| Isodon amethystoides(Benth)Cy Wu et Hsuan(I.amethystoids),a Chinese herbal medicine,possess a variety of pharmacological activity including antibacterial activity,anti-inflammatory and detoxification.As a member of Isodon species,I.amethystoides is notable for interesting chemical diversity of the composition.The phytochemical investigation of I.amethystoides has been extensively conducted,and variety of diterpenoids(mainly ent-kauranoids)have been isolated and characterized.In the previous researches,the most commonly used method for the extraction of terpenoids from I.amethystoids is heat reflux extraction.This traditional method has low extraction efficiency and the bio-active components could be interrupted by continuous heating.Therefore,a press-share assisted interaction technology combined with ultrasonic extraction was applied for the extraction of active components from I.amethystoids in this study.The chemical components in the extracts were separated and purified,and the anti-cancer activities of these compounds were evaluated.Furthermore,the potential mechanism for the cytotoxicity of the chemical constituents from I.amethystoids was also explored.A press-share assisted interaction(PAI)technology was applied to aqueous extraction of chemical components from I.amethystoids by using glaucocalyxinA as the quality control of the extracts.Firstly,the effects of ethanol concentration,extraction temperature,extraction time and cycles on glaucocalyxin A yield were investigated using single factor experiment;and then orthogonal experiments were designed to optimize extraction efficiency by varying the five extraction variables.Optimal extraction was obtained using 4 extraction cycles with 80% ethanol for 30 min at 55 °C.Na2CO3,NaHCO3 and ?-cyclodextrin were selected as the assistant reagents in the grinding of I.amethystoids materials,respectively.The results indicated that the highest glaucocalyxinA yield of 4.99% was observed using superfine powdered plant material with Na2CO3 content of 3.0%(w/w).Compared with soaking extraction,the glaucocalyxinA yield was improved by 1.39% and the extraction time was reduced from 24 h to 1.5 h.The chemical components in the extracts from I.amethystoids were isolated and purified by using column chromatography on silica gel,thin-layer chromatography and recrystal methods.Two compounds,glaucocalyxin A and inflexuside A,were isolated and characterized,and this was the first report of the presence of inflexuside A in I.amethystoides.The antitumor activities of the components from I.amethystoids were investigated by MTT,lactate dehydrogenase(LDH)and reactive oxygen species(ROS)assays.The three extracts(20 ?g/m L)obtained using soaking extraction,ultrasonic extraction and press-share assisted interaction(PAI)assisted ultrasonic extraction from I.amethystoides were coincubated with HepG2 cells for 24 h.The results indicated that the extract using press-share assisted extraction from I.amethystoids displayed the highest antitumor activities.Moreover,glaucocalyxin A and inflexuside A demonstrated significant antitumor activities with the IC50 of 7.09 ± 0.21 ?mol/L and 5.04 ± 0.32 ?mol/L,respectively,and the cytotoxicity of these two compounds may be explained by the overproduction of ROS induced oxidant damage.In summary,compared with traditional immersion extraction,press-share assisted interaction technology combined with ultrasonic extraction has been successfully applied to the extraction of active components from I.amethystoids.As a green and efficient extraction technology,this method could potentially be used for large-scale industrial production.In this study,inflexuside A was isolated from I.amethystoids for the first time,which would expand the class of diterpenoids and enrich the biological diversities from Isodon,and provide a possibility and theoretical basis for the continuous investigation of the potential antitumor medicine. |
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