星点设计-效应面法优化牛樟芝总三萜提取工艺及其抗肿瘤活性研究

Optimization of extracting technology of total triterpenoids in Antrodia camphorata by central composite design-response surface methodology and study on antitumor activity of total triterpenoids

目的研究牛樟芝总三萜（ACTT）提取工艺及其抗肿瘤活性。方法采用星点设计-效应面法（CCD-RSM）优化ACTT的提取工艺；通过MTT法检测其对人肝癌HepG2细胞体外增殖抑制作用；以最佳质量浓度的ACTT提取物（ACTTE）处理HepG2细胞，应用荧光显微镜进行形态学观察；应用AnnexinV凋亡检测试剂盒进行流式细胞分析，应用流式细胞仪检测细胞凋亡率。结果优化的工艺为加20倍量的90％乙醇，超声提取2次，每次40min，超声功率400W，ACTT提取率达11．87％，实际值与预测值的偏差为1．56％；不同质量浓度（12．5-200μg／mL）的ACTTE对HepG2细胞的增殖均有明显的抑制作用（P〈0．01）；直接形态显微观察及Hoechst33342荧光染色可观察到核浓缩及核碎裂等典型细胞凋亡特征及凋亡小体；流式细胞仪结果显示，给药组细胞凋亡率与空白组相比，有显著差异（P〈0．01）。结论CCD-RSM优化牛樟芝的超声醇提工艺，实际值与预测值吻合度高，预测性良好，是可行的；ACTTE体外对HepG2细胞有明显的增殖抑制及诱导凋亡的作用。

Objective To study the extracting technology ofAntrodia camphorata total triterpenoids （ACTT） and their anti-tumor activity. Methods To optimize the ACTT extraction process by central composite design of response surface methodology （CCD-RSM）. To compare the inhibition of ACTT extract at different concentration and different time on in vitro proliferation of cultured HepG2 cell using MTT assay; To observe the HepG2 cell morphology by fluorescence microscopy and flow cytometry and to detect the cell apoptosis rate by AnnexinV apoptosis assay kit. Results The optimized process was plus 20 times amount of 90% ethanol, ultrasonic extraction twice, 40 min each time, ultrasonic power of 400 W. ACTT extracting rate was 11.87%, deviation between the actual and predicted values of ACTT extraction rate was 1.56%. MTT assay showed that in 12.5-200 μg/mL, ACTT extract at different concentration （12.5--200 μg/mL） on the proliferation of HepG2 cells showed significant inhibition （P 〈 0.01）. Microscopic observation and direct form Hoechst 33342 staining were used to observe the nuclear enrichment and nuclear fragmentation and other typical features of apoptosis and apoptotic bodies; Flow cytometry showed that there was significant difference of apoptosis rate in the administration group （P 〈 0.01） compared with the control group. Conclusion Using CCD-RSM to optimize antrodia ultrasonic alcohol extraction process, the actual and predicted values are consistent with high and good predictability which is feasible. ACTT extract can significantly inhibit the proliferation and induce apoptosis on HepG2 cells in vitro.