Isolation,Structural Identification And Anti-hepafibrosis Activity Of The Saponins From Gynostemma Pentaphyllum

Liver Cirrhosis(LC).a common chronic progressive liver disease in clinic,has a high incidence,long course and poor prognosis,which seriously endangers human health and life safety.Hepatic Fibrosis(HF),which has histological reversibility,is a pivotal stage in the development of various chronic liver diseases to LC.It is particularly important to prevent or reverse HF for the prevention and treatment of LC.At present,the search and development of drugs for the prevention and treatment of HF is still a hotspot because no effective therapeutic drug for HF in clinic.The activation of hepatic stellate cells(HSC)and its continued secretion of extracellular matrix,especially with I type collagen protein α1(COL1A1),is the key process of liver fibrosis.Targeted therapy to inhibit the proliferation and activation of HSC and the secretion of extracellular matrix and induce its apoptosis can be used as a clinical treatment strategy for HF,and can also be used as a detection method and evaluation index to evaluate the activity of anti-hepatic fibrosis drugs.Gynostemma pentaphyllum,belonging to the genus Gynostemma Blume of the family Cucurbitaceae,is a perennial herbaceous plant which has the functions of clearing heat and detoxifying,relieving cough and resolving phlegm,nourishing qi and invigorating the spleen and tranquilizing the mind,and commonly used in the treatment of infectious hepatitis.Our previous studies have confirmed that gypenosides had high anti-fibrosis activity,and more than 70 new compounds have been isolated.Through the experiment of inhibition of HSC activation and proliferation in vitro,we screened the compound NPLC-0393,and its mechanism research demonstrated that NPLC-0393 is a protein phosphatase 2Cα(PP2Cα)agonists(EC50=6.43μM).We supposed that gypenosides have a definite inhibition of HSC activation and proliferation,showing good potential anti liver fibrosis.Based on the previous research in our group,in this paper we used silica gel column,MCI column,reversed phase prep-HPLC and other methods to isolate and purify the total saponin extract of Gynostemma pentaphylum.The effects of compounds on the cell viability of human hepatic stellate cells(LX-2)were investigated by CCK-8 assay,and their structure-activity relationships were preliminarily analyzed.And the effects on COL1A1 gene expression in LX-2 cells of compounds was analyzed by RT-PCR.1.Eighteen gypenosides(G1～G18)were isolated and purified from the total saponin extract of Gynostemma pentaphyllum,and their structures were identified by spectral analysis combined with literature comparison.Compounds G1～G4 are novel compounds,namely Gypenoside J1～J4.And compounds G5 and G6 were isolated from this plant for the first time.2.Through analysing the results of compounds influencing on LX-2 cell viability and COL1A1 gene expression,it was found that G6 could inhibit LX-2 cell viability and COL1A1 gene expression,G14 could reduce the expression of COL1A1,suggesting that both of them may have good anti-hepatic fibrosis activity,which would be worth to further research.3.The structure-activity relationships of gypenosides on LX-2 cell viability were preliminarily analyzed,and it was speculated that the difference of the sugar moiety in C-3 and C-21,the substituent in C-19 and C-26 and the configuration in C-21 and C-23 could affect the viability of LX-2 cells.