基于灰色关联度分析和聚类分析的3种滇产重楼抗肝癌作用谱效关系探索

目的:研究长柱重楼、滇重楼和南重楼的HPLC指纹图谱与其抗肝癌作用的谱效关系,为明确重楼抗肝癌作用的物质基础提供实验依据。方法:采用HPLC建立3种重楼提取物的指纹图谱,流动相乙腈(A)-水(B)梯度洗脱(0～10 min,20%A; 10～20 min,20%～25%A; 20～30 min,25%～30%A; 30～40 min,30%～35%A; 40～50 min,35%～40%A; 50～60 min,40%A; 60～75 min,40%～45%A; 75～80 min,45%～60%A),流速0. 9 m L·min~(-1),检测波长203 nm;利用噻唑蓝(MTT)比色法测定3种重楼提取物对肝癌HepG2细胞的增殖抑制作用,计算半数抑制浓度(IC_(50));运用聚类分析(HCA)和灰色关联度分析(GRA)研究3种重楼指纹图谱和抗肝癌作用的关系,找出对抗肝癌作用贡献较大的成分。结果:在3种重楼的HPLC指纹图谱中,确定其中11个色谱峰为共有峰。作用时间72 h时长柱重楼、滇重楼、南重楼的IC_(50)分别为148. 33,178. 87,208. 09 mg·L~(-1),其中长柱重楼的抗肝癌活性最强。灰色关联度结果显示,滇重楼共有峰中关联度较高的为1～10号峰,长柱重楼共有峰关联度较高的为1～7号峰,南重楼共有峰中关联度较高的为1～4,6～10,N1号峰,与IC_(50)关联度均0. 7。各重楼变量的聚类分析结果显示,可与IC_(50)聚为一类的色谱峰的关联度均 0. 7。结论:建立了3种重楼的HPLC指纹图谱,重复性良好。3种重楼中的1～4,6和7号色谱峰对抗肝癌药效贡献最大。

Spectrum-effect Relationship of Anti-hepatoma Activity from 3 Kinds of Genus Paris Based on Cluster Analysis and Grey Relational Analysis

Objective: To study the spectrum-effect relationship between HPLC fingerprints and anti-hepatoma activity of Paris polyphylla var. yunnanensis, P. forrestii and P. vietnamensis, and to elucidate its effective substance. Method: HPLC was used to establish the fingerprint of three extracts from the plant. The mobile phase was consisted of acetonitrile (A)-water (B) for gradient elution (0-10 min, 20% A; 10-20 min, 20% -25% A; 20-30 min, 25% -30% A; 30-40 min, 30% -35% A; 40-50 min, 35% -40% A; 50-60 min, 40% A; 60-75 min, 40% -45% A; 75-80 min, 45% -60% A), and the flow rate was 0.9 mL·min^-1. The UV detection wavelength was 203 nm. Thiazolyl blue tetrazolium bromide (MTT) array was used to detect the inhibitory effects of three extracts on the proliferation of HepG2 cells, and the half inhibitory concentration (IC₅₀) was calculated. Cluster analysis and grey relational analysis were used to analyze the data of spectrum and efficacy, and to find out the components that contributed a lot to the anti-liver cancer effect. Result: A total of 11 common peaks were identified as common peaks among HPLC fingerprints of three kinds of Paris. After treated 72 h, P. forrestii has the highest inhibitory effect on the HepG2 cells, the IC₅₀ of P. forrestii, P. polyphylla var. yunnanensis and P. vietnamensis were 148.33, 178.87, 208.09 mg·L^-1, respectively. According to the grey relational analysis, the common peaks 1-10 from P. polyphylla var. yunnanensis had great correlation to anti-tumor effect, and the common peaks 1-7 for P. forrestii, the common peaks 1-4, 6-10, N1 for P. vietnamensis, all the correlation degrees with IC₅₀ were >0.7.Cluster analysis of variables in each Paris showed that peaks with correlation degree >0.7 could cluster with IC₅₀. Conclusion: The established HPLC fingerprint method is reliable with good reproducibility. The peaks 1-4, 6 and 7 from three kinds of Paris have the greatest contribution to the anti-hepatoma effect.