The Study Of Discrimination Of Proliferation Inhibiting Ingredients Of Corydalis Soxicola Bunting Extract On HSC-T6 Based On Composition-activity Relationship

Objective:Hepatic fibrosis?HF?occurs as the consequence of asustained wound healing response of liver to toxic with long-term,which further development can lead to cirrhosis and hepatoma carcinoma.However,there is lack of effective drugs for the treatment of HF.According to the research,the total alkaloids of Corydalis saxicola Bunting with function of anti-hepatic fibrosis and liver protective activities on chronic hepatic fibrosis rats.To date,however,there were no previous studies on anti-hepatic fibrosis activity of Corydalis saxicola Bunting.Thus,in the present study,we have attempted to establish the composition-activity relationship?CAR?model based on study of chemical composition and relating inhibiting rate of the proliferation of HSC-T6and recognize the potential anti-hepatic fibrosis active compounds of Corydalis soxicola Bunting.Methods:1.To analyse the compounds from Corydalis soxicola Bunting by ultra-performance liquid chromatography quadrupole/time of flight mass spectrometry?UPLC-Q/TOF MS?.The compounds from Corydalis soxicola Bunting were identified according to the molecular ion peaks/quasi-molecular ion peaks and corresponding ion fragments,and comparing of standard reference substance and literatures.2.Nine orthogonal Corydalis soxicola Bunting extract were analyzed by HPLC,and identified the common peaks by traditional medicine fingerprint,and anti-hepatic fibrosis activity of extract was investigated by MTT assays on HSC-T6.3.The potential active components of Corydalis soxicola Bunting extract were identified by scores plot and variable importance in the projection?VIP?values by means of orthogonal partial least squares?OPLS?analysis,meanwhile,some compounds were identified by comparing standard reference substance.4.LDH kit was used to detect the effect of dehydrocavidine,palmatine and berberine on LDH activity of HL-7702.Then the activities of identified components were verified by MTT,flow cytometry?FCM?and transmission electron microscope?TEM?.Western blotting was used to detect the protein of caspase-3?bax and bcl-2.Results:1.Among these 19 compounds of extract,dehydrocavidine,palmatine,berberine and coptisine were identified by contrasting their molecular ion peaks and corresponding ion fragments combined with above standard reference substance.Then the other 15 compounds were tentatively identified by comparing ion fragment information and literatures.Subsequently,we analyzed the fragmentation pathways of the identified compounds in Corydalis soxicola Bunting extract.2.The 9 Corydalis soxicola Bunting extract were semiquantitative by HPLC,and 21 characteristic peaks were profiled.The results showed that quality differed a lot from sample to sample;the results of MTT indicated that the same amount of extracts inhibition activity on HSC-T6cells fluctuated between 90.1%and 5.3%,due to the different extraction conditions.3.The results showed that 6 peaks were significantly interrelated inhibitive activity on HSC-T6 employing the OPLS among nine orthogonal Corydalis soxicola Bunting extract,which including peaks 13,14,16,18,19and 20.Peaks 18,19 and 20 were characterized as dehydrocavidine,palmatine and berberine,respectively.4.The results of LDH showed that no obvious cytotoxic effect was found when the final concentration of dehydrocavidine,palmatine and berberine were less than 0.15,0.10 and 0.10 mg·mL^-1,respectively.The proliferation inhibiting rate of dehydrocavidine,palmatine and berberineon HSC-T6 cells in a dose-dependent manner,furthermore,drug combination showed more potent inhibitory activity than single agent on HSC-T6 cells viability.Flow cytometry detection showed that 0.10 mg·mL^-11 of dehydrocavidine,palmatine and berberine stimulated apoptosis of HSC-T6 that was obviously higher than control,and typical morphological changes of apoptosis were observed by TEM.Furthermore,Western blotting confirmed that dehydrocavidine,palmatine and berberine could induce apoptosis of HSC-T6cells which by down regulation bcl-2,up regulation of bax and caspase-3.CONCLUSION:1.In this study,we identified 19 compounds and analyzed mass spectral fragmentation pathways of them in Corydalis soxicola Bunting extract,which can effectively inhibit the proliferation of HSC-T6 cells.2.It was the first time study about anti-hepatic fibrosis active components in Corydalis soxicola Bunting by composition-activity relationship?CAR?,and found that there were 6 potential active components related to inhibit proliferation of HSC-T6,3 of them were characterized as dehydrocavidine,palmatine and berberine,respectively.At the same time,CAR provided a reference to search active components between traditional Chinese medicine and characteristic national medicine.3.0.10mg·mL^-11 of dehydrocavidine,palmatine,berberine and their combination showed obviously inhibit proliferation and induce apoptosis on HSC-T6,which related to the expression of inhibiting bcl-2,and promoting bax and caspase-3 protein.4.Interestingly,the effect of the combination showed more potent inhibitory activity than single drug on HSC-T6,which suggested the synergistic effect of multiple components may be the effective mode of promoting apoptosis of HSC-T6 cells.