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.
引文
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