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OBJECTIVE：To optimize ultrasonic-assisted ethanol-（NH4）2SO4 aqueous two-phase extraction technology of citru- sinol from Desmodium caudatum . METHODS ：Using the content of citrusinol as indexes ，with ethanol volume fraction ， solid-liquid ratio ，（NH4）2SO4 addition amount ，ultrasonic time and ultrasonic temperature as factors ，based on the single factor tests，Box-Behnken design-response surface methodology was used to optimize the extraction technology of citrusinol. RESULTS ： The optimized extraction technology of citrusinol included that ethanol volume fraction was 95.35％，the solid-liquid ratio was 1∶50.35 （g/mL），（NH4）2SO4 addition amount was 4.49 g，ultrasonic time was 48.7 min，ultrasonic temperature was 57.6 ℃. In 3 times of validation tests ，the extraction rates of citrusinol were 0.637 8，0.638 4，0.625 4 mg/g，respectively，which was close to predicted value（0.630 5 mg/g）. CONCLUSIONS ：The optimized ultrasonic-assisted ethanol- （NH4）2SO4 aqueous two-phase extraction technology is stable and feasible ，and can be used for the extraction of citrusinol from D. caudatum .
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OBJECTIVE：To study the effects of citrusinol on proliferation ，migration and the expression of skeleton-related proteins of human hepatocellular cells HepG 2，and to investigate its interaction mode with skeleton-related proteins. METHODS ： CCK-8 assay was used to detect the effects of different concentrations （12.5，25，50，100，200 μmol/L）of citrusinol on the proliferation of HepG 2 cells for 24 h. HepG 2 cells were divided into negative control group ，citrusinol low-concentration and high-concentration groups （50，100 μmol/L citrusinol）. After treated for 24 h，the migration of HepG 2 cells was detected by cell scratch test ；cell migration rate was calculated. mRNA and protein expression of F-actin ，β-tubulin and Ezrin in HepG 2 cells were determined by RT-PCR and Western blotting assay. Molecular docking software Schrodinger 2015 was used to analyze the interaction mode between citrusinol and above 3 kinds of proteins. RESULTS ：Citrusinol showed significant inhibition effect on the proliferation of HepG 2 cells （P＜0.05 or P＜0.01），in dose-dependent trend. Compared with negative control group ，cell migration， mRNA and protein expression levels of F-actin ， β-tubulin， Ezrin were decreased significantly in citrusinol low-concentration and high-concentration groups （P＜0.05 or P＜0.01）. Molecular docking results showed that the citrusinol could form hydrogen bond and hydrophobic bond with the above 3 skeleton-related proteins. CONCLUSIONS ：Citrusinol can inhibit the proliferation and migration of HepG 2 cells，the mechanism may be associated with the down-regulation of mRNA and protein expression of F-actin ，β-tubulin and Ezrin. The mode of its interaction with skeleton-related proteins may be the formation of hydrogen bond or hydrophobic bond.
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Objective To study the inhibitory effect and mechanism of citrusinol on proliferation of human hepatocellular carcinoma cells HepG2.Methods The inhibitory rate of HepG2 cells cultured in vitro was measured by MTT assay.The morphology and distribution of the ceils were observed by HE and acridine orange staining.The cell division cycle was detected by flow cytometry.The expression of F-actin protein was observed by fluorescent chromogenic method.Results Citrusinol could inhibit the growth of HepG2 cells,and the IC50 of the inhibitory rate was 76.46 μmol/L.Citrusinol could make the HepG2 cells shrink,arrest the cell division cycle to G2/M,and inhibit the expression of F-actin.Conclusion Citrusinol can prevent cell proliferation by arresting cell division cycle in G2/M phase and inhibiting the formation of cytoskeleton,thus inhibiting the growth of G2/M.