药效组分:橙皮苷-橙皮素-柚皮素(2.92:1:2.99)。
(2)韩国枳实的药效组分
70%乙醇提取物中的药效组分:柚皮苷-橙皮苷-枸橘苷-柚皮素-橙皮素(514.55:16.37:2454.23:20:1);其中水层的药效组分:柚皮苷-橙皮苷-枸橘苷(28.78:1:144.24);氯仿层的药效组分:柚皮素-橙皮素(19.27:1)。
3.2体内降血脂生物效应
3.2.1枳实
能降低高血脂模型SD大鼠血清中的总胆固醇(TC)、甘油三酯(TG)和低密度脂蛋白胆固醇(LDL-C)含量、天门冬氨酸转移酶(AST)活性并升高高密度脂蛋白胆固醇(HDL-C)含量。
对高血脂模型SD大鼠血清中丙氨酸氨基转移酶(ALT)活性和血糖无影响。
3.2.2韩国枳实
能降低高血脂模型SD大鼠血清中的TC、TG和LDL-C含量并升高HDL-C含量。
对高血脂模型SD大鼠血清中ALT活性,AST活性和血糖含量无影响。对于高脂饲料喂养造成的高血脂模型SD大鼠,韩国枳实对其降脂效应比枳实稍好。
3.3枳实及其药效组分体外降血脂生物效应
3.3.1枳实
(1)饮片
①抑制Ox-LDL损伤HUVEC ICAM-1表达;②恢复Ox-LDL诱导的HUVEC培养液中NO含量至正常水平;③提高H2O2损伤的HepG2细胞活性;④可抑制t-BOOH诱导的HepG2细胞LDH的漏出;提取物氯仿层可抑制H2O2诱导的HepG2细胞LDH的漏出;⑤降低氧化损伤的HepG2细胞中的MDA含量;⑥提高氧化损伤的HepG2细胞的T-AOC;⑦提高t-BOOH诱导的HepG2细胞Cu-Zn-SOD活力;提取物氯仿层可提高H2O2诱导的HepG2细胞Cu-Zn-SOD活力;⑧提高氧化损伤的HepG2细胞GSH-Px活力;⑨提高氧化损伤的HepG2细胞CAT活力;⑩降低氧化损伤的HepG2细胞MMP;○11对氧化损伤的HepG2细胞DNA有保护作用。
(2)药效组分
①抑制Ox-LDL损伤HUVEC ICAM-1表达;②降低氧化损伤的HepG2细胞中的MDA含量;③提高氧化损伤的HepG2细胞的T-AOC;④提高t-BOOH诱导的HepG2细胞Cu-Zn-SOD活力;⑤提高氧化损伤的HepG2细胞GSH-Px活力;⑥提高氧化损伤的HepG2细胞CAT活力;⑦降低氧化损伤的HepG2细胞MMP。
3.3.2韩国枳实
(1)饮片
对Ox-LDL损伤HUVEC ICAM-1表达,H2O2损伤的HepG2细胞活性,氧化损伤的HepG2细胞中的MDA含量和T-AOC以及GSH-Px活力和CAT活力,t-BOOH诱导的HepG2细胞Cu-Zn-SOD活力,氧化损伤的HepG2细胞MMP的生物活性均和中国枳实饮片的相同。
可以提高Ox-LDL诱导的HUVEC培养液中NO含量但也显著高于正常水平。提取物氯仿层可抑制H2O2和t-BOOH诱导的HepG2细胞LDH的漏出。
(2)药效组分
对Ox-LDL损伤HUVEC ICAM-1表达,氧化损伤的HepG2细胞中的MDA含量和T-AOC以及GSH-Px活力和CAT活力,t-BOOH诱导的HepG2细胞Cu-Zn-SOD活力,氧化损伤的HepG2细胞MMP影响均和中国枳实药效组分的相同。
3.4枳实降血脂质量评价方法建立
(1)生物效应指标:枳实及其药效组分对降血脂相关效应指标的生物效应比(BER)均在0.9~1.1之间并且除了韩国枳实70%乙醇提取物氯仿层药效组分对氧化损伤的HepG2细胞GSH-Px活性影响与韩国枳实70%乙醇提取物氯仿层相比有显著差异(t-test)外,枳实和其相应的药效组分对降血脂相关的效应指标t检验均无显著差异。药效组分的降血脂相关多个指标的标准生物效应作为效应标准,建立了枳实降血脂生物效应评价方法。
(2)药效组分指标:枳实的药效组分为柚皮苷-橙皮苷-枸橘苷-新橙皮苷-柚皮素-橙皮素(223.66:18.50:254.49:321.00:4.46:1)。韩国枳实的药效组分为柚皮苷-橙皮苷-枸橘苷-柚皮素-橙皮素(514.55:16.37:2454.23:20:1)。
4.结论
4.1枳实及其所确定的药效组分降血脂生物效应具有等效性,二者没有显著性差异(t检验)即该药效组分可以表述枳实降血脂临床疗效的品质特征。
4.2枳实生物效应评价方法
(1)降血脂相关的生物活性指标
①抗氧化指标:H2O2和t-BOOH氧化损伤的HepG2细胞的MDA含量,T-AOC,GSH-Px活力,CAT活力和MMP的变化;t-BOOH氧化损伤的HepG2细胞Cu-Zn-SOD活力。
②对HUVEC ICAM-1表达的影响。
(2)生物效应评价方法建立
①抗氧化指标法:枳实对H2O2和t-BOOH氧化损伤的HepG2细胞的MDA含量的BER分别为0.9879和1.0258;对H2O2和t-BOOH氧化损伤的HepG2细胞T-AOC的BER分别为1.00558和0.99777;对H2O2和t-BOOH氧化损伤的HepG2细胞GSH-Px活力的BER分别为1.026和1.044;对H2O2和t-BOOH氧化损伤的HepG2细胞CAT活力的BER分别为0.991和1.023;对H2O2和t-BOOH氧化损伤的HepG2细胞MMP降低的BER分别为0.921743和0.922297;对t-BOOH氧化损伤的HepG2细胞Cu-Zn-SOD活力的BER为1.01546。
②细胞间黏附分子表达法:枳实对Ox-LDL诱导的HUVEC的ICAM-1的表达抑制的BER为0.9781。
枳实及其药效组分的降血脂相关生物活性指标的BER在0.9~1.1之间并且枳实与其药效组分的生物效应无显著差异。故可确定枳实药效组分的标准生物效应作为效应标准。
4.3枳实降血脂药效组分
降血脂药效组分能够做为枳实的质量评价指标之一。
4.4枳实降血脂质量标准建议
4.4.1抗氧化指标法和细胞间黏附分子表达法
①用与药效组分完全相同的生物效应测定方法检测待测枳实生物效应。
②计算BER并做相关数理统计检验。
③生物效应评价方法标准建议:
1≥BERS≥0.9且经数理学比较与效应标准无差异,为合格药品; BERS>1且经数理学比较与效应标准无差异为优质药品; BERS<0.9或经数理学比较与效应标准有差异为不合格药品;
4.4.2药效组分分析
采用HPLC法。
待测样品枳实所得药效组分比例和含量应在以柚皮苷-橙皮苷-枸橘苷-新橙皮苷-柚皮素-橙皮素(223.66:18.50:254.49:321.00:4.46:1)为基准的一个范围内。
5.创新点
(1)首次研究了枳实和韩国枳实降血脂(血浊证)的生物效应。
(2)确定了枳实降血脂的药效组分。
(3)建立了枳实药效组分生物效应评价方法。
(4)创立了生物效应比的计算公式:BERsample=Xsample/XACA。
1. Objectives
The research is about the active components alignment (ACA) of Fructus Aurantii Immaturus reducing hyperlipoidemia, Fructus Aurantii Immaturus and its ACA’s biological effects of reducing hyperlipoidemia, meanwhile, compared with Fructus Ponciri; and also about establishing the method of biological-effect evaluation about reducing hyperlipoidemia ACA of Fructus Aurantii Immaturus and Fructus Ponciri, discussing the biologic quality standard of Fructus Aurantii Immaturus ACA for settling the foundation of new quality evaluation system on Chinese Medicine (CM) ACA.
2. Methods
The reducing hyperlipodemia ACA of Fructus Aurantii Immaturus and Fructus Ponciri were analyzed by analyzing the concentration according to HPLC and determination of biological effect about reducing hyperlipoidemia.
The reducing hyperlipoidemia effect was validated by using hyperlipoidemia model of SD rat induced by high-fat food and Triton WR-1339. By combining HPLC technique and determination of biological effects on Fructus Aurantii Immaturus ACA and Fructus Ponciri ACA the reducing hyperlipoidemia ACA of Fructus Aurantii Immaturus and Fructus Ponciri were found. The in vitro indexes about hyperlipoidemia were chosen to study the effect on reducing hyperlipoidemia of Fructus Aurantii Immaturus and Fructus Ponciri and their ACAs. That is choosing HUVEC model induced by Ox-LDL to study the effect on ICAM-1 expression and NO concentration in supernate of Fructus Aurantii Immaturus and Fructus Ponciri and their ACAs. The effects of Fructus Aurantii Immaturus and Fructus Ponciri and their ACAs on activity of LDH in supernate, concentration of MDA, T-AOC, activity of Cu-Zn-SOD, activity of GSH-Px, activity of CAT in HepG2 induced by H2O2 and t-BOOH were studied. The effects of Fructus Aurantii Immaturus and Fructus Ponciri and their ACAs about MMP of HepG2 induced by H2O2 and t-BOOH were researched by flow cytometry(FCM). The protective effect of Fructus Aurantii Immaturus and Fructus Ponciri and their ACAs to DNA of HepG2 induced by H2O2 and t-BOOH was studied by using SCGE.
3. Results
3.1 Reducing Hyperlipoidemia ACA of Fructus Aurantii Immaturus
(1) ACA of Fructus Aurantii Immaturus
Extraction of 70%EtOH which was recorded in pharmacopoeia was the researchful object and its ACA (CJSE ACA) is Naringin- Hesperidin- Poncirin- Neohesperidin- Naringenin- Hesperetin(223.66:18.50:254.49:321.00:4.46:1); And the ACA of its water fraction (CJSW ACA) is Naringin- Hesperidin- Neohesperidin- Naringenin(178.51:15.18:257.67:1); The ACA of its chloroform fraction (CJSC ACA) is Hesperidin- Hesperetin- Naringenin(2.92:1:2.99).
(2) ACA of Fructus Ponciri
ACA of Extraction of 70%EtOH (KJSE ACA) is Naringin-Hesperidin-Poncirin- Naringenin- Hesperetin(514.55:16.37:2454.23:20:1); And the ACA of its water fraction (KJSW ACA) is Naringin- Hesperidin- Poncirin(28.78:1:144.24); The ACA of its chloroform fraction (KJSC ACA) is Naringenin- Hesperetin(19.29:1)
3.2 Reducing Hyperlipoidemia Effect of Fructus Aurantii Immaturus in vivo
3.2.1 Fructus Aurantii Immaturus
The contents of TG, TC, LDL-C in serum of hyperlipoidemia model SD rat were decreased while the content of HDL-C in serum of hyperlipoidemia model SD rat was increased after being cured by Fructus Aurantii Immaturus, when the activity of AST can be reduced and the activity of ALT and the content of glucose can not be affected.
3.2.2 Fructus Ponciri
The contents of TG, TC, LDL-C in serum of hyperlipoidemia model SD rat were decreased while the content of HDL-C in serum of hyperlipoidemia model SD rat was increased after being cured by Fructus Ponciri, when activity of ALT, AST and the content of glucose in serum of hyperlipoidemia model SD rat can not be affected.
The reducing hyperlipoidemia effect of Fructus Ponciri is a little better than that of Fructus Aurantii Immaturus in the hyperlipoidemia model SD rat induced by high-fat diet.
3.3 Biological Effect about Reducing Hyperlipoidemia in vitro of Fructus Aurantii Immaturus and Its ACA
3.3.1 Fructus Aurantii Immaturus
(1) Decoction Pieces
①ICAM-1 expression of HUVEC induced by Ox-LDL was inhibited.②The content of NO in supernate medium of HUVEC induced by Ox-LDL can be regulated to normal level;③The viability of HepG2 induced by H2O2 can be elevated.④Leakage of LDH in HepG2 induced by t-BOOH can be inhibited and leakage of LDH in HepG2 induced by H2O2 can be inhibited by CJSC.⑤The content of MDA in HepG2 induced by H2O2 and t-BOOH can be decreased.⑥The T-AOC of HepG2 induced by H2O2 and t-BOOH can be elevated.⑦Activity of Cu-Zn-SOD in HepG2 induced by H2O2 can be increased by CJSC. Activity of Cu-Zn-SOD in HepG2 induced by H2O2 and t-BOOH can be increased.⑧Activity of GSH-Px in HepG2 induced by H2O2 and t-BOOH can be increased.⑨Activity of CAT in HepG2 induced by H2O2 and t-BOOH can be increased.⑩MMP of HepG2 induced by H2O2 and t-BOOH can be reduced.○11 The oxidative damage on DNA of HepG2 which was induced by H2O2 and t-BOOH can be protected
(2)ACA
①ICAM-1 expression of HUVEC induced by Ox-LDL was inhibited.②The content of MDA in HepG2 induced by H2O2 and t-BOOH can be decreased.③The T-AOC of HepG2 induced by H2O2 and t-BOOH can be elevated.④Activity of Cu-Zn-SOD in HepG2 induced by t-BOOH can be increased.⑤Activity of GSH-Px in HepG2 induced by H2O2 and t-BOOH can be increased.⑥Activity of CAT in HepG2 induced by H2O2 and t-BOOH can be increased.⑦MMP of HepG2 induced by H2O2 and t-BOOH can be reduced.
3.3.2 Fructus Ponciri
(1) Decoction Pieces
The biological effects about ICAM-1 expression of HUVEC induced by Ox-LDL, the viability of HepG2 induced by H2O2, the content of MDA, T-AOC, activity of GSH-Px and CAT in HepG2 induced by H2O2 and t-BOOH, activity of Cu-Zn-SOD in HepG2 induced by t-BOOH, MMP of HepG2 induced by H2O2 and t-BOOH are the same with those of Fructus Aurantii Immaturus decoction pieces.
The content of NO in supernate medium of HUVEC induced by Ox-LDL can be increased to the level which was higher than normal level. Leakage of LDH in HepG2 induced by H2O2 and t-BOOH can be inhibited by KJSC.
3.4 Establishment of Biological-Effect Evaluation Method about Reducing Hyperlipoidemia of Fructus Aurantii Immaturus and Fructus Ponciri
(1) Index of biological effects: BERs of Fructus Aurantii Immaturus and its ACA, Fructus Ponciri and its ACA about the biological effect of reducing hyperlipoidemia are between 0.9 and 1.1. And only the difference between the biological effect of activity of GSH-Px from KJSC ACA and that from KJSC is significant (t-test). The differences between the biological effect from Fructus Aurantii Immaturus and its ACA, Fructus Ponciri and its ACA are not significant (t-test). The standard biological effects which are about reducing hyperlipoidemia of ACA are as a standard of effect and then the biological-effect evaluation method was established.
(2) Index of ACA: Fructus Aurantii Immaturus: Naringin- Hesperidin- Poncirin- Neohesperidin- Naringenin- Hesperetin(223.66:18.50:254.49:321.00:4.46 : 1 ) ;Fructus Ponciri: Naringin-Hesperidin-Poncirin- Naringenin- Hesperetin(514.55:16.37:2454.23:20:1).
4. Conclusions
4.1 The effects of reducing hyperlipoidemia of Fructus Aurantii Immaturus and its ACA, Fructus Ponciri and its ACA are equivalent. The difference between the two is not significant according to t-test. It shows Fructus Aurantii Immaturus ACA can represent the quality characteristics about reducing hyperlipoidemia of Fructus Aurantii Immaturus.
4.2 Biological-Effect Evaluation Method of Fructus Aurantii Immaturus
(1) Biological effect indexes about reducing hyperlipoidemia
①Antioxidant Index: The contents of MDA, T-AOC, activity of GSH-Px and CAT, the change of MMP in HepG2 induced by H2O2 and t-BOOH; The activity of Cu-Zn-SOD in HepG2 induced by t-BOOH;
②Expression of HUVEC ICAM-1
(2) Establishment of Biological-Effect Evaluation Method
①Method of Antioxidant index: BERs about content of MDA in HepG2 induced by H2O2 and t-BOOH of Fructus Aurantii Immaturus are 0.9879 and 1.0258 respectively. BERs of T-AOC in HepG2 induced by H2O2 and t-BOOH of Fructus Aurantii Immaturus are 1.00558 and 0.99777 respectively; BERs of GSH-Px in HepG2 induced by H2O2 and t-BOOH of Fructus Aurantii Immaturus are 1.026 and 1.044; BERs of CAT in HepG2 induced by H2O2 and t-BOOH of Fructus Aurantii Immaturus are 0.991 and 1.023; BERs of reducing MMP of HepG2 induced by H2O2 and t-BOOH. BER of Cu-Zn-SOD activity is 1.01546.
②Method of ICAM-1 Expression: BER of inhibition to ICAM-1 expression induced by Ox-LDL is 0.9781.
The BERs of Fructus Aurantii Immaturus and its ACA are all between 0.9 and 1.1. At the same time the difference between the biological effect from Fructus Aurantii Immaturus and its ACA is not significant according to mathematical statistics calculation. So the standard biological effects of ACAs of Fructus Aurantii Immaturus can be as standards of effect.
4.3 Reducing hyperlipoidemia ACA of Fructus Aurantii Immaturus The reducing hyperlipoidemia ACA of Fructus Aurantii Immaturus could be one of indexes for evaluating the qulity of Fructus Aurantii Immaturus.
4.4 Suggestions of Quality Standards to the Effects about Reducing Hyperlipoidemia of Fructus Aurantii Immaturus
4.4.1 Method of Antioxidant Index and Expression of ICAM-1
①The biological effects of the sample should be checked by using the same method which was used to checked the biological effect of ACA.
②BER should be calculated and Statistical Test should be done.
③Suggestions about Standard of Biological-Effect Evaluation Method Eligible Sample: 1≥BERS≥0.9 and the difference is not significant between the biological effect and standards of effect.
High Quality Sample: BERS>1 and the difference is not significant between the biological effect and standards of effect.
Unqualified Sample: BERS<0.9 or the difference is significant between the biological effect and standards of effect.
4.4.2 Analysis of ACA
HPLC is used in the analysis of ACA.
Ratio and concentration of ACA of samples should be in the range which benchmark is Naringin- Hesperidin- Poncirin- Neohesperidin- Naringenin- Hesperetin(223.66:18.50:254.49:321.00:4.46:1)
5. Innovations
(1) Reducing hyperlipoidemia effect (Xue Zhuo Zheng)of Fructus Aurantii Immaturus and Fructus Ponciri were validated by modern medical experiment first.
(2) ACAs of Fructus Aurantii Immaturus and Fructus Ponciri were determined.
(3) Biological-effect evaluation method about reducing hyperlipoidemia of Fructus Aurantii Immaturus and Fructus Ponciri was established.
(4) Formula of calculating BER was established: BERsample=Xsample/XACA
研究枳实(Fructus Aurantii Immaturus)降血脂药效组分及其生物效应,并与韩国枳实(Fructus Ponciri)比较;建立枳实降血脂药效组分的生物效应评价方法,探讨枳实药效组分生物质量标准,为建立中药药效组分质量标准评价新体系奠定基础。
2.研究方法
采用高效液相色谱法(HPLC)测定枳实降血脂的药效组分,并进行与降血脂相关的生物效应指标分析。
利用高脂饲料和Triton WR-1339诱导SD大鼠造高血脂模型,验证枳实降血脂生物效应。利用HPLC-药效组分生物活性测定结合法,确定枳实降血脂药效组分。选取和高血脂相关的体外生物效应指标研究枳实及其药效组分的降血脂效应,即采用氧化低密度脂蛋白(Ox-LDL)损伤人脐静脉内皮细胞株(HUVEC)模型,研究枳实及其药效组分对HUVEC细胞间黏附分子-1(ICAM-1)表达和细胞培养液中一氧化氮(NO)含量的影响;采用过氧化氢(H2O2)和叔丁基过氧化氢(t-BOOH)诱导的人肝癌细胞株(HepG2)氧化损伤模型,研究枳实及其药效组分对乳酸脱氢酶(LDH)漏出、丙二醛(MDA)含量、总抗氧化能力(T-AOC)、Cu-Zn-超氧化物歧化酶(Cu-Zn-SOD)活力、谷胱甘肽过氧化物酶(GSH-Px)活力、过氧化氢酶(CAT)活力影响;利用流式细胞技术,研究枳实及其药效组分对线粒体膜电位(MMP)的影响;利用单细胞凝胶电泳技术,研究枳实对H2O2和t-BOOH诱导的HepG2细胞脱氧核糖核酸(DNA)氧化损伤的保护作用。
3.研究结果
3.1枳实降血脂的药效组分
(1)枳实的药效组分
以药典规定的70%乙醇提取物为标准中药的研究样品,其药效组分为:柚皮苷-橙皮苷-枸橘苷-新橙皮苷-柚皮素-橙皮素(223.66:18.50:254.49:321.00:4.46:1);其中水层部分的药效组分:柚皮苷-橙皮苷-新橙皮苷-柚皮素(178.51:15.18:257.67:1);氯仿层部分的药效组分:橙皮苷-橙皮素-柚皮素(2.92:1:2.99)。
(2)韩国枳实的药效组分
70%乙醇提取物中的药效组分:柚皮苷-橙皮苷-枸橘苷-柚皮素-橙皮素(514.55:16.37:2454.23:20:1);其中水层的药效组分:柚皮苷-橙皮苷-枸橘苷(28.78:1:144.24);氯仿层的药效组分:柚皮素-橙皮素(19.27:1)。
3.2体内降血脂生物效应
3.2.1枳实
能降低高血脂模型SD大鼠血清中的总胆固醇(TC)、甘油三酯(TG)和低密度脂蛋白胆固醇(LDL-C)含量、天门冬氨酸转移酶(AST)活性并升高高密度脂蛋白胆固醇(HDL-C)含量。
对高血脂模型SD大鼠血清中丙氨酸氨基转移酶(ALT)活性和血糖无影响。
3.2.2韩国枳实
能降低高血脂模型SD大鼠血清中的TC、TG和LDL-C含量并升高HDL-C含量。
对高血脂模型SD大鼠血清中ALT活性,AST活性和血糖含量无影响。对于高脂饲料喂养造成的高血脂模型SD大鼠,韩国枳实对其降脂效应比枳实稍好。
3.3枳实及其药效组分体外降血脂生物效应
3.3.1枳实
(1)饮片
①抑制Ox-LDL损伤HUVEC ICAM-1表达;②恢复Ox-LDL诱导的HUVEC培养液中NO含量至正常水平;③提高H2O2损伤的HepG2细胞活性;④可抑制t-BOOH诱导的HepG2细胞LDH的漏出;提取物氯仿层可抑制H2O2诱导的HepG2细胞LDH的漏出;⑤降低氧化损伤的HepG2细胞中的MDA含量;⑥提高氧化损伤的HepG2细胞的T-AOC;⑦提高t-BOOH诱导的HepG2细胞Cu-Zn-SOD活力;提取物氯仿层可提高H2O2诱导的HepG2细胞Cu-Zn-SOD活力;⑧提高氧化损伤的HepG2细胞GSH-Px活力;⑨提高氧化损伤的HepG2细胞CAT活力;⑩降低氧化损伤的HepG2细胞MMP;○11对氧化损伤的HepG2细胞DNA有保护作用。
(2)药效组分
①抑制Ox-LDL损伤HUVEC ICAM-1表达;②降低氧化损伤的HepG2细胞中的MDA含量;③提高氧化损伤的HepG2细胞的T-AOC;④提高t-BOOH诱导的HepG2细胞Cu-Zn-SOD活力;⑤提高氧化损伤的HepG2细胞GSH-Px活力;⑥提高氧化损伤的HepG2细胞CAT活力;⑦降低氧化损伤的HepG2细胞MMP。
3.3.2韩国枳实
(1)饮片
对Ox-LDL损伤HUVEC ICAM-1表达,H2O2损伤的HepG2细胞活性,氧化损伤的HepG2细胞中的MDA含量和T-AOC以及GSH-Px活力和CAT活力,t-BOOH诱导的HepG2细胞Cu-Zn-SOD活力,氧化损伤的HepG2细胞MMP的生物活性均和中国枳实饮片的相同。
可以提高Ox-LDL诱导的HUVEC培养液中NO含量但也显著高于正常水平。提取物氯仿层可抑制H2O2和t-BOOH诱导的HepG2细胞LDH的漏出。
(2)药效组分
对Ox-LDL损伤HUVEC ICAM-1表达,氧化损伤的HepG2细胞中的MDA含量和T-AOC以及GSH-Px活力和CAT活力,t-BOOH诱导的HepG2细胞Cu-Zn-SOD活力,氧化损伤的HepG2细胞MMP影响均和中国枳实药效组分的相同。
3.4枳实降血脂质量评价方法建立
(1)生物效应指标:枳实及其药效组分对降血脂相关效应指标的生物效应比(BER)均在0.9~1.1之间并且除了韩国枳实70%乙醇提取物氯仿层药效组分对氧化损伤的HepG2细胞GSH-Px活性影响与韩国枳实70%乙醇提取物氯仿层相比有显著差异(t-test)外,枳实和其相应的药效组分对降血脂相关的效应指标t检验均无显著差异。药效组分的降血脂相关多个指标的标准生物效应作为效应标准,建立了枳实降血脂生物效应评价方法。
(2)药效组分指标:枳实的药效组分为柚皮苷-橙皮苷-枸橘苷-新橙皮苷-柚皮素-橙皮素(223.66:18.50:254.49:321.00:4.46:1)。韩国枳实的药效组分为柚皮苷-橙皮苷-枸橘苷-柚皮素-橙皮素(514.55:16.37:2454.23:20:1)。
4.结论
4.1枳实及其所确定的药效组分降血脂生物效应具有等效性,二者没有显著性差异(t检验)即该药效组分可以表述枳实降血脂临床疗效的品质特征。
4.2枳实生物效应评价方法
(1)降血脂相关的生物活性指标
①抗氧化指标:H2O2和t-BOOH氧化损伤的HepG2细胞的MDA含量,T-AOC,GSH-Px活力,CAT活力和MMP的变化;t-BOOH氧化损伤的HepG2细胞Cu-Zn-SOD活力。
②对HUVEC ICAM-1表达的影响。
(2)生物效应评价方法建立
①抗氧化指标法:枳实对H2O2和t-BOOH氧化损伤的HepG2细胞的MDA含量的BER分别为0.9879和1.0258;对H2O2和t-BOOH氧化损伤的HepG2细胞T-AOC的BER分别为1.00558和0.99777;对H2O2和t-BOOH氧化损伤的HepG2细胞GSH-Px活力的BER分别为1.026和1.044;对H2O2和t-BOOH氧化损伤的HepG2细胞CAT活力的BER分别为0.991和1.023;对H2O2和t-BOOH氧化损伤的HepG2细胞MMP降低的BER分别为0.921743和0.922297;对t-BOOH氧化损伤的HepG2细胞Cu-Zn-SOD活力的BER为1.01546。
②细胞间黏附分子表达法:枳实对Ox-LDL诱导的HUVEC的ICAM-1的表达抑制的BER为0.9781。
枳实及其药效组分的降血脂相关生物活性指标的BER在0.9~1.1之间并且枳实与其药效组分的生物效应无显著差异。故可确定枳实药效组分的标准生物效应作为效应标准。
4.3枳实降血脂药效组分
降血脂药效组分能够做为枳实的质量评价指标之一。
4.4枳实降血脂质量标准建议
4.4.1抗氧化指标法和细胞间黏附分子表达法
①用与药效组分完全相同的生物效应测定方法检测待测枳实生物效应。
②计算BER并做相关数理统计检验。
③生物效应评价方法标准建议:
1≥BERS≥0.9且经数理学比较与效应标准无差异,为合格药品; BERS>1且经数理学比较与效应标准无差异为优质药品; BERS<0.9或经数理学比较与效应标准有差异为不合格药品;
4.4.2药效组分分析
采用HPLC法。
待测样品枳实所得药效组分比例和含量应在以柚皮苷-橙皮苷-枸橘苷-新橙皮苷-柚皮素-橙皮素(223.66:18.50:254.49:321.00:4.46:1)为基准的一个范围内。
5.创新点
(1)首次研究了枳实和韩国枳实降血脂(血浊证)的生物效应。
(2)确定了枳实降血脂的药效组分。
(3)建立了枳实药效组分生物效应评价方法。
(4)创立了生物效应比的计算公式:BERsample=Xsample/XACA。
1. Objectives
The research is about the active components alignment (ACA) of Fructus Aurantii Immaturus reducing hyperlipoidemia, Fructus Aurantii Immaturus and its ACA’s biological effects of reducing hyperlipoidemia, meanwhile, compared with Fructus Ponciri; and also about establishing the method of biological-effect evaluation about reducing hyperlipoidemia ACA of Fructus Aurantii Immaturus and Fructus Ponciri, discussing the biologic quality standard of Fructus Aurantii Immaturus ACA for settling the foundation of new quality evaluation system on Chinese Medicine (CM) ACA.
2. Methods
The reducing hyperlipodemia ACA of Fructus Aurantii Immaturus and Fructus Ponciri were analyzed by analyzing the concentration according to HPLC and determination of biological effect about reducing hyperlipoidemia.
The reducing hyperlipoidemia effect was validated by using hyperlipoidemia model of SD rat induced by high-fat food and Triton WR-1339. By combining HPLC technique and determination of biological effects on Fructus Aurantii Immaturus ACA and Fructus Ponciri ACA the reducing hyperlipoidemia ACA of Fructus Aurantii Immaturus and Fructus Ponciri were found. The in vitro indexes about hyperlipoidemia were chosen to study the effect on reducing hyperlipoidemia of Fructus Aurantii Immaturus and Fructus Ponciri and their ACAs. That is choosing HUVEC model induced by Ox-LDL to study the effect on ICAM-1 expression and NO concentration in supernate of Fructus Aurantii Immaturus and Fructus Ponciri and their ACAs. The effects of Fructus Aurantii Immaturus and Fructus Ponciri and their ACAs on activity of LDH in supernate, concentration of MDA, T-AOC, activity of Cu-Zn-SOD, activity of GSH-Px, activity of CAT in HepG2 induced by H2O2 and t-BOOH were studied. The effects of Fructus Aurantii Immaturus and Fructus Ponciri and their ACAs about MMP of HepG2 induced by H2O2 and t-BOOH were researched by flow cytometry(FCM). The protective effect of Fructus Aurantii Immaturus and Fructus Ponciri and their ACAs to DNA of HepG2 induced by H2O2 and t-BOOH was studied by using SCGE.
3. Results
3.1 Reducing Hyperlipoidemia ACA of Fructus Aurantii Immaturus
(1) ACA of Fructus Aurantii Immaturus
Extraction of 70%EtOH which was recorded in pharmacopoeia was the researchful object and its ACA (CJSE ACA) is Naringin- Hesperidin- Poncirin- Neohesperidin- Naringenin- Hesperetin(223.66:18.50:254.49:321.00:4.46:1); And the ACA of its water fraction (CJSW ACA) is Naringin- Hesperidin- Neohesperidin- Naringenin(178.51:15.18:257.67:1); The ACA of its chloroform fraction (CJSC ACA) is Hesperidin- Hesperetin- Naringenin(2.92:1:2.99).
(2) ACA of Fructus Ponciri
ACA of Extraction of 70%EtOH (KJSE ACA) is Naringin-Hesperidin-Poncirin- Naringenin- Hesperetin(514.55:16.37:2454.23:20:1); And the ACA of its water fraction (KJSW ACA) is Naringin- Hesperidin- Poncirin(28.78:1:144.24); The ACA of its chloroform fraction (KJSC ACA) is Naringenin- Hesperetin(19.29:1)
3.2 Reducing Hyperlipoidemia Effect of Fructus Aurantii Immaturus in vivo
3.2.1 Fructus Aurantii Immaturus
The contents of TG, TC, LDL-C in serum of hyperlipoidemia model SD rat were decreased while the content of HDL-C in serum of hyperlipoidemia model SD rat was increased after being cured by Fructus Aurantii Immaturus, when the activity of AST can be reduced and the activity of ALT and the content of glucose can not be affected.
3.2.2 Fructus Ponciri
The contents of TG, TC, LDL-C in serum of hyperlipoidemia model SD rat were decreased while the content of HDL-C in serum of hyperlipoidemia model SD rat was increased after being cured by Fructus Ponciri, when activity of ALT, AST and the content of glucose in serum of hyperlipoidemia model SD rat can not be affected.
The reducing hyperlipoidemia effect of Fructus Ponciri is a little better than that of Fructus Aurantii Immaturus in the hyperlipoidemia model SD rat induced by high-fat diet.
3.3 Biological Effect about Reducing Hyperlipoidemia in vitro of Fructus Aurantii Immaturus and Its ACA
3.3.1 Fructus Aurantii Immaturus
(1) Decoction Pieces
①ICAM-1 expression of HUVEC induced by Ox-LDL was inhibited.②The content of NO in supernate medium of HUVEC induced by Ox-LDL can be regulated to normal level;③The viability of HepG2 induced by H2O2 can be elevated.④Leakage of LDH in HepG2 induced by t-BOOH can be inhibited and leakage of LDH in HepG2 induced by H2O2 can be inhibited by CJSC.⑤The content of MDA in HepG2 induced by H2O2 and t-BOOH can be decreased.⑥The T-AOC of HepG2 induced by H2O2 and t-BOOH can be elevated.⑦Activity of Cu-Zn-SOD in HepG2 induced by H2O2 can be increased by CJSC. Activity of Cu-Zn-SOD in HepG2 induced by H2O2 and t-BOOH can be increased.⑧Activity of GSH-Px in HepG2 induced by H2O2 and t-BOOH can be increased.⑨Activity of CAT in HepG2 induced by H2O2 and t-BOOH can be increased.⑩MMP of HepG2 induced by H2O2 and t-BOOH can be reduced.○11 The oxidative damage on DNA of HepG2 which was induced by H2O2 and t-BOOH can be protected
(2)ACA
①ICAM-1 expression of HUVEC induced by Ox-LDL was inhibited.②The content of MDA in HepG2 induced by H2O2 and t-BOOH can be decreased.③The T-AOC of HepG2 induced by H2O2 and t-BOOH can be elevated.④Activity of Cu-Zn-SOD in HepG2 induced by t-BOOH can be increased.⑤Activity of GSH-Px in HepG2 induced by H2O2 and t-BOOH can be increased.⑥Activity of CAT in HepG2 induced by H2O2 and t-BOOH can be increased.⑦MMP of HepG2 induced by H2O2 and t-BOOH can be reduced.
3.3.2 Fructus Ponciri
(1) Decoction Pieces
The biological effects about ICAM-1 expression of HUVEC induced by Ox-LDL, the viability of HepG2 induced by H2O2, the content of MDA, T-AOC, activity of GSH-Px and CAT in HepG2 induced by H2O2 and t-BOOH, activity of Cu-Zn-SOD in HepG2 induced by t-BOOH, MMP of HepG2 induced by H2O2 and t-BOOH are the same with those of Fructus Aurantii Immaturus decoction pieces.
The content of NO in supernate medium of HUVEC induced by Ox-LDL can be increased to the level which was higher than normal level. Leakage of LDH in HepG2 induced by H2O2 and t-BOOH can be inhibited by KJSC.
3.4 Establishment of Biological-Effect Evaluation Method about Reducing Hyperlipoidemia of Fructus Aurantii Immaturus and Fructus Ponciri
(1) Index of biological effects: BERs of Fructus Aurantii Immaturus and its ACA, Fructus Ponciri and its ACA about the biological effect of reducing hyperlipoidemia are between 0.9 and 1.1. And only the difference between the biological effect of activity of GSH-Px from KJSC ACA and that from KJSC is significant (t-test). The differences between the biological effect from Fructus Aurantii Immaturus and its ACA, Fructus Ponciri and its ACA are not significant (t-test). The standard biological effects which are about reducing hyperlipoidemia of ACA are as a standard of effect and then the biological-effect evaluation method was established.
(2) Index of ACA: Fructus Aurantii Immaturus: Naringin- Hesperidin- Poncirin- Neohesperidin- Naringenin- Hesperetin(223.66:18.50:254.49:321.00:4.46 : 1 ) ;Fructus Ponciri: Naringin-Hesperidin-Poncirin- Naringenin- Hesperetin(514.55:16.37:2454.23:20:1).
4. Conclusions
4.1 The effects of reducing hyperlipoidemia of Fructus Aurantii Immaturus and its ACA, Fructus Ponciri and its ACA are equivalent. The difference between the two is not significant according to t-test. It shows Fructus Aurantii Immaturus ACA can represent the quality characteristics about reducing hyperlipoidemia of Fructus Aurantii Immaturus.
4.2 Biological-Effect Evaluation Method of Fructus Aurantii Immaturus
(1) Biological effect indexes about reducing hyperlipoidemia
①Antioxidant Index: The contents of MDA, T-AOC, activity of GSH-Px and CAT, the change of MMP in HepG2 induced by H2O2 and t-BOOH; The activity of Cu-Zn-SOD in HepG2 induced by t-BOOH;
②Expression of HUVEC ICAM-1
(2) Establishment of Biological-Effect Evaluation Method
①Method of Antioxidant index: BERs about content of MDA in HepG2 induced by H2O2 and t-BOOH of Fructus Aurantii Immaturus are 0.9879 and 1.0258 respectively. BERs of T-AOC in HepG2 induced by H2O2 and t-BOOH of Fructus Aurantii Immaturus are 1.00558 and 0.99777 respectively; BERs of GSH-Px in HepG2 induced by H2O2 and t-BOOH of Fructus Aurantii Immaturus are 1.026 and 1.044; BERs of CAT in HepG2 induced by H2O2 and t-BOOH of Fructus Aurantii Immaturus are 0.991 and 1.023; BERs of reducing MMP of HepG2 induced by H2O2 and t-BOOH. BER of Cu-Zn-SOD activity is 1.01546.
②Method of ICAM-1 Expression: BER of inhibition to ICAM-1 expression induced by Ox-LDL is 0.9781.
The BERs of Fructus Aurantii Immaturus and its ACA are all between 0.9 and 1.1. At the same time the difference between the biological effect from Fructus Aurantii Immaturus and its ACA is not significant according to mathematical statistics calculation. So the standard biological effects of ACAs of Fructus Aurantii Immaturus can be as standards of effect.
4.3 Reducing hyperlipoidemia ACA of Fructus Aurantii Immaturus The reducing hyperlipoidemia ACA of Fructus Aurantii Immaturus could be one of indexes for evaluating the qulity of Fructus Aurantii Immaturus.
4.4 Suggestions of Quality Standards to the Effects about Reducing Hyperlipoidemia of Fructus Aurantii Immaturus
4.4.1 Method of Antioxidant Index and Expression of ICAM-1
①The biological effects of the sample should be checked by using the same method which was used to checked the biological effect of ACA.
②BER should be calculated and Statistical Test should be done.
③Suggestions about Standard of Biological-Effect Evaluation Method Eligible Sample: 1≥BERS≥0.9 and the difference is not significant between the biological effect and standards of effect.
High Quality Sample: BERS>1 and the difference is not significant between the biological effect and standards of effect.
Unqualified Sample: BERS<0.9 or the difference is significant between the biological effect and standards of effect.
4.4.2 Analysis of ACA
HPLC is used in the analysis of ACA.
Ratio and concentration of ACA of samples should be in the range which benchmark is Naringin- Hesperidin- Poncirin- Neohesperidin- Naringenin- Hesperetin(223.66:18.50:254.49:321.00:4.46:1)
5. Innovations
(1) Reducing hyperlipoidemia effect (Xue Zhuo Zheng)of Fructus Aurantii Immaturus and Fructus Ponciri were validated by modern medical experiment first.
(2) ACAs of Fructus Aurantii Immaturus and Fructus Ponciri were determined.
(3) Biological-effect evaluation method about reducing hyperlipoidemia of Fructus Aurantii Immaturus and Fructus Ponciri was established.
(4) Formula of calculating BER was established: BERsample=Xsample/XACA
引文
[1] 国家药典委员会.中华人民共和国药典,2005 版一部:172
[3] 中国科学院植物研究所[M].中国经济植物志(下册). 北京: 科学技术出版社, 1961:1364
[4] 高士彦,郑宏钧.柑橘属果皮及幼果中挥发油的气相色谱分析[J].药物分析 杂志,1986,6(2):833
[5] Kekelidae NA. Citrus wilsonii leaves essential oils[J]. Khim Prir Soedin,1982(6):785
[6] Matsubara. Studies on physio logical active substances in citrus fruit peel[J]. Yukagaku, 1988, 37(1):13
[7] 中国科学院植物研究所.中国经济植物志(下册) [M].北京:科学技术出版社,1961:1366
[8] 李仪奎,姜名英.中药药理学[M].北京:中国中医药出版社,1992:169 - 179
[9] 李岩,陈苏宁,李宇权,等.芍药甘草汤、四逆散对胃排空及小肠推进功能影响的拆方研[J].中华消化杂志,1996,16(2):20
[10] 陈琦,杨雪梅,徐成贺.厚朴三物汤拆方及其药量变化的实验研究[J].时珍国医国药,2001,12(9):776~778
[11] 李新旺,任钧国.枳实白术配伍的实验研究[J].中医研究,2002,15(6):23~24
[12] 况玲.枳实对绵羊小肠电活动的影响[J].中医药研究,1997,13(3):49~50
[13] 王翠芬,杨德治,魏义全,等.枳实对大鼠胃肠电活动影响的初步研究[J].东南大学学报,2001,20(3):153~154
[14] 黄子荷,杨德治,魏义全,等.阿托品对枳实增强犬小肠电活动效应的影响[J].中国中西医结合杂志,1996,16(5):293~294
[15] 郑天珍,李伟,瞿颂义,等.枳实、白术对大鼠离体胃各部位平滑肌条的作用[J].甘肃科学学报,1998,10(3):65~67
[16] 杨颖丽,郑天珍,瞿颂义,等.枳实对大鼠离体小肠平滑肌条的作用[J].西北师范大学学报(自然科学版),1998,34(2):70~71
[17] 谢冬萍,李伟,郑天珍,等.枳实对大鼠结肠肌条运动的影响[J].山东医科大学学报,2001,39(5):438
[18] 毕庆和,杨德治,应崇智,等.枳实对小肠电活动的影响[J].中国医药学报,1991,6(1):39~40
[19] 胡 盛 珊 , 王 大 元 , 邱 萍 , 等 . 枳 实 有 效 成 分 的 药 理 活 性 比 较 [J]. 中 草药,1994,25(8):419~420
[20] 胡盛珊,王大元,邱萍,等.枳实、枳壳挥发油对动物胃肠道的作用[J].东南大学学报,2001,20(3):144~145
[21] 刘波,李雪驼,徐和利,等.5 种中药制剂杀灭幽门螺杆菌的实验研究[J].中国新药杂志,2002,11(6):457
[22] 李红芳,李丹明,瞿颂义,等.枳实和陈皮对兔离体主动脉平滑肌条作用机理探讨[J].中成药,2001,23(9):659~660
[23] 倪艺,陈梦麟.枳实薤白桂枝汤治疗冠心病 58 例[J].国医论坛,2000,15(1):8
[24] 欧兴长,丁家欣,张玲.枳实等 11 种中药体外抗血栓作用研究[J].中西医结合杂志,1989,9(6):358~359
[25] 吉中强,宋鲁卿,高晓昕,等.11 种中药对大鼠血小板聚集和红细胞流变性的影响[J].山东中医杂志,2000,(2)19:107
[26] 陈庆平,王诗雅.名医印会河教授临床抓主经症经验集粹(十二)[J].中国乡村医药,2001,8(8):28
[27] 杨颖丽.枳实、青皮对平滑肌运动的影响[J].西北师范大学学报(自然科学版),2001,38(2):115~116
[28] 汤容.枳实和苦参对离体家兔阴道平滑肌收缩作用的研究[J].时珍国医国药,2001,12(11):973
[29] 王长虹,张冰,张宇,等.复方枳实合剂对实验动物的抗疲劳作用研究[J].牡丹江医学院学报,2001,22(1):15~17
[30] 周 海 均 , 等 . 生 物 检 定 统 计 方 法 ( 第 二 版 )[M]. 北 京 : 人 民 卫 生 出 版社,1988:1~4
[31] 张贵君.常用中药生物鉴定[M].北京:化学工业出版社 2006,1:1,7~8
[32] 封士兰,陈立仁 .中药质量标准研究新进展 [J].兰州医学院学报,2002,28(4):85~86
[33] 李 亚 萍 . 浅 议 我 国 中 药 质 量 标 准 的 进 展 [J]. 医 学 理 论 与 实 践 ,2006,19(2):227~228
[34] 王 跃 生 . 中 药 质 量 标 准 现 代 化 发 展 现 状 与 思 考 [J]. 中 国 中 药 杂志,2003,28(12):1108~1109
[35] 雷 茂 华 . 谈 中 药 制 剂 质 量 标 准 制 定 中 的 几 点 误 区 [J]. 江 苏 中医,1998,19(2):48
[36] 罗国安,王义明,曹进,杨学东.建立我国现代中药质量标准体系的研究[J].世界科学技术,2002,4(4):6
[37] 赖小平.高效毛细管电泳及其在中药质量分析中的应用[J].中药新药与临床药理, 1998,9(3):186
[38] 王 宝 琴 . 中 药 质 量 检 验 方 法 应 用 研 究 现 状 [J]. 中 药 新 药 与 临 床 药理,1997,8(2):110
[39] 王 宝 琴 . 中 药 质 量 标 准 研 究 含 量 测 定 的 选 定 原 则 刍 议 [J]. 中 成药,1995,17(3):50
[40] EMEA. Final proposals for Revision of the Note for Guidance on Quality ofHerbal Remedies.1998
[41] 李晓燕 .中成药质量及质量标准有关问题的探讨 [J]. 广东药学院学报,1996,12(2):106
[42] 杨曾让.中成药质量标准隐患初探[J].中成药,1995,17(4):48
[43] 夏柳影.中成药质量标准的演变和展望[J].中成药,1994,16(9):43
[44] 郑志华.加强中药质量标准研究是实现中药现代化的必要前提[J].中国药业,2001,10(2):4~5
[45] 董丽丽,李野.加速完善我国中药饮片质量标准[J].中国药房,2004, 15 (7):444
[46] 朱红宏.关于《中国药典》2005 年版新增中药饮片质量标准之浅见[J].中国药品标准, 2005,6(1):38
[47] 陈皎.对中药饮片质量工作的探讨[J].中国中药杂志,1995,20(4):214
[48] 陈吉炎,陈黎,安志斌,等.中药饮片质控乏力的原因与对策[J].中药材, 2003,26(1):43
[49] 张志国,殷剑.中药饮片质量的现状与思考[J].中药研究与信息,2003, 5(8):13
[50] 贾维臣,等.中药炮制品质量的影响因素剖析[J].时珍国医国药2003,14 (8):489
[51] 董洪根.浅论影响中药饮片质量与疗效的因素[J].江苏中医药2003,24 (1) :41
[52] 刘以霞.影响中药饮片质量的问题及其解决方法[J].江苏药学与临床研究, 2002,10(4):39
[53] 李敏,等.提高中药饮片质量应把好六关[J].湖南中医药导报,1996,2(5):39
[54] 雷永军,夏明衍,孟黎明,朱建.我国现行中药材质量标准中的若干问题分析[J].中国医院药学杂志,2001,21(1):46
[55] 谢培山.中医药文化与现代质量控制[J].中国中西医结合杂志,1998,18(11):654
[56] 祝国光.中药如何进入欧共体市场[M].北京,中国中医药科技,2000:54
[57] 张焱.浅谈规范中药质量标准的迫切性[J].山西中医,1996,5:34
[58] 贺兰英.再谈中药现代化—中药质量标准是中药现代化的前提[J].中医药学刊,2004,22(1): 148,171
[59] Cheung MC, Albers JJ. Characterization of lipoprotein particles isolated by immunoaffinity chromatography: particles containing AI and A Ⅱ and particles containing AI but no AⅡ[J]. J Biol Chem, 1984, 259:12201-12209
[60] Aarai K, Maguchi S. Glycation and inactivation of human Cu-Zn-Superoxide dismutase: Identification of the in vitro glycated sites [J]. J Biol Chem, 1989, 262:16969-16972
[61] Vlassara H, Brownle M, Manogue K, et al. Cachectin TNF and IL-1inducedby glucose-modified protein: role in normal tissue remodeling [J]. Science, 1988, 240:156
[62] Kirstein M, Aston C, Hints R et al. Receptor-specific induction of insulin-slide growth factor I in human monocytes by advanced glycosylation end product modified protein [J]. J Clin Invest , 1988, 90:439-446
[63] Fievetc, Theret N, Shojaee N, et al. Apolipoprotein Ai containing particles and reverse cholesterol transport in IDDM [J]. Diabetes, 1992, 41 (sappl.2) :81-85
[64] Takata K, Horiuchi S, Araki N, et al. Endocytic uptake of nonenzymatically glycosylated proteins is mediated by scavenger receptor for aldehyde-modified proteins [J]. J Biol Chem, 1988,263:14819-14825
[65] Kannel WB, Hypertension and other risk factors in coronary heart disease [J]. Am Heart J, 1987,114:918-925
[66] Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990 [J]. Nature, 1993, 362:81-808
[67] Kihara S, Matsuzawa Y, Kubo M, et al. Autoimmune hyperchylomicroemia [J]. New Engl J Med 1989,320:1255-1259
[68] Soria LF, Ludwig EH, Clark HRG, et.al. Association between a specific apolipoprotein B mutation and familial defective apolipoprotein B [J]. Pro Nat Acad Sci USA, 1989, 86:587-591
[69] Shimomura I, Tokunaga K, Jiaos, et al. Marded enhancement of acyl-CoA synthetase activity and mRNA paralleled to lipoprotein lipase mRNA, in adipose tissue of zucker obese rats [J]. Biochim Biophys Acta, 1992, 1124:112-118
[70] Fujioka S, Matsuzawa Y,T okunaga K, et al. Contribution of intra-abdominal fat accumulation to the impairment of glucose and lipid metabolism in human obesity [J].Metabolism, 1987, 36:54-59
[71] Yamamoto A, Matsuzawa Y, Kishino B, et al. Effects of probucol on homozygous case of familial hypercholesterolemia [J]. Atheroscterosis, 1983, 48:157-166
[72] Kanawa T, Tanaka M , Fukush i Y et al. Effect of low density lipoprotein from normal rabbits on hypercholesterolemia and the development of atherosclerosis[J]. Pathobildogy, 1991; 59 (2):85
[73] Steinberg D. Low density lipoprotein oxidation and its pathobiological significance [J]. J Biol Chem. 1997, 272(34):20963-20966
[74] Ross R. The pathagensis of atherosclerosis: a perspective for the 1990s [J]. Nature, 1993, 362(6423):801-809
[75] Mycks PR, Wright TF, Tanner M et al. The effects of native LDL andoxidized LDL on EDRF bioactivity and nitric oxide production in vascular endothelium[J]. J Lab Clin Med, 1994, 24 (5):627
[76] 吴卫平,匡培根,金泽武道,等.Ox-LDL对血管内皮细胞PECAM基因表达的影响[J].中国神经免疫和神经病学杂志,1995,2(2):69
[77] Biffl WL ,Moore EE, Moore FA et al. Nitric oxide reduces endothelial expression of intercellular adhesion molecule ( ICAM-1) [J]. J Surg Res, 1996, 63: 328
[78] 杜怡峰,申黎青,孙圣刚,童萼塘.氧化修饰低密度脂蛋白对血管内皮细胞DNA合成的影响[J].中国老年学杂志2000,20(6):368-369
[79] 樊蓉,刘虹,薛龙增,等.氧化型低密脂蛋白对培养的内皮细胞一氧化氮合成和释放的影响[J].中国动脉硬化杂志,1999, 7: 345-347
[80] 陶津,陈瑷,周玫.实验性动脉粥样硬化中脂质过氧化物在脂蛋白中的分布变化[J].中华医学杂志,1991,71(6):345
[81] 刘娟,刘尚喜,周玫,等.血浆LDL的快速提取和脂质过氧化物含量的测定[J].第一军医大学学报,1994,14(1):56
[82] Hennig B, Chow CK.Lipid peroxidation and endothelial cell injury: implication in atherosclerosis [J]. Free Radical Bio Med, 1988,4: 992108
[83] Printseva OY, Peclo MM, Gown AM. Various cell types in human atherosclerotic lesions express ICAM-1: Further immunocytochemical and immunochemical studies employing monoclonal antibody [J]. Am J Pathol, 1992, 140 (4):8892896
[84] 夏春枝,邓仲端.脂质过氧化诱导培养的内皮细胞表达巨噬细胞炎性蛋白-α和血管细胞黏附分子1[J].中国动脉硬化杂志, 2000, 8(3 ): 2022
[85] Kokura S, Wolf RE, Yoshikawa T, et al. Molecular mechanisms of neutrophil-endothelial cell adhesion induced by Redox imbalance [J]. Circulation Research, 1999, 84: 5162524
[86] Ross R. Atherosclerosis----As inflammatory disease [M]. New Engl Med, 1999
[87] Green LC , Wagncr DA ,Glogowski J . Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids [J]. Anal Bichem , 1982 ,126 (1) :131
[88] Ross R. The pathogenesis of atherosclerosis-an update [J].. N Engl J Med , 1986 , 314(8) :488
[89] Takami S, Yamashita S, Kihara S, et al. High concentration of glucose induces the expression of intercellular adhesion molecule-1 in human umbilical vein endothelial cells [J]. Atherosclerosis, 1998 ,138(1) :35～41
[90] Orem C, Orem A , Uydu HA , et al . The effects of lipid – lowering therapy on low- density lipoprotein auto - antibodies: relationship with low- density lipoprotein oxidation and plasma total antioxidant status[J] Coron Artery Dis.2002 ,13(1) :65～71
[91] Sawamura T, Kume N , Aoyama T et al . An endothelial receptor for low density lipoprotein [J] . Nature, 1997 ,386(6) :73
[92] Lehr HA , Becker M, Marklund SL , et al . Superoxide-dependent stimulation of leukocyte adhesion by oxidatively modified LDL in vivo [J]. Aarterioscler Thromb. 1992 ,12(7) :824～829
[93] Cominacini L, Garbin U, Fratta PA, et al. Lacidipine inhibits the activation of transcripition factor NF -κ appaB and the expression of adhesion molecules induced by pro - oxidant signal on endothelial cells [J]. J Hypertens. 1997 , 15 (12 pt2) :1633～1640
[94] Thorne SA , Abbot SE, Stevens CR , et al . Modified low density lipoprotein and cytokine mediate monocyte adhesion to smooth muscle cells [J] .Atherosclerosis. 1996 ,127(2) :167～176
[95] 王斌, 管思明.氧化型低密度脂蛋白对人脐静脉内皮细胞ICAM- 1 及NO 表达的影响[J].中国微循环2004,8(3):142
[96] 尹鸿操,陈铁真,董玉兰,等. 脂质过氧化对培养内皮细胞表达细胞黏附分子的影响[J].中国动脉硬化杂志, 1998 , 6(2) :1202124.
[97] Rubbo H, Parthasarathy S, Barnes S, et al . Nitric oxide inhibition of lipoxygenase-dependent liposome and low- density lipoprotein oxidation: termination of radical chain propagation reactions and formation of nitrogen-containing oxidized lipid derivatives [J]. Arch Biochem Biophys .1995 ,324(1) :15～25
[98] Mukherjee S, Coaxum SD , Maleque M, et al . Effects of oxidized low density lipoprotein on nitric oxide synthetase and protein kinase C activities in bovine endothelial cells [J] . Cell Mol Biol, 2001, 47(6):1051-1058
[99] Aliaga A, Saacleh R , Assian E, et al . Insuline inhibits the expression of intercellular adhesion molecule-1 by human aortic endothelial cells through stimulation of nitric oxide[J] . J Clin Endocrinol Metab. 2000 ,85(7) :2572～2575
[100] Moncada S , Higgs A. The L-arginine nitric oxide pathway[J] . N Engl J Med ,1993 ,329(27):2002-2012
[101] Basseneg E. Antiplateleteffects of endothelium2derived relaxing factor and nitric oxide donors [J] . Eur Heart, 1991 ,12 (Suppl E):12-16
[102] Lefer AM, Ma XL. Decreased basal nitric oxide release in hypercholesterolemia increase neutrophil adherence to rabbit coronary artery endothelium [J]. Arterioscler Throms ,1993 ,13(6) :771-774
[103] Scott Burden T , vanhoutte Pm. The endothelium as a regulator of vascular smooth muscle proliferation [J]. Circulation, 1993 , 87 ( suppl V) : 51 -55
[104] Zhao B , Zhang Y, Lin B et al . Endothelial cells injured by oxidized low density lipoproteins [J]. Am J Hematol, 1995, 49(3):250-252
[105] Lin Li. Ben jam in HS. Protection of vascular endothelial cell from hydrogen peroxide induced oxidant injury by pyenosides saponins of gynostemma pentaphyllum [J]. Phytotherapy Research, 1993 ,7 (1) :299-307
[106] 林秋雄,余细勇,单志新,等.氧化型低密度脂蛋白对血管内皮细胞ICAM-1 mRNA表达的影响[J].岭南心血管病杂志,2003,9(4):284
[107] Liao J K, Shin WS, Lee WY et al. Oxidized lowdensity lipoprotein decrease the expression of endothelial nitric oxide synthase [J]. Biod Chem, 1995 , 270 (1):319-324
[108] 吴松鹰,林求诚,杨持,等.老年脂质代谢紊乱中医易患因素的临床研究[J].中国中西医结合杂志,1993,13(5):273
[109] 宋剑南,刘东远,牛小红,高血脂症与中医痰浊关系的实验研究[J].辽宁中医基础医学杂志,1995,1(1)49
[110] 贾钰华,吕志平.老年前期高血脂症的中医辨证与微循环改变[J]. 辽宁中医杂志,1992,(4):3
[111] 黄可儿,方永奇,李小兵,等.高脂血症血液流变学改变与中医分型的关系[J]. 新中医,1993,(2):6
[112] 张兆云,张安静,毕俊英.中医药防治高脂血症信息分析[J].中国中医药信息杂志,1994(6):33
[113] 程小曲.痰浊型冠心病与血脂,脂蛋白,载脂蛋白的关系及痰浊形成机理的探讨[J].新中医,1994,(3):7
[114] 叶少伟.蝮蛇抗栓酶结合辨证治疗 80 例高脂血症临床观察[J].新中医,1994,26(2):29
[115] 熊文生.辩治高脂血症重在理脾化痰[J].新中医,1996,28(11):4
[116] 周文全.安脂舒胶囊治疗肾阴虚血瘀型老年高脂血症的临床研究[J].中国中医药科技,1995,2(4):12
[1] Kusama, H, Nishiyama, M. Ikeda, S. Pharmacological invetigation of bezafibrate, a hypolipidemic agent(I).Effect of bezafibrate on normol and experimental hyperlipidemia in rats[J]. Folia Pharmacol. Japan, 1998, 92:175-180
[2] Niiho, H., Yamazaki, Y., Itoh, H., Tkeshita, T., Kinjo, Jun-ei Nohar,T. Pharmacological Studies on Puerariae Flos.II. The effects of Puerariae Flos on Alcohol-induced Unusual Metabolism and Experimental Liver Injury in Mice[J]. Yakugaku Zasshi, 1990, 110:604-611
[3] Jeon SM, Bok SH, Jang MK, Lee MK, Nam KT, Park YB, Rhee SJ, Choi MS. Antioxidative activity of naringin and lovastatin in high cholesterol-fed rabbits[J]. Life Sciences, 2001, 69:2856-2866
[4] Kim HJ, Oh GT, Park YB, Lee MK, Seo HJ, Chio MS. Naringin alters the cholesterol biosyntheseis and antioxidant enzyme activities in LDL receptor-knockout mice under cholesterol fed condition[J]. Life Sciences, 2004, 74:1621-1634
[5] Kim DH, Bac EA, Han MJ. Anti-Helicobacter pylori of the metabolites of poncirin from Poncirus trifoliata by human intestinal bacteria[J]. Biol Pharm Bull, 1999, 22(4):422-424
[6] 余艳辉,文军,郭兆贵. Triton WR-1339 对小鼠血脂水平的影响 [ J] . 中国药理学通报, 2002, 18( 5) : 599 - 600.
[7] 左绍远,钱金. 复方螺旋藻片对高血脂症小鼠血脂的影响[J]. 中国现代应用药学杂志, 2001,18(2):92-94
[8] 倪鸿昌,李俊. 高血脂症实验性动物模型的研究进展[J]. 安徽医药,2004,8(6):402-404
[9] 何冰,陈小夏,李娟,等. 茶多酚对高血脂症动物血脂调节、脂质过氧化及血小板聚集功能的影响[J]. 广东药学院学报, 1996, 12 (1): 29-32
[10] 林成仁,王敏,周亚伟,等. 调脂降糖胶囊对家兔实验性高血脂及动脉粥样硬化的影响[J]. 中药新药与临床药理,1997,8(2):90-92
[11] 孟甄,孙立红,陈芸芸,等. 石榴叶鞣质对高血脂高血糖模型动物脂代谢的影响[J]. 中国实验方剂学杂志,2005,11(1):22-23
[12] 段淑芳,周国英. 高血脂症与2型糖尿病关系的研究进展,现代中西医结合杂志,2004,13(10):1391
[1] 潘馨. 绿衣枳壳中柚皮苷含量测定方法的研究[J]. 中南药学,2005,3(2):82
[2] 童荣生,孙世明,吴齐贤. RP-HPLC测定枳实中橙皮苷的含量[J]. 华西药学杂志,2000 ,15(6) :454-455
[3] 刘向前,魏圣淑,白完淑. 中国产和韩国产芸香科植物的成分比较研究[J]. 中草药,2003,34(7):586-588
[4] 贾强,白杨,马燕,等. 枳壳和枳实化学成分的HPLC-ESI-MS分析[J]. 中草药,2005,36(2):169-171
[5] 杨武亮,杨世林,张敏. RP-HPLC法测定枳壳中柚皮苷和新橙皮苷的含量[J].中药新药与临床药理,2005,16(4):261-263
[6] 肖培根主编. 新编中药志.第二卷[M]. 北京:化学工业出版社,2002,1:453
[1] 江振友,杨春华,江丽芳. 人脐静脉内皮细胞的继代培养[J]. 蚌埠医学院学报,1997,22(4):211-213
[2] 陈樱,刘德敏,孟东. 人脐静脉内皮细胞体外培养方法的改进[J]. 天津医科大学学报,2003,9(3):318-320
[3] 梁永钜,符立梧. MTS法细胞毒试验的非线性关系研究[J]. 中国药理学通报,2004, 20 (6) :623-627
[4] 王琳,贾琴,范能全. 评价细胞毒性方法的探讨[J]. 泸州医学院报,2005, 28(2) :132-134
[5] 杜怡峰,申黎青,孙圣刚.氧化修饰低密度脂蛋白对血管内皮细胞DNA合成的影响[J].中国老年学杂志,2000,20(6):368-369
[6] Kanawa T, Tanaka M , Fukush i Y et al1Effect of low density lipoprotein from normal rabbits on hypercholesterolemia and the development of atherosclerosis [J]. Pathobildogy, 1991; 59 (2): 85
[7] 张俊玲,刘赛,王瑞臣. 扇贝裙边糖胺聚糖对Ox-LDL致血管内皮细胞损伤的保护作用[J]. 中国药理学通报,2004, 20 (12) :1389-1392
[8] 林秋雄,余细勇,单志新. 氧化型低密度脂蛋白对血管内皮细胞ICAM-1mRNA表达的影响[J]. 岭南心血管病杂志,2003,9(4):283
[9] 左云飞,张耀铮,贾红龙. 芪参降脂饮对ApoB、LDL2c 、Ox-LDL水平的影响[J]. 中药药理与临床,2000, 16 (6):30
[10] 刘梦琼,郑义. 阿魏酸钠对氧化低密度脂蛋白所致的内皮细胞释放NO的影响[J]. 华中医学杂志,2002,26(1):15
[11] 韩峰,谢梅林,朱路佳. 百草降脂灵对动脉粥样硬化兔Ox-LDL和LDL mRNA表达的影响[J]. 中国新药与临床杂志,2002,21 (7) :389-393
[12] 王斌, 管思明. 氧化型低密度脂蛋白对人脐静脉内皮细胞ICAM-1及NO表达的影响[J]. 中国微循环, 2004,8(3):140-142
[13] 张红梅,田辉凯,姚小皓,等. 绿谷灵芝对人脐静脉内皮细胞ICAM-1和VCAM-1表达的影响[J]. 中国药理学通报,2002, 18 (4):461-463
[14] 曾玉,邓红,王筠. 茶多酚对高糖时内皮细胞产生NO、NOS和ICAM-1的影响[J]. 南京医科大学学报(自然科学版),2005,25(7):460-463
[15] Cory, A.H. et al. Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture[J]. Cancer Comm., 1991, 3:207-212
[16] Riss, T.L., Moravec, R.A. Comparison of MTT, XTT and a novel tetrazolium compound MTS for in vitro proliferation and chemosensitivity assays[J]. Mol. Biol. Cell, 1992, (Suppl.) 3: 184
[17] 唐孟萱,周万军,胡佳元. HepG2 细胞培养方法与条件的探讨. 实用预防医学,200512(1):71-72
[18] 张卓然. 实用细胞培养技术[M]. 北京:人民卫生出版社,1999:46
[19] Arshag D, Mooradian Michael J. Haas, Khuram Wadud. Ascorbic acid and α-tocopherol down-regulate apolipoprotein A-I gene expression in HepG2 and Caco-2 cell lines[J]. Metabolism Clinical and Experimental, 2006,5 5 : 159–167
[20] Javier Garc?′a-Alonso, Gaspar Ros, M, Jesu′s Periago. Antiproliferative and cytoprotective activities of a phenolic-rich juice in HepG2 cells [J]. Food Research International, 2006, 39: 982–991
[21] Shin-Pei Yang, Kimberly Wilson, Abdul Kawa. Effects of green tea extracts on gene expression in HepG2 and Cal-27 cells [J]. Food and Chemical Toxicology, 2006, 44: 1075–1081
[22] Paolo Mondola, Rosalb Seru, Mariarosaria Santillo, et al. Effect of Cu,Zn superoxide dismutase on cholesterol metabolism in human hepatocarcinoma (HepG2) cells[J].Biochemical and Biophysical Research Communications, 2002, 295: 603–609
[23] Kazuo Shimizu, Hitoshi Ashida,aYukinaga Matsuura. Antioxidative bioavailability of artepillin C in Brazilian propolis[J]. Archives of Biochemistry and Biophysics, 2004, 424: 181–188
[24] Zhanna Shutenko, Yann Henry, Elisabeth Pinard, et al. Influence of the Antioxidant Quercetin In Vivo on the Level of Nitric Oxide Determined by Electron Paramagnetic Resonance in Rat Brain during Global Ischemia and Reperfusion[J]. Biochemical Pharmacology, 1999, 57(98):199-208
[25] 李 彦 , 谢 明 . 单 细 胞 凝 胶 电 泳 技 术 操 作 方 法 介 绍 [J]. 生 物 学 通报,2005,40(7):52-55
[26] 张显忠,苗苗,李艳玲. 改良单细胞凝胶电泳技术在DNA损伤与修复检测中的应用[J]. 泰山医学院学报,2005,26(3):206-208
[27] 高建军,丰盛梅,金慰芳. 碱性单细胞凝胶电泳铺胶技术的改进[J]. 复旦学报(医学版),2004, 31(3):311-313
[28] Singh NP, Mccoy MT, Tice RR, et al. A simple technique for quantitation of low levels of DNA damage in individual cells [J]. Exp. Cell Res, 1998, 175:184
[29] Hartmann A, Agurell E, Beevers C, et al. Recommendations for conducting the in vivo alkalline comet assay-The 4th international comet assay workshop[J]. Mutagenesis, 2003, 18(1):45
[30] 张遵真,衡正,李蕊,等. 单细胞凝胶电泳试验的最适条件研究[J].卫生毒理学杂志,1998,12(4):249
[31] 张建平,田源,陈道达.单细胞凝胶电泳制胶方法的改进[J]. 癌变畸变突变,1998,10(3):189
[3] 中国科学院植物研究所[M].中国经济植物志(下册). 北京: 科学技术出版社, 1961:1364
[4] 高士彦,郑宏钧.柑橘属果皮及幼果中挥发油的气相色谱分析[J].药物分析 杂志,1986,6(2):833
[5] Kekelidae NA. Citrus wilsonii leaves essential oils[J]. Khim Prir Soedin,1982(6):785
[6] Matsubara. Studies on physio logical active substances in citrus fruit peel[J]. Yukagaku, 1988, 37(1):13
[7] 中国科学院植物研究所.中国经济植物志(下册) [M].北京:科学技术出版社,1961:1366
[8] 李仪奎,姜名英.中药药理学[M].北京:中国中医药出版社,1992:169 - 179
[9] 李岩,陈苏宁,李宇权,等.芍药甘草汤、四逆散对胃排空及小肠推进功能影响的拆方研[J].中华消化杂志,1996,16(2):20
[10] 陈琦,杨雪梅,徐成贺.厚朴三物汤拆方及其药量变化的实验研究[J].时珍国医国药,2001,12(9):776~778
[11] 李新旺,任钧国.枳实白术配伍的实验研究[J].中医研究,2002,15(6):23~24
[12] 况玲.枳实对绵羊小肠电活动的影响[J].中医药研究,1997,13(3):49~50
[13] 王翠芬,杨德治,魏义全,等.枳实对大鼠胃肠电活动影响的初步研究[J].东南大学学报,2001,20(3):153~154
[14] 黄子荷,杨德治,魏义全,等.阿托品对枳实增强犬小肠电活动效应的影响[J].中国中西医结合杂志,1996,16(5):293~294
[15] 郑天珍,李伟,瞿颂义,等.枳实、白术对大鼠离体胃各部位平滑肌条的作用[J].甘肃科学学报,1998,10(3):65~67
[16] 杨颖丽,郑天珍,瞿颂义,等.枳实对大鼠离体小肠平滑肌条的作用[J].西北师范大学学报(自然科学版),1998,34(2):70~71
[17] 谢冬萍,李伟,郑天珍,等.枳实对大鼠结肠肌条运动的影响[J].山东医科大学学报,2001,39(5):438
[18] 毕庆和,杨德治,应崇智,等.枳实对小肠电活动的影响[J].中国医药学报,1991,6(1):39~40
[19] 胡 盛 珊 , 王 大 元 , 邱 萍 , 等 . 枳 实 有 效 成 分 的 药 理 活 性 比 较 [J]. 中 草药,1994,25(8):419~420
[20] 胡盛珊,王大元,邱萍,等.枳实、枳壳挥发油对动物胃肠道的作用[J].东南大学学报,2001,20(3):144~145
[21] 刘波,李雪驼,徐和利,等.5 种中药制剂杀灭幽门螺杆菌的实验研究[J].中国新药杂志,2002,11(6):457
[22] 李红芳,李丹明,瞿颂义,等.枳实和陈皮对兔离体主动脉平滑肌条作用机理探讨[J].中成药,2001,23(9):659~660
[23] 倪艺,陈梦麟.枳实薤白桂枝汤治疗冠心病 58 例[J].国医论坛,2000,15(1):8
[24] 欧兴长,丁家欣,张玲.枳实等 11 种中药体外抗血栓作用研究[J].中西医结合杂志,1989,9(6):358~359
[25] 吉中强,宋鲁卿,高晓昕,等.11 种中药对大鼠血小板聚集和红细胞流变性的影响[J].山东中医杂志,2000,(2)19:107
[26] 陈庆平,王诗雅.名医印会河教授临床抓主经症经验集粹(十二)[J].中国乡村医药,2001,8(8):28
[27] 杨颖丽.枳实、青皮对平滑肌运动的影响[J].西北师范大学学报(自然科学版),2001,38(2):115~116
[28] 汤容.枳实和苦参对离体家兔阴道平滑肌收缩作用的研究[J].时珍国医国药,2001,12(11):973
[29] 王长虹,张冰,张宇,等.复方枳实合剂对实验动物的抗疲劳作用研究[J].牡丹江医学院学报,2001,22(1):15~17
[30] 周 海 均 , 等 . 生 物 检 定 统 计 方 法 ( 第 二 版 )[M]. 北 京 : 人 民 卫 生 出 版社,1988:1~4
[31] 张贵君.常用中药生物鉴定[M].北京:化学工业出版社 2006,1:1,7~8
[32] 封士兰,陈立仁 .中药质量标准研究新进展 [J].兰州医学院学报,2002,28(4):85~86
[33] 李 亚 萍 . 浅 议 我 国 中 药 质 量 标 准 的 进 展 [J]. 医 学 理 论 与 实 践 ,2006,19(2):227~228
[34] 王 跃 生 . 中 药 质 量 标 准 现 代 化 发 展 现 状 与 思 考 [J]. 中 国 中 药 杂志,2003,28(12):1108~1109
[35] 雷 茂 华 . 谈 中 药 制 剂 质 量 标 准 制 定 中 的 几 点 误 区 [J]. 江 苏 中医,1998,19(2):48
[36] 罗国安,王义明,曹进,杨学东.建立我国现代中药质量标准体系的研究[J].世界科学技术,2002,4(4):6
[37] 赖小平.高效毛细管电泳及其在中药质量分析中的应用[J].中药新药与临床药理, 1998,9(3):186
[38] 王 宝 琴 . 中 药 质 量 检 验 方 法 应 用 研 究 现 状 [J]. 中 药 新 药 与 临 床 药理,1997,8(2):110
[39] 王 宝 琴 . 中 药 质 量 标 准 研 究 含 量 测 定 的 选 定 原 则 刍 议 [J]. 中 成药,1995,17(3):50
[40] EMEA. Final proposals for Revision of the Note for Guidance on Quality ofHerbal Remedies.1998
[41] 李晓燕 .中成药质量及质量标准有关问题的探讨 [J]. 广东药学院学报,1996,12(2):106
[42] 杨曾让.中成药质量标准隐患初探[J].中成药,1995,17(4):48
[43] 夏柳影.中成药质量标准的演变和展望[J].中成药,1994,16(9):43
[44] 郑志华.加强中药质量标准研究是实现中药现代化的必要前提[J].中国药业,2001,10(2):4~5
[45] 董丽丽,李野.加速完善我国中药饮片质量标准[J].中国药房,2004, 15 (7):444
[46] 朱红宏.关于《中国药典》2005 年版新增中药饮片质量标准之浅见[J].中国药品标准, 2005,6(1):38
[47] 陈皎.对中药饮片质量工作的探讨[J].中国中药杂志,1995,20(4):214
[48] 陈吉炎,陈黎,安志斌,等.中药饮片质控乏力的原因与对策[J].中药材, 2003,26(1):43
[49] 张志国,殷剑.中药饮片质量的现状与思考[J].中药研究与信息,2003, 5(8):13
[50] 贾维臣,等.中药炮制品质量的影响因素剖析[J].时珍国医国药2003,14 (8):489
[51] 董洪根.浅论影响中药饮片质量与疗效的因素[J].江苏中医药2003,24 (1) :41
[52] 刘以霞.影响中药饮片质量的问题及其解决方法[J].江苏药学与临床研究, 2002,10(4):39
[53] 李敏,等.提高中药饮片质量应把好六关[J].湖南中医药导报,1996,2(5):39
[54] 雷永军,夏明衍,孟黎明,朱建.我国现行中药材质量标准中的若干问题分析[J].中国医院药学杂志,2001,21(1):46
[55] 谢培山.中医药文化与现代质量控制[J].中国中西医结合杂志,1998,18(11):654
[56] 祝国光.中药如何进入欧共体市场[M].北京,中国中医药科技,2000:54
[57] 张焱.浅谈规范中药质量标准的迫切性[J].山西中医,1996,5:34
[58] 贺兰英.再谈中药现代化—中药质量标准是中药现代化的前提[J].中医药学刊,2004,22(1): 148,171
[59] Cheung MC, Albers JJ. Characterization of lipoprotein particles isolated by immunoaffinity chromatography: particles containing AI and A Ⅱ and particles containing AI but no AⅡ[J]. J Biol Chem, 1984, 259:12201-12209
[60] Aarai K, Maguchi S. Glycation and inactivation of human Cu-Zn-Superoxide dismutase: Identification of the in vitro glycated sites [J]. J Biol Chem, 1989, 262:16969-16972
[61] Vlassara H, Brownle M, Manogue K, et al. Cachectin TNF and IL-1inducedby glucose-modified protein: role in normal tissue remodeling [J]. Science, 1988, 240:156
[62] Kirstein M, Aston C, Hints R et al. Receptor-specific induction of insulin-slide growth factor I in human monocytes by advanced glycosylation end product modified protein [J]. J Clin Invest , 1988, 90:439-446
[63] Fievetc, Theret N, Shojaee N, et al. Apolipoprotein Ai containing particles and reverse cholesterol transport in IDDM [J]. Diabetes, 1992, 41 (sappl.2) :81-85
[64] Takata K, Horiuchi S, Araki N, et al. Endocytic uptake of nonenzymatically glycosylated proteins is mediated by scavenger receptor for aldehyde-modified proteins [J]. J Biol Chem, 1988,263:14819-14825
[65] Kannel WB, Hypertension and other risk factors in coronary heart disease [J]. Am Heart J, 1987,114:918-925
[66] Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990 [J]. Nature, 1993, 362:81-808
[67] Kihara S, Matsuzawa Y, Kubo M, et al. Autoimmune hyperchylomicroemia [J]. New Engl J Med 1989,320:1255-1259
[68] Soria LF, Ludwig EH, Clark HRG, et.al. Association between a specific apolipoprotein B mutation and familial defective apolipoprotein B [J]. Pro Nat Acad Sci USA, 1989, 86:587-591
[69] Shimomura I, Tokunaga K, Jiaos, et al. Marded enhancement of acyl-CoA synthetase activity and mRNA paralleled to lipoprotein lipase mRNA, in adipose tissue of zucker obese rats [J]. Biochim Biophys Acta, 1992, 1124:112-118
[70] Fujioka S, Matsuzawa Y,T okunaga K, et al. Contribution of intra-abdominal fat accumulation to the impairment of glucose and lipid metabolism in human obesity [J].Metabolism, 1987, 36:54-59
[71] Yamamoto A, Matsuzawa Y, Kishino B, et al. Effects of probucol on homozygous case of familial hypercholesterolemia [J]. Atheroscterosis, 1983, 48:157-166
[72] Kanawa T, Tanaka M , Fukush i Y et al. Effect of low density lipoprotein from normal rabbits on hypercholesterolemia and the development of atherosclerosis[J]. Pathobildogy, 1991; 59 (2):85
[73] Steinberg D. Low density lipoprotein oxidation and its pathobiological significance [J]. J Biol Chem. 1997, 272(34):20963-20966
[74] Ross R. The pathagensis of atherosclerosis: a perspective for the 1990s [J]. Nature, 1993, 362(6423):801-809
[75] Mycks PR, Wright TF, Tanner M et al. The effects of native LDL andoxidized LDL on EDRF bioactivity and nitric oxide production in vascular endothelium[J]. J Lab Clin Med, 1994, 24 (5):627
[76] 吴卫平,匡培根,金泽武道,等.Ox-LDL对血管内皮细胞PECAM基因表达的影响[J].中国神经免疫和神经病学杂志,1995,2(2):69
[77] Biffl WL ,Moore EE, Moore FA et al. Nitric oxide reduces endothelial expression of intercellular adhesion molecule ( ICAM-1) [J]. J Surg Res, 1996, 63: 328
[78] 杜怡峰,申黎青,孙圣刚,童萼塘.氧化修饰低密度脂蛋白对血管内皮细胞DNA合成的影响[J].中国老年学杂志2000,20(6):368-369
[79] 樊蓉,刘虹,薛龙增,等.氧化型低密脂蛋白对培养的内皮细胞一氧化氮合成和释放的影响[J].中国动脉硬化杂志,1999, 7: 345-347
[80] 陶津,陈瑷,周玫.实验性动脉粥样硬化中脂质过氧化物在脂蛋白中的分布变化[J].中华医学杂志,1991,71(6):345
[81] 刘娟,刘尚喜,周玫,等.血浆LDL的快速提取和脂质过氧化物含量的测定[J].第一军医大学学报,1994,14(1):56
[82] Hennig B, Chow CK.Lipid peroxidation and endothelial cell injury: implication in atherosclerosis [J]. Free Radical Bio Med, 1988,4: 992108
[83] Printseva OY, Peclo MM, Gown AM. Various cell types in human atherosclerotic lesions express ICAM-1: Further immunocytochemical and immunochemical studies employing monoclonal antibody [J]. Am J Pathol, 1992, 140 (4):8892896
[84] 夏春枝,邓仲端.脂质过氧化诱导培养的内皮细胞表达巨噬细胞炎性蛋白-α和血管细胞黏附分子1[J].中国动脉硬化杂志, 2000, 8(3 ): 2022
[85] Kokura S, Wolf RE, Yoshikawa T, et al. Molecular mechanisms of neutrophil-endothelial cell adhesion induced by Redox imbalance [J]. Circulation Research, 1999, 84: 5162524
[86] Ross R. Atherosclerosis----As inflammatory disease [M]. New Engl Med, 1999
[87] Green LC , Wagncr DA ,Glogowski J . Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids [J]. Anal Bichem , 1982 ,126 (1) :131
[88] Ross R. The pathogenesis of atherosclerosis-an update [J].. N Engl J Med , 1986 , 314(8) :488
[89] Takami S, Yamashita S, Kihara S, et al. High concentration of glucose induces the expression of intercellular adhesion molecule-1 in human umbilical vein endothelial cells [J]. Atherosclerosis, 1998 ,138(1) :35～41
[90] Orem C, Orem A , Uydu HA , et al . The effects of lipid – lowering therapy on low- density lipoprotein auto - antibodies: relationship with low- density lipoprotein oxidation and plasma total antioxidant status[J] Coron Artery Dis.2002 ,13(1) :65～71
[91] Sawamura T, Kume N , Aoyama T et al . An endothelial receptor for low density lipoprotein [J] . Nature, 1997 ,386(6) :73
[92] Lehr HA , Becker M, Marklund SL , et al . Superoxide-dependent stimulation of leukocyte adhesion by oxidatively modified LDL in vivo [J]. Aarterioscler Thromb. 1992 ,12(7) :824～829
[93] Cominacini L, Garbin U, Fratta PA, et al. Lacidipine inhibits the activation of transcripition factor NF -κ appaB and the expression of adhesion molecules induced by pro - oxidant signal on endothelial cells [J]. J Hypertens. 1997 , 15 (12 pt2) :1633～1640
[94] Thorne SA , Abbot SE, Stevens CR , et al . Modified low density lipoprotein and cytokine mediate monocyte adhesion to smooth muscle cells [J] .Atherosclerosis. 1996 ,127(2) :167～176
[95] 王斌, 管思明.氧化型低密度脂蛋白对人脐静脉内皮细胞ICAM- 1 及NO 表达的影响[J].中国微循环2004,8(3):142
[96] 尹鸿操,陈铁真,董玉兰,等. 脂质过氧化对培养内皮细胞表达细胞黏附分子的影响[J].中国动脉硬化杂志, 1998 , 6(2) :1202124.
[97] Rubbo H, Parthasarathy S, Barnes S, et al . Nitric oxide inhibition of lipoxygenase-dependent liposome and low- density lipoprotein oxidation: termination of radical chain propagation reactions and formation of nitrogen-containing oxidized lipid derivatives [J]. Arch Biochem Biophys .1995 ,324(1) :15～25
[98] Mukherjee S, Coaxum SD , Maleque M, et al . Effects of oxidized low density lipoprotein on nitric oxide synthetase and protein kinase C activities in bovine endothelial cells [J] . Cell Mol Biol, 2001, 47(6):1051-1058
[99] Aliaga A, Saacleh R , Assian E, et al . Insuline inhibits the expression of intercellular adhesion molecule-1 by human aortic endothelial cells through stimulation of nitric oxide[J] . J Clin Endocrinol Metab. 2000 ,85(7) :2572～2575
[100] Moncada S , Higgs A. The L-arginine nitric oxide pathway[J] . N Engl J Med ,1993 ,329(27):2002-2012
[101] Basseneg E. Antiplateleteffects of endothelium2derived relaxing factor and nitric oxide donors [J] . Eur Heart, 1991 ,12 (Suppl E):12-16
[102] Lefer AM, Ma XL. Decreased basal nitric oxide release in hypercholesterolemia increase neutrophil adherence to rabbit coronary artery endothelium [J]. Arterioscler Throms ,1993 ,13(6) :771-774
[103] Scott Burden T , vanhoutte Pm. The endothelium as a regulator of vascular smooth muscle proliferation [J]. Circulation, 1993 , 87 ( suppl V) : 51 -55
[104] Zhao B , Zhang Y, Lin B et al . Endothelial cells injured by oxidized low density lipoproteins [J]. Am J Hematol, 1995, 49(3):250-252
[105] Lin Li. Ben jam in HS. Protection of vascular endothelial cell from hydrogen peroxide induced oxidant injury by pyenosides saponins of gynostemma pentaphyllum [J]. Phytotherapy Research, 1993 ,7 (1) :299-307
[106] 林秋雄,余细勇,单志新,等.氧化型低密度脂蛋白对血管内皮细胞ICAM-1 mRNA表达的影响[J].岭南心血管病杂志,2003,9(4):284
[107] Liao J K, Shin WS, Lee WY et al. Oxidized lowdensity lipoprotein decrease the expression of endothelial nitric oxide synthase [J]. Biod Chem, 1995 , 270 (1):319-324
[108] 吴松鹰,林求诚,杨持,等.老年脂质代谢紊乱中医易患因素的临床研究[J].中国中西医结合杂志,1993,13(5):273
[109] 宋剑南,刘东远,牛小红,高血脂症与中医痰浊关系的实验研究[J].辽宁中医基础医学杂志,1995,1(1)49
[110] 贾钰华,吕志平.老年前期高血脂症的中医辨证与微循环改变[J]. 辽宁中医杂志,1992,(4):3
[111] 黄可儿,方永奇,李小兵,等.高脂血症血液流变学改变与中医分型的关系[J]. 新中医,1993,(2):6
[112] 张兆云,张安静,毕俊英.中医药防治高脂血症信息分析[J].中国中医药信息杂志,1994(6):33
[113] 程小曲.痰浊型冠心病与血脂,脂蛋白,载脂蛋白的关系及痰浊形成机理的探讨[J].新中医,1994,(3):7
[114] 叶少伟.蝮蛇抗栓酶结合辨证治疗 80 例高脂血症临床观察[J].新中医,1994,26(2):29
[115] 熊文生.辩治高脂血症重在理脾化痰[J].新中医,1996,28(11):4
[116] 周文全.安脂舒胶囊治疗肾阴虚血瘀型老年高脂血症的临床研究[J].中国中医药科技,1995,2(4):12
[1] Kusama, H, Nishiyama, M. Ikeda, S. Pharmacological invetigation of bezafibrate, a hypolipidemic agent(I).Effect of bezafibrate on normol and experimental hyperlipidemia in rats[J]. Folia Pharmacol. Japan, 1998, 92:175-180
[2] Niiho, H., Yamazaki, Y., Itoh, H., Tkeshita, T., Kinjo, Jun-ei Nohar,T. Pharmacological Studies on Puerariae Flos.II. The effects of Puerariae Flos on Alcohol-induced Unusual Metabolism and Experimental Liver Injury in Mice[J]. Yakugaku Zasshi, 1990, 110:604-611
[3] Jeon SM, Bok SH, Jang MK, Lee MK, Nam KT, Park YB, Rhee SJ, Choi MS. Antioxidative activity of naringin and lovastatin in high cholesterol-fed rabbits[J]. Life Sciences, 2001, 69:2856-2866
[4] Kim HJ, Oh GT, Park YB, Lee MK, Seo HJ, Chio MS. Naringin alters the cholesterol biosyntheseis and antioxidant enzyme activities in LDL receptor-knockout mice under cholesterol fed condition[J]. Life Sciences, 2004, 74:1621-1634
[5] Kim DH, Bac EA, Han MJ. Anti-Helicobacter pylori of the metabolites of poncirin from Poncirus trifoliata by human intestinal bacteria[J]. Biol Pharm Bull, 1999, 22(4):422-424
[6] 余艳辉,文军,郭兆贵. Triton WR-1339 对小鼠血脂水平的影响 [ J] . 中国药理学通报, 2002, 18( 5) : 599 - 600.
[7] 左绍远,钱金. 复方螺旋藻片对高血脂症小鼠血脂的影响[J]. 中国现代应用药学杂志, 2001,18(2):92-94
[8] 倪鸿昌,李俊. 高血脂症实验性动物模型的研究进展[J]. 安徽医药,2004,8(6):402-404
[9] 何冰,陈小夏,李娟,等. 茶多酚对高血脂症动物血脂调节、脂质过氧化及血小板聚集功能的影响[J]. 广东药学院学报, 1996, 12 (1): 29-32
[10] 林成仁,王敏,周亚伟,等. 调脂降糖胶囊对家兔实验性高血脂及动脉粥样硬化的影响[J]. 中药新药与临床药理,1997,8(2):90-92
[11] 孟甄,孙立红,陈芸芸,等. 石榴叶鞣质对高血脂高血糖模型动物脂代谢的影响[J]. 中国实验方剂学杂志,2005,11(1):22-23
[12] 段淑芳,周国英. 高血脂症与2型糖尿病关系的研究进展,现代中西医结合杂志,2004,13(10):1391
[1] 潘馨. 绿衣枳壳中柚皮苷含量测定方法的研究[J]. 中南药学,2005,3(2):82
[2] 童荣生,孙世明,吴齐贤. RP-HPLC测定枳实中橙皮苷的含量[J]. 华西药学杂志,2000 ,15(6) :454-455
[3] 刘向前,魏圣淑,白完淑. 中国产和韩国产芸香科植物的成分比较研究[J]. 中草药,2003,34(7):586-588
[4] 贾强,白杨,马燕,等. 枳壳和枳实化学成分的HPLC-ESI-MS分析[J]. 中草药,2005,36(2):169-171
[5] 杨武亮,杨世林,张敏. RP-HPLC法测定枳壳中柚皮苷和新橙皮苷的含量[J].中药新药与临床药理,2005,16(4):261-263
[6] 肖培根主编. 新编中药志.第二卷[M]. 北京:化学工业出版社,2002,1:453
[1] 江振友,杨春华,江丽芳. 人脐静脉内皮细胞的继代培养[J]. 蚌埠医学院学报,1997,22(4):211-213
[2] 陈樱,刘德敏,孟东. 人脐静脉内皮细胞体外培养方法的改进[J]. 天津医科大学学报,2003,9(3):318-320
[3] 梁永钜,符立梧. MTS法细胞毒试验的非线性关系研究[J]. 中国药理学通报,2004, 20 (6) :623-627
[4] 王琳,贾琴,范能全. 评价细胞毒性方法的探讨[J]. 泸州医学院报,2005, 28(2) :132-134
[5] 杜怡峰,申黎青,孙圣刚.氧化修饰低密度脂蛋白对血管内皮细胞DNA合成的影响[J].中国老年学杂志,2000,20(6):368-369
[6] Kanawa T, Tanaka M , Fukush i Y et al1Effect of low density lipoprotein from normal rabbits on hypercholesterolemia and the development of atherosclerosis [J]. Pathobildogy, 1991; 59 (2): 85
[7] 张俊玲,刘赛,王瑞臣. 扇贝裙边糖胺聚糖对Ox-LDL致血管内皮细胞损伤的保护作用[J]. 中国药理学通报,2004, 20 (12) :1389-1392
[8] 林秋雄,余细勇,单志新. 氧化型低密度脂蛋白对血管内皮细胞ICAM-1mRNA表达的影响[J]. 岭南心血管病杂志,2003,9(4):283
[9] 左云飞,张耀铮,贾红龙. 芪参降脂饮对ApoB、LDL2c 、Ox-LDL水平的影响[J]. 中药药理与临床,2000, 16 (6):30
[10] 刘梦琼,郑义. 阿魏酸钠对氧化低密度脂蛋白所致的内皮细胞释放NO的影响[J]. 华中医学杂志,2002,26(1):15
[11] 韩峰,谢梅林,朱路佳. 百草降脂灵对动脉粥样硬化兔Ox-LDL和LDL mRNA表达的影响[J]. 中国新药与临床杂志,2002,21 (7) :389-393
[12] 王斌, 管思明. 氧化型低密度脂蛋白对人脐静脉内皮细胞ICAM-1及NO表达的影响[J]. 中国微循环, 2004,8(3):140-142
[13] 张红梅,田辉凯,姚小皓,等. 绿谷灵芝对人脐静脉内皮细胞ICAM-1和VCAM-1表达的影响[J]. 中国药理学通报,2002, 18 (4):461-463
[14] 曾玉,邓红,王筠. 茶多酚对高糖时内皮细胞产生NO、NOS和ICAM-1的影响[J]. 南京医科大学学报(自然科学版),2005,25(7):460-463
[15] Cory, A.H. et al. Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture[J]. Cancer Comm., 1991, 3:207-212
[16] Riss, T.L., Moravec, R.A. Comparison of MTT, XTT and a novel tetrazolium compound MTS for in vitro proliferation and chemosensitivity assays[J]. Mol. Biol. Cell, 1992, (Suppl.) 3: 184
[17] 唐孟萱,周万军,胡佳元. HepG2 细胞培养方法与条件的探讨. 实用预防医学,200512(1):71-72
[18] 张卓然. 实用细胞培养技术[M]. 北京:人民卫生出版社,1999:46
[19] Arshag D, Mooradian Michael J. Haas, Khuram Wadud. Ascorbic acid and α-tocopherol down-regulate apolipoprotein A-I gene expression in HepG2 and Caco-2 cell lines[J]. Metabolism Clinical and Experimental, 2006,5 5 : 159–167
[20] Javier Garc?′a-Alonso, Gaspar Ros, M, Jesu′s Periago. Antiproliferative and cytoprotective activities of a phenolic-rich juice in HepG2 cells [J]. Food Research International, 2006, 39: 982–991
[21] Shin-Pei Yang, Kimberly Wilson, Abdul Kawa. Effects of green tea extracts on gene expression in HepG2 and Cal-27 cells [J]. Food and Chemical Toxicology, 2006, 44: 1075–1081
[22] Paolo Mondola, Rosalb Seru, Mariarosaria Santillo, et al. Effect of Cu,Zn superoxide dismutase on cholesterol metabolism in human hepatocarcinoma (HepG2) cells[J].Biochemical and Biophysical Research Communications, 2002, 295: 603–609
[23] Kazuo Shimizu, Hitoshi Ashida,aYukinaga Matsuura. Antioxidative bioavailability of artepillin C in Brazilian propolis[J]. Archives of Biochemistry and Biophysics, 2004, 424: 181–188
[24] Zhanna Shutenko, Yann Henry, Elisabeth Pinard, et al. Influence of the Antioxidant Quercetin In Vivo on the Level of Nitric Oxide Determined by Electron Paramagnetic Resonance in Rat Brain during Global Ischemia and Reperfusion[J]. Biochemical Pharmacology, 1999, 57(98):199-208
[25] 李 彦 , 谢 明 . 单 细 胞 凝 胶 电 泳 技 术 操 作 方 法 介 绍 [J]. 生 物 学 通报,2005,40(7):52-55
[26] 张显忠,苗苗,李艳玲. 改良单细胞凝胶电泳技术在DNA损伤与修复检测中的应用[J]. 泰山医学院学报,2005,26(3):206-208
[27] 高建军,丰盛梅,金慰芳. 碱性单细胞凝胶电泳铺胶技术的改进[J]. 复旦学报(医学版),2004, 31(3):311-313
[28] Singh NP, Mccoy MT, Tice RR, et al. A simple technique for quantitation of low levels of DNA damage in individual cells [J]. Exp. Cell Res, 1998, 175:184
[29] Hartmann A, Agurell E, Beevers C, et al. Recommendations for conducting the in vivo alkalline comet assay-The 4th international comet assay workshop[J]. Mutagenesis, 2003, 18(1):45
[30] 张遵真,衡正,李蕊,等. 单细胞凝胶电泳试验的最适条件研究[J].卫生毒理学杂志,1998,12(4):249
[31] 张建平,田源,陈道达.单细胞凝胶电泳制胶方法的改进[J]. 癌变畸变突变,1998,10(3):189