1.Separation and Purification of Alhagi sparsifolia n -butanol Extract Monomeric Compounds and Study on Their Effects on Human Cervical Cancer HeLa Cells
Xuesong LIU ; Xiaoling MA ; Leiling SHI ; ALETENGTUYA ; Dapeng ZHANG ; Ning LI ; Hongyan WEI
China Pharmacy 2019;30(4):506-512
OBJECTIVE: To separate and purify Alhagi sparsifolia n-butanol extract monomeric compounds, and to investigate its effects on the proliferation and metastasis of human cervical cancer HeLa cells. METHODS: The n-butanol extract was separated and purified by silica gel column, Sephadex LH-20 gel column and prep-HPLC. The structures of compounds were analyzed and identified according to physicochemical properties and spectrum (mass spectrum, hydrogen spectrum, carbon spectrum) data. Using human cervical cancer HeLa cells as objects, 5-FU as positive control, MTT assay was used to detect the inhibitory rate of HeLa cells pretreated with different doses of compounds (6.25, 12.5, 25, 50, 100, 200 μg/mL); IC50 was calculated to screen active monomers. Scratch test was used to investigate the effects of above active monomers (all 50 μg/mL) on the migration ability of HeLa cells. Kim’s formula was used to evaluate the effects of 5-FU separately combined with above active monomers [(3.125+6.25),(6.25+12.5),(12.5+25),(25+50)μg/mL]. RESULTS: Six compounds were isolated from the n-butanol extract part of A. sparsifolia and identified as butin (Ⅰ), 3′,4′,7-trihydroxyisoflavone (Ⅱ), p-methoxyphenylacetic acid (Ⅲ), 4-hydroxyacetophenone (Ⅳ), aurantiamide acetate (Ⅴ), protocatechualdehydea (Ⅵ). Compared with blank control group, 5-FU and each compound (5-FU:6.25-200 μg/mL, compound Ⅰ: 12.5-200 μg/mL; compound Ⅱ: 25, 50, 200 μg/mL; compound Ⅲ: 6.25, 100, 200 μg/mL; compound Ⅳ: 50, 100, 200 μg/mL; compound Ⅴ: 12.5, 25, 200 μg/mL; compound Ⅵ: 6.25-200 μg/mL) could significantly increase the cell inhibition rate. IC50 of compound Ⅰ, Ⅴ, Ⅵ were decreased significantly (P<0.05 or P<0.01), and those of compound Ⅰ and Ⅵ were lower relatively. The migration distance of cells in 5-FU and compound Ⅰ and Ⅵgroups were decreased significantly, compared with blank control group (P<0.05 or P<0.01). 5-FU separately combined with compound Ⅰ and Ⅵ showed additive and enhanced inhibitory effects on the proliferation of HeLa cells (synergistic index>0.9). CONCLUSIONS: Compounds Ⅰ-Ⅵ are isolated from Alhagi for the first time. Butin and protocatechualdehydea are active monomers of its n-butanol extract part. Above two monomers can inhibit the proliferation and migration of human cervical cancer Hela cells, with strong inhibitory effect in vitro, and stronger inhibitory effect combined with 5-FU than any compound alone.