ABSTRACT
OBJECTIVE：To study the spectrum-ef fect relationship of anti-hepatoma effect of C Ⅱ-3 extract from Periplaneta americana，and to preliminarily clarify the anti-hepatoma active components of it. METHODS ：Based on UHPLC fingerprints of 10 batches of C Ⅱ-3 samples，with the help of UHPL C-Q-TOF/MS，the compounds corresponding to each chromatographic peak were identified qualitatively by standard substances and related literatures. Using the IC 50 value of each batch of C Ⅱ-3 sample against human hepatoma cells HepG 2 as anti-hepatoma activity index ，the spectrum-effect relationship of fingerprint and anti-hepatoma effect was established and analyzed by the combination of grey relational analysis （GRA）and orthogonal partial least squares（OPLS）. RESULTS ：There were 25 common peaks in UHPLC fingerprints of 10 batches of C Ⅱ-3 samples，and 10 chemical compounds were identified ，which were cyclo- （Tyr-Pro）（peak 24），cyclo-（Gly-Phe）（peak 15），hypoxanthine（peak 3）， adenine（peak 7），phenylalanine（peak 10），inosine（peak 11），N-acetyldopamine（peak 16），cyclo-（Pro-Ala）（peak 13），2-hydroxy propionyl（peak 22），cyclo-（Pro-Ser）（peak 6）. The IC 50 of 10 batches of C Ⅱ-3 samples to HepG 2 cells was 70.550-200.303 μg/mL. Among 25 common peaks ，the order of GRA correlation （r）of anti-hepatoma activity was peak 20＞23＞24＞15，all of r values were greater than 0.7；the order of variable importance projection （VIP）of OPLS analysis was peak 23＞18＞15＞24＞7＞14＞6＞ 2＞20，all of VIP values were greater than 1. The standard regression coefficients of peak 7，15，20，23，24 were all greater than 0；while the standard regression coefficients of peak 2，6，14，18 were all less than 0. Conjoint analysis shows that the order of anti-hepatoma activity was peak 20＞23＞24＞15. CONCLUSIONS：unknown chemical ingredients （peak 20， ），cyclo-（Tyr-Pro）（peak 24）and cyclo- （Gly-Phe）（peak 15） in C Ⅱ-3 may be main anti-hepatoma active components.
ABSTRACT
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.