不同方法提取的芹菜子挥发油的GC-MS分析及其抗肝纤维化作用

GC-MS Analysis and Anti-hepatic Fibrosis Effect of Volatile Oil from Celery Seed by Different Extraction Methods

关键词:  芹菜子  提取方法  气质联用  主成分分析  肝纤维化

Key words:  celery seed  extraction methods  GC-MS  principal component analysis(PCA)  hepatic fibrosi

摘要:

目的 分析比较2种不同方法提取的挥发油对TGF-β1诱导的HSC-LX2增殖和肝纤维化指标的影响。方法 分别采用索氏提取法和水蒸气蒸馏法提取芹菜子挥发油，通过GC-MS对其挥发油成分进行分析，实验数据通过主成分分析法进行综合评价。CCK-8法检测2种方法提取的芹菜子挥发油对TGF-β1诱导的HSC-LX2增殖的影响；酶联免疫吸附法测定HSC-LX2细胞上清液中肝纤维化指标人层黏连蛋白、人透明质酸、Ⅲ型前胶原肽含量。结果 索氏提取法提取的挥发油成分共鉴定出36种，水蒸气蒸馏法为26种，其中共有成分13种。CCK-8法结果显示索氏提取法提取的芹菜子挥发油对TGF-β1诱导的HSC-LX2增殖抑制作用强于水蒸气蒸馏法。酶联免疫吸附法测得水蒸气蒸馏和索氏提取的芹菜子挥发油高剂量组细胞上清液中肝纤维化指标，与模型组相比含量显著性降低（P<0.01），而索氏提取法组肝纤维化指标更接近于阴性对照组的正常细胞。主成分分析结果显示，选用第一个作为主成分，包含原指标95.277%信息量，芹菜子挥发油中2-丁炔基-1，4-二醇基双醋酸酯、5-甲基-1-己醇、2，5-二氢-1-亚硝基吡咯、2-羟基-反式环己甲腈、2-甲基-1，6-庚二烯这几个成分对抗肝纤维化作用贡献比较大。结论 2种方法提取的芹菜子挥发油在类别和含量上存在较大差异，体外抗肝纤维化作用强弱也有所不同，为后续深入研究芹菜子挥发油成分及其抗肝纤维化机制奠定基础。

Abstract:

OBJECTIVE To analyze and compare volatile oils from celery seed by different extraction methods and their effects on proliferation of HSC-LX2 induced by TGF-β1. METHODS The volatile oils were extracted by Soxhlet extraction method and steam distillation, their volatile oils were identified by gas chromatography-mass spectrometry(GC-MS), and experimental data were comprehesively evaluated by using principal component analysis(PCA). CCK-8 assay was used to detect the effect of volatile oil from celery seed by different extraction methods on proliferation of HSC-LX2 induced by TGF-β1. Determination of laminin, hyaluronic acid and type Ⅲ procollagen peptide in hepatic fibrosis indicators of HSC-LX2 cells by enzyme-linked immunosorbent assay. RESULTS Thirty-six kinds of volatile oils extracted by Soxhlet extraction method were separated and identified, while there were twenty-six kinds of volatile oils extracted by stream distillation. There were thirteen volatile oils in common. CCK-8 method showed that the volatile oil of celery seed extracted by Soxhlet extraction method had a stronger inhibitory effect on proliferation of HSC-LX2 induced by TGF-b1 than that by steam distillation. The hepatic fibrosis indicators in the cell supernatant of high-dose volatile oil from celery seed extracted by steam distillation and Soxhlet extraction method was significantly reduced compared with the model group (P<0.01), while the hepatic fibrosis index of Soxhlet extraction method group was closer to that of the negative control group. PCA results showed that the first one was selected as the main component, containing 95.277% information of the original index. 2-butyny-1,4-diol diacetate, 5-methyl-1-hexanol, 2,5-dihydro-1-nitroso pyrrole, 2-hydroxy-trans-cyclohexanonitrile and 2-methyl-1,6-heptadiene were contributed to anti-hepatic fibrosis a lot. CONCLUSION The volatile oil extracted from celery seed by the two methods has great differences in type and content, and the anti-hepatic fibrosis effect is also different in vitro, which lays a foundation for further study on the volatile oil of celery seed and its anti-hepatic fibrosis mechanism.