Chemical Constituents Of Scutellaria Barbata And Their Antitumor Mechanism

Scutellaria barbata D.Don is one of the commonly used heat clearing and detoxifying drugs in clinic,and it is often combined with other drugs for tumor treatment.In recent years,a series of novel neo-clerodane diterpenoids have been found showing strong antitumor effect,and being one of the research focuses.The structure-activity relationship and mechanism of antitumor activity of neo-clerodane diterpenoids are not clear,and the synergistic relationship between neo-clerodane diterpenoids and flavonoids in antitumor activity should be further explored.Objective:To isolate a series of flavonoids and neo-clerodane diterpenoids from Scutellaria barbata,and study the structure-activity relationship and mechanism of neo-clerodane diterpenoids in vitro;based on the theory of group distribution,to explore the synergistic relationship between flavonoids and diterpenoids in anti-tumor effect.Methods:1.The acetone extract of Scutellaria barbata was systematically separated by solvent extraction;the extract was separated and purified by silica gel column chromatography,liquid phase preparation and recrystallization;the structures of the compounds were identified by NMR,MS,UV-IR and their physicochemical properties;2.MTT method was used to evalute the cytotoxic activity of neo-clerodane diterpenoids with different structures to different human cancer cell lines LoVo,HCT-116(human colorectal cancer),MCF-7(breast cancer),SMMC-7721(liver cancer)and A549(lung cancer)in vitro,and the structure-activity relationship of these compounds in vitro was preliminarily analyzed by comparing their IC5o values;3.Human colorectal cancer LoVo cells were treated with neo-clerodane diterpenoid scutebata A.The antitumor activity of neo-clerodane diterpenoid in vitro was further studied by plate cloning,cell scratch healing,Transwell cell migration and invasion assay.The influence of drugs on LoVo cell cycle was detected by flow cytometry,Hoechst 33342 fluorescence staining.The apoptosis of Lovo cells was detected by caspase 3,8,9 kits,and the cell cycle,apoptosis related protein cyclin A,Bax and Bcl-2 were detected by Western blot;4.Network pharmacology method was used to predict the anti-tumor and anti-inflamlatory targets of Scutellaria barbata flavonoids and diterpenoids.The component target network was constructed by Cytoscape 3.6.1 software,and the combined targets were screened out.Autodock 4.2 molecular docking software was used to simulate the binding ability of compounds and target proteins.5.MTT method was used to study the effect of different ratio of diterpenoid scutebata A and flavone apigenin on the cytotoxic activity of LoVo,and the interaction relationship between them was judged by calculating the synergistic index of the two;Western blot was used to further study the effect of the two compounds on the expression level of GSTP1 and CBR1 protein.Results:Using 1H-NMR,13C-NMR,HSQC,HMBC,HRES1-MS and IR analyses methods combined with their physical and chemical properties to identify the structure of the isolated compounds,finally 49 compounds were isolated from the acetone extract of Scutellaria barbata,including 12 flavonoids and 29 neo-clerodane diterpenoids(including 4 new compounds).The study of structure-activity relationship showed that the neo-clerodane diterpenoids were less sensitive to lung cancer A549 cells,while senstiive to human colorectal cancer LoVo cells.Moreovrer,the toxicity of the compounds is related to the type of C-1,C-6 and C-7 substituents and the prencence of double bond of C-11/C-12 in the structure.In vitro,scutebata A can inhibit the scratch healing and plate clone formation of Lovo cells.Transwell experiment showed that it can inhibit the migration and invasion of Lovo cells.Scutebata A can also induce S-phase arrest and apoptosis of Lovo cells.The results of western blotting showed that it could regulate cyclin A,Bax,Bcl-2 and caspase-3 activity,and two high binding targets of GSTP and CBR1 ware screened by network pharmacology combined with molecular docking technology.The combination of scutebata A and apigenin can down regulate GSTP1 and up regulate CBR1 protein expression.Conclusion:The antitumor activities of the neo-clerodane diterpenoids with different structures in Scutellaria barbata are remarkable in vitro,and they have certain selectivity to different tumor cells,which is mainly related to the type of oxygen-containing substituents at positions C-1,C-6 and C-7 in the structure.These diterpenoids can induce S-phase arrest of LoVo cell cycle,up regulate Bax/Bc1-2 and activate caspase 3 in vitro by down regulating cyclin A.In vitro,flavonoids and diterpenes may play a synergistic role in antitumor activity by regulating the levels of GSTP1 and CBR1 protein.