栀子苷、京尼平、栀子蓝的体内外肝毒性对比研究
|
摘要
目的:通过动物与细胞实验,比较栀子苷与京尼平、栀子蓝体内外肝毒性,进一步明确中药栀子致肝毒性的物质基础,为栀子临床安全应用提供科学依据。方法:(1)体内实验部分:采用动物实验方法测定大鼠血清中丙氨酸氨基转移酶(ALT),天门冬氨酸氨基转移酶(AST),碱性磷酸酶(ALP),总胆红素(TBIL);(2)体外实验部分:采用Cell Counting Kit-8(CCK-8)法检测不同浓度栀子苷、京尼平及栀子蓝的细胞毒作用;试剂盒法测定细胞上清液中ALT,AST的活力。结果:(1)与空白组比较,栀子苷灌胃给药0.4、1.2 g·kg~(-1) 24 h后都表现为ALT、AST、TBIL值明显升高(P<0.05,P<0.01),腹腔注射0.4、1.2 g·kg~(-1) 24 h后生化指标未表现出异常。京尼平腹腔注射0.1 g·kg~(-1) 24 h后AST、TBIL明显升高(P<0.05,P<0.01),0.2、0.4 g·kg~(-1)组全部死亡,京尼平灌胃0.2、0.4 g·kg~(-1)组ALT、AST、TBIL肝功能指标不同程度升高(P<0.05,P<0.01)。栀子蓝腹腔注射1.5 g·kg~(-1) TBIL明显升高(P<0.05),栀子蓝灌胃6 g·kg~(-1) ALP、ALT、AST、TBIL均无明显变化。(2)细胞毒性实验结果显示,与阴性对照组相比,栀子苷给药组800、1000μg·mL~(-1)抑制人体肝细胞L-O2细胞的增殖(P<0.01,P<0.05),同时ALT测定结果显示,800、1000μg·mL~(-1)可以显著提高肝细胞培养上清液中ALT的活力(P<0.01,P<0.05)。栀子蓝给药组200、600、800、1000μg·mL~(-1)可抑制人体肝细胞L-O2细胞的增殖(P<0.01),ALT测定结果显示800、1000μg·mL~(-1)可显著增加ALT活力(P<0.01)。京尼平给药组50、100、150、200、250、3000μg·mL~(-1)具有显著的细胞毒作用(P<0.01),ALT测定结果显示100、150μg·mL~(-1)明显提高肝细胞培养上清液中ALT活力(P<0.05)。结论:京尼平具有明确的体内外肝毒性,推测京尼平可能是栀子苷在体内的毒性物质基础。
Objective: To compare the geniposide, genipin, gardenia blue hepatotoxicity in vivo and vitro through animal and cell experiment and further clarify the toxicity material basis of gardenia,providing scientific a basis for the clinical use of gardenia. Methods:(1) In vivo: Animal experimental method was used for determining the serum alanine aminotransferase(ALT), aspartate aminotransferase(AST) and alkaline phosphatase(ALP), total bilirubin(TBIL).(2)In vitro experiment: Cell counting kit-8(cck-8) method was used to detect the cytotoxic effects of different concentrations of gardenoside, genipin and gardenia blue.The activities of ALT and AST were determined by kit method.Results:(1)Compared with the normal group, the biochemical indexes of gardenoside intragastric 0.4 g·kg~(-1),1.2 g·kg~(-1) for 24 h and the levels of ALT, AST and TBIL increased obviously(P<0.05, P<0.01). The gardenoside intraperitoneal injection with 0.4 g·kg~(-1),1.2 g·kg~(-1) showed no abnormality after 24 h.Genipin intraperitoneal injection with 0.1 g·kg~(-1),the levels of AST and TBIL increased obviously(P<0.05, P<0.01).After the death of all,genipin intragastric injection with 0.2 g·kg~(-1) and 0.4 g·kg~(-1) groups' ALT, AST and TBIL increased(P<0.05, P<0.01). After gardenia blue intraperitoneal injection with 1.5 g·kg~(-1),the level of TBIL significantly increased(P<0.05). The results in the gardenia blue peritoneal injection 6 g·kg~(-1) group had no significant change.(2)Compared with negative control group. The cytotoxicity experiment results showedgardenoside dosage groups(including 800 μg·mL~(-1), 1000 μg·mL~(-1)) inhibited the cell proliferation of L-02(P<0.01, P<0.05). The level of ALT increased(P<0.01, P<0.05).The proliferation of L-02 cells(P<0.01) was inhibited by gardenia blue group of 200, 600, 800, 1000 μg·mL~(-1). The results showed that 800 and 1000 μg·mL~(-1) could significantly increase ALT activity(P<0.01).Genipin group of 50, 100, 150, 200, 250, and 3000 μg·mL~(-1) has a significant cytotoxic effect(P<0.01). The determination of ALT results showed that ALT activity was improved obviously at the dose of 100 μg·mL~(-1) and 150 μg·mL~(-1)(P<0.05).Conclusion:Genipin has a clear hepatocyte toxicity invivoandvitro, indicating that it may be the basis of the toxic substance of gardenoside in vivo.
Objective: To compare the geniposide, genipin, gardenia blue hepatotoxicity in vivo and vitro through animal and cell experiment and further clarify the toxicity material basis of gardenia,providing scientific a basis for the clinical use of gardenia. Methods:(1) In vivo: Animal experimental method was used for determining the serum alanine aminotransferase(ALT), aspartate aminotransferase(AST) and alkaline phosphatase(ALP), total bilirubin(TBIL).(2)In vitro experiment: Cell counting kit-8(cck-8) method was used to detect the cytotoxic effects of different concentrations of gardenoside, genipin and gardenia blue.The activities of ALT and AST were determined by kit method.Results:(1)Compared with the normal group, the biochemical indexes of gardenoside intragastric 0.4 g·kg~(-1),1.2 g·kg~(-1) for 24 h and the levels of ALT, AST and TBIL increased obviously(P<0.05, P<0.01). The gardenoside intraperitoneal injection with 0.4 g·kg~(-1),1.2 g·kg~(-1) showed no abnormality after 24 h.Genipin intraperitoneal injection with 0.1 g·kg~(-1),the levels of AST and TBIL increased obviously(P<0.05, P<0.01).After the death of all,genipin intragastric injection with 0.2 g·kg~(-1) and 0.4 g·kg~(-1) groups' ALT, AST and TBIL increased(P<0.05, P<0.01). After gardenia blue intraperitoneal injection with 1.5 g·kg~(-1),the level of TBIL significantly increased(P<0.05). The results in the gardenia blue peritoneal injection 6 g·kg~(-1) group had no significant change.(2)Compared with negative control group. The cytotoxicity experiment results showedgardenoside dosage groups(including 800 μg·mL~(-1), 1000 μg·mL~(-1)) inhibited the cell proliferation of L-02(P<0.01, P<0.05). The level of ALT increased(P<0.01, P<0.05).The proliferation of L-02 cells(P<0.01) was inhibited by gardenia blue group of 200, 600, 800, 1000 μg·mL~(-1). The results showed that 800 and 1000 μg·mL~(-1) could significantly increase ALT activity(P<0.01).Genipin group of 50, 100, 150, 200, 250, and 3000 μg·mL~(-1) has a significant cytotoxic effect(P<0.01). The determination of ALT results showed that ALT activity was improved obviously at the dose of 100 μg·mL~(-1) and 150 μg·mL~(-1)(P<0.05).Conclusion:Genipin has a clear hepatocyte toxicity invivoandvitro, indicating that it may be the basis of the toxic substance of gardenoside in vivo.
引文
[1] 杨洪军,付梅红,吴子伦,等.栀子对大鼠肝毒性的实验研究[J].中国中药杂志,2006,31(13):1091-1093.
[2] 王波,杨洪军,高双荣,等.栀子对大鼠肝肾毒性的病理学观察[J].中国实验方剂学杂志,2007,13(5):45-48.
[3] 张海燕,邬伟魁,李芳,等.栀子保肝利胆作用及其肝毒性研究[J].中国中药杂志,2011,36(19):2610-2614.
[4] Ding Y, Zhang T, Tao J S, et al. Potential hepatotoxicity of geniposide, the major iridoid glycoside in dried ripe fruits of Gardenia jasminoides (Zhi-zi)[J]. Natural Product Research, 2013,27(10):929-933.
[5] 谢志忻,钟云,黄丽萍,等.栀子的降脂作用与安全性评价研究[J].中药药理与临床,2014,30(5):105-108.
[6] 胡燕珍,罗光明,卫军营.栀子中京尼平苷对肝脏的保护与过量毒性[J].中国现代中药,2015,17(10):1113-1116.
[7] 程生辉,张妍妍,李会芳,等.基于黄疸模型大鼠的栀子苷急性肝肾毒性研究[J].中国实验方剂学杂志,2015,21(4):174-178.
[8] 杨全军,范明松,孙兆林,等.栀子化学成分、药理作用及体内过程研究进展[J].中国现代中药,2010,12(9):7-12.
[9] 张燕,朱华旭,郭立玮.栀子中环烯醚萜类化合物的体内过程及其对相关酶的影响[J].中国中药杂志,2012,37(3):269-273.
[10] 杨全军,范明松,孙兆林,等.栀子化学成分、药理作用及体内过程研究进展[J].中国现代中药,2010,12(9):7-12.
[11] 王智勇,张海燕,杨明,等.京尼平苷四种不同给药途径的肝毒性初步研究[J].现代生物医学进展,2013(5):824-827.
[12] 王清然,邓中平.栀子肝脏毒性研究[J].中成药,2016,38(6):1351-1354.
[13] 程生辉,唐超,李会芳,等.栀子苷对正常大鼠急性肝、肾毒性的时-毒关系分析[J].中国实验方剂学杂志,2016,22(1):162-165.
[14] 林庆勋,徐列明.栀子水提液对小鼠的肝毒性及健脾保肝方的预防作用[J].上海中医药大学学报,2009(6):59-63.
[15] 丁越,张彤,项乐源,等.栀子苷致大鼠肝毒性时-量关系及其机理研究[C].中华中医药学会药用植物化学与中药资源可持续发展学术研讨会,2009.
[16] 任艳青,田宇柔,李琛,等.京尼平苷及其体内代谢产物京尼平对HepG2细胞毒性的比较及机制研究[J].中国药理学通报,2016,32(12):1755-1761.
[17] Gao LN, Zhang Y, Cui YL, et al. Evaluation of genipin on human cytochrome P450 isoenzymes and P-glycoprotein in vitro[J]. Fitoterapia, 2014, 98:130-136.
[18] 王坤,金若敏,陈长勋.栀子与黄连解毒汤肝毒性的比较研究[J].中国中药杂志,2013,38(14):2365-2369.
[19] Watanabe T, Jono H, Han J, et al. Synergistic Activation of NF-κB by Nontypeable Haemophilus influenzae and Tumor Necrosis Factor α[J]. Proceedings of the National Academy of Sciences of the United States of America, 2004, 101(10):3563.
[20] Nagaki M, Iwai H, Naiki T, et al. High levels of serum interleukin-10 and tumor necrosis factor-alpha are associated with fatality in fulminant hepatitis[J]. Journal of Infectious Diseases, 2000, 182(4):1103-1108.
[21] Wang JY, Liu P. Abnormal immunitive and gene mutation in pa-tientswith severe hepatitis-B[J]. Gastroenterology, 2003,9(9): 2009.
[2] 王波,杨洪军,高双荣,等.栀子对大鼠肝肾毒性的病理学观察[J].中国实验方剂学杂志,2007,13(5):45-48.
[3] 张海燕,邬伟魁,李芳,等.栀子保肝利胆作用及其肝毒性研究[J].中国中药杂志,2011,36(19):2610-2614.
[4] Ding Y, Zhang T, Tao J S, et al. Potential hepatotoxicity of geniposide, the major iridoid glycoside in dried ripe fruits of Gardenia jasminoides (Zhi-zi)[J]. Natural Product Research, 2013,27(10):929-933.
[5] 谢志忻,钟云,黄丽萍,等.栀子的降脂作用与安全性评价研究[J].中药药理与临床,2014,30(5):105-108.
[6] 胡燕珍,罗光明,卫军营.栀子中京尼平苷对肝脏的保护与过量毒性[J].中国现代中药,2015,17(10):1113-1116.
[7] 程生辉,张妍妍,李会芳,等.基于黄疸模型大鼠的栀子苷急性肝肾毒性研究[J].中国实验方剂学杂志,2015,21(4):174-178.
[8] 杨全军,范明松,孙兆林,等.栀子化学成分、药理作用及体内过程研究进展[J].中国现代中药,2010,12(9):7-12.
[9] 张燕,朱华旭,郭立玮.栀子中环烯醚萜类化合物的体内过程及其对相关酶的影响[J].中国中药杂志,2012,37(3):269-273.
[10] 杨全军,范明松,孙兆林,等.栀子化学成分、药理作用及体内过程研究进展[J].中国现代中药,2010,12(9):7-12.
[11] 王智勇,张海燕,杨明,等.京尼平苷四种不同给药途径的肝毒性初步研究[J].现代生物医学进展,2013(5):824-827.
[12] 王清然,邓中平.栀子肝脏毒性研究[J].中成药,2016,38(6):1351-1354.
[13] 程生辉,唐超,李会芳,等.栀子苷对正常大鼠急性肝、肾毒性的时-毒关系分析[J].中国实验方剂学杂志,2016,22(1):162-165.
[14] 林庆勋,徐列明.栀子水提液对小鼠的肝毒性及健脾保肝方的预防作用[J].上海中医药大学学报,2009(6):59-63.
[15] 丁越,张彤,项乐源,等.栀子苷致大鼠肝毒性时-量关系及其机理研究[C].中华中医药学会药用植物化学与中药资源可持续发展学术研讨会,2009.
[16] 任艳青,田宇柔,李琛,等.京尼平苷及其体内代谢产物京尼平对HepG2细胞毒性的比较及机制研究[J].中国药理学通报,2016,32(12):1755-1761.
[17] Gao LN, Zhang Y, Cui YL, et al. Evaluation of genipin on human cytochrome P450 isoenzymes and P-glycoprotein in vitro[J]. Fitoterapia, 2014, 98:130-136.
[18] 王坤,金若敏,陈长勋.栀子与黄连解毒汤肝毒性的比较研究[J].中国中药杂志,2013,38(14):2365-2369.
[19] Watanabe T, Jono H, Han J, et al. Synergistic Activation of NF-κB by Nontypeable Haemophilus influenzae and Tumor Necrosis Factor α[J]. Proceedings of the National Academy of Sciences of the United States of America, 2004, 101(10):3563.
[20] Nagaki M, Iwai H, Naiki T, et al. High levels of serum interleukin-10 and tumor necrosis factor-alpha are associated with fatality in fulminant hepatitis[J]. Journal of Infectious Diseases, 2000, 182(4):1103-1108.
[21] Wang JY, Liu P. Abnormal immunitive and gene mutation in pa-tientswith severe hepatitis-B[J]. Gastroenterology, 2003,9(9): 2009.