Isolation,Purification And Antitumor Activity Of Antrodia Cinnamomea Triterpenoids

Antrodia cinnamomea is a rare medicinal fungus growing in Taiwan,contains various active compounds,such as polysaccharides and triterpenoids.It has many physiological activities,including anti-inflammation,anti-tumor,liver protection and immune enhancement and other physiological activities.Due to such physiological activities and outstanding medicinal value,Antrodia cinnamomea has attracted much attention.Triterpenoids,a kind of the active compounds,lead to a bitter taste of A.cinnamomea fruit body,and then they become one of the most important research and development objects of the medicinal resources of A.cinnamomea.At present,the research on triterpenoids mainly focuses on the optimization of culture medium,production technology and the composition of the fruiting body.However,there are few studies on the separation and purification and the structure-activity relationship of triterpenoids,which limit their application in industries.In order to solve this problem,based on the optimization of solid-state fermentation in our previous study,the mycelial triterpenoids were isolated and purified,and their anti-tumor activity and mechanism were studied in vitro.The main findings are as follows:1.Using 80% ethanol solution at the solid-liquid ratio of 1:60 at 80 ?,triterpenoids were extracted from solid-fermented A.cinnamomea mycelia.The extract was then treated to remove lipids using petroleum ether.And the resulting product,A.cinnamomea triterpenoids(ACTs),was obtained,and the content was determined as 15.90 ± 0.90 mg/g by vanillin and perchloric acid detection method.2.The components of ACTs were separated by Silica gel column chromatography.After optimization,the eluent was determined to be the mixture of isopropanol and petroleum ether(volume ratio 1:4)and distilled water,and the optimal diameter height ratio was 1:8.According to vanillin-perchloric acid method,two components were collected and termed as ACTs-a and ACTs-b,respectively.3.Subsequent purification of ACTs,ACTs-a and ACTs-b was performed by HPLC.The results showed that the HPLC profile of ACTs have most quantities of peaks,followed by those of ACTs-a and ACTs-b.Three obvious peaks were found in ACTs-a profile at 12,14 and 15.5min respectively.The substances with peaks at 12 and 15.5min were unstable,therefore,the peak appearing at the 14 min was collected and purified repeatedly by HPLC,and the resulting product was termed as ACTs-c.4.The composition of ACTs-c was detected by LC-MS,and then analyzed by database comparison.The results showed that ACTs-c was mainly composed of limonin derivative,and contains trace flavonoids,lipids,sugars and other impurities.5.The activities of ACTs,ACTs-a,ACTs-b and ACTs-c were analyzed and compared by in vitro antitumor test.The results showed that ACTs,ACTs-a and ACTs-c could significantly inhibit the proliferation of He La cells,with the maximum inhibition rates of 84.37%,93.51% and 97.00%,respectively.IC50(half inhibitory concentration)values were 492.29 ?g/m L,17.81 ?g/m L,35.91 ?g/m L,respectively.6.On the basis of the above,the antitumor mechanism was further studied.He La cells were treated with annexinv-fitc /PI double staining,and cell cycle arrest was detected by flow cytometry.The results showed that the DNA replication process of He La cells treated with ACTs was blocked in G2/M phase,and the proportion of cells staying in S phase decreased.After treatment with ACTs-c,the DNA replication process of He La cells was blocked in S phase,and the proportion of cells staying in G1 phase decreased.Western Blot assay indicated that the expression level of Cyclin B1 in He La cells was significantly down-regulated,while the expression level of CDK1 protein remained unchanged.This suggests that G0/G1 phase arrest is associated with downregulation of the Cyclin B1/CDK1 complex.Compared with the control group,the expression of bcl-2 protein in He La cells treated with ACTs-c was down-regulated,and the expression of Bax and P53 proteins was up-regulated,which showed a concentration-dependent effect.In conclusion,a compound,ACTs-c,was obtained and purified from from A.cinnamomea mycelial tripernoids,and showed strong anti-proliferation activity against tumor cells through cell cycle arrest and apoptosis.The results indicate that it has the potential to develop an anti-tumor agent or health food.In further study on its structural characterization and in vivo activities will be conducted to promote its application.