柳蒿化学成分的含量测定及肝损伤保护作用的研究

Determination the Constituents of Artemisia integrifolia Linn.and Study the Protective Effect of Liver Injury

目的建立HPLC同时测定柳蒿(Artemisia integrifolia Linn.)中绿原酸、芦丁、木犀草苷、槲皮素含量的方法,并研究柳蒿提取液对肝脏的保护作用,探讨其可能机理。方法采用高效液相色谱法。色谱柱为Inertsil ODS-3柱,流动相为乙腈-0.2%磷酸水溶液(梯度洗脱),流速为1.0 mL·min-1,检测波长270 nm,柱温25℃,进样量为10μL;60只雄性小鼠随机分为6组,每组10只,除正常组外,其余各组均腹腔注射0.1%的CCl4橄榄油溶液致小鼠急性肝损伤,测定小鼠血清中谷丙转氨酶(ALT)和谷草转氨酶(AST)活力;肝组织中丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)、总抗氧化能力(T-AOC)和总超氧化物歧化酶(T-SOD)。结果绿原酸、芦丁、木犀草苷、槲皮素检测进样量线性范围分别为1.015 2~16.243 2μg(r=0.999 9),0.257 1~4.113 6μg(r=0.999 9),0.081 6~1.305 6μg(r=0.999 8),0.027 3~0.436 8μg(r=0.999 9);精密度、稳定性、重复性试验的RSD<2.0%;加样回收率分别为98.73%~100.42%(RSD=0.52%,n=5),95.64%~102.07%(RSD=1.52%,n=5),94.05%~99.79%(RSD=1.73%,n=5),96.22%~101.89%(RSD=2.45%,n=5)。柳蒿提取液可明显抑制CCl4致小鼠肝损伤后血清中ALT和AST升高,肝组织中MDA含量升高,提高肝组织中T-SOD、GSH-PX、T-AOC活性。结论建立的HPLC方法,简便、精确、灵敏度高,可用于柳蒿药材中4种成分含量的同时测定。柳蒿对CCl4所致小鼠急性肝损伤具有保护作用。该结果可为今后柳蒿的开发利用和深入研究提供一定的参考。

Objective To establish a method for determination of chlorogenic acid,rutin,luteoloside and quercetin in Artemisia integrifolia Linn.by HPLC,investigate the protective effect of liver injury and discuss the possible mechanism.Methods HPLC method was adopted.The determination was performed on Inertsil ODS-3 column with mobile phase consisted of acetonitrile-0.2% phosphoric acid solution(gradient elution) at a flow rate of 1.0 mL·min-1.The detection wavelength was set at 270 nm.The column temperature was 25℃,and sample size was 10 μL;60 male mice were separated into six groups and each group had ten mice except for the normal group,all groups were injected intraperitoneally with0.1% CC14 olive oil solution to induce acute liver injury.The activity of Alanine aminotransferase(ALT)and aspartate aminotransferase(AST) in serum were determined.MDA,Glutathione peroxidase(GSH-Px),Total antioxidant capacity(T-AOC) and total superoxide dismutase(T-SOD) in liver tissue were also determined.Results It showed that the linear ranges of chlorogenic acid,rutin,luteoloside and quercetin were 1.015 2-16.243 2 μg(r=0.999 9),0.257 1-4.113 6 μg(r=0.999 9),0.081 6-1.305 6 μg(r=0.9998),0.027 3~0.436 8 μg(r=0.999 9),respectively.RSDs of precision,stability and reproducibility tests were all lower than 2.0%.The recoveries were 98.73%~100.42%(RSD=0.52%,n=5),95.64%~102.07%(RSD=1.52%,n=5),94.05%~99.79%(RSD=1.73%,n=5),96.22%~101.89%(RSD=2.45%,n=5),respectively;Total Flavonoids of Artemisia integrifolia L.could significantly decreased the content of ALT and AST in the serum with liver injury induced by CCl4,moreover,Artemisia integrifolia L.could inhibited the increase of MDA and decrease the activity of T-SOD,GSH-Px and T-AOC in liver tissue.Conclusion The method is simple,accurate and sensitive,which can be used for simultaneous determination of four constituents in Artemisia integrifolia Linn..Meanwhile,Artemisia integrifolia Linn.have protective effects on acute liver injury induced by CC14 in mice.It provided certain reference significance for development and further study of the resources in Artemisia integrifolia Linn..