Study On The Chemical Constituents And Bioactivities Of Ficus Hirta Vahl

Wuzhimaotao is the roots of Ficus hirta which belongs to Ficus genus of Moraceae family.It has the similar effect to Radix astragali on Qi-benefiting and deficiency reinforcing.Chemical research showed that F.hirta possessed various constituents,including phenylpropanoids,lignins,coumarins,flavonoids,triterpenes,steroids,anthraquinones,and phenolic acids.Pharmacological studies showed that F.hirta had a variety of pharmacological activities,including antioxidant and anti-aging effects,anti-inflammatory and anti-microbial effects,anti-tumor effect and improved memory function of AD rats.Up to now,the study on chemical component of F.hirta was not comprehensive and the pharmacological study was merely focused on crude extracts and limited compounds.Thus,the chemical and pharmacological study needed to be further explored.In order to better study on the bioactive material basis of F.hirta,the chemical composition,the constituents into blood,anti-AD activity,anti-neuroinflammatory activity and anti-tumor activity of F.hirta have been carried out in the article.First of all,102 compounds have been isolated from F.hirta through silica gel column chromatography,Sephadex LH-20,ODS,and preparative HPLC.The structures of all isolates,including 27 phenolic glycosides,11 flavonoids,15 coumarins,16 phenylpropionic acids and lignanss,15 amides and alkaloids,and 18 other compounds have been identified according to UV,1R,HRESIMS,NMR,CD,ECD,X-ray single crystal diffraction,and other technological methods.Compounds 1 to 11,28a/b,38a/b,59,68 and 69 are new compounds,and compound 65 is a new natural product.Secondly,the constituents into blood of F.hirta have been studied by LC-MS,and 24 compounds into blood have been detected.The structures of 12 compounds have been identified by literatures and comparison with isolated compounds.Thirdly,biological activity including anti-AD activity,anti-neuroinflammatory activity and anti-tumor activity of F.hirta have been conducted in the article.In the part of study on the anti-AD activity of isolated compounds,the western blotting showed that compound 38 can down-regulate the expression of Aβ,BACE1 and Appoptosin proteins at the concentration of 20 μM,and compounds 34 and 35 could significantly down-regulat Aβ,APP-β-CTF and Appoptosin proteins at the concentration of 40 μM.In the part of study on the anti-neuroinflammatory activity of isolated compounds,the key proteins of NF-κB(p65),MAPKs(JNK and ERK1/2)and AKT signaling pathways were tested by western blotting.As a result,compound 11 markedly inhibited the phosphorylation of NF-κB p65,JNK,ERK,and AKT,while compound 1 significantly reduced the protein levels of p-JNK,p-AKT,and partly restrained the phosphorylation of ERK,but had no obvious effects on NF-κB p65 at the concentration of 20 μM.In the part of study on the anti-tumor activity of isolated compounds,the MTT method was used for detecting the viability of isolates to tumor cells.The results showed that compounds 31 and 65 possessed significant cytotoxic activity on H460,HeLa,HepG2 and MCF-7 cells,compounds 35,36,37,43,44 and 49 had obvious cytotoxic activity on HeLa cell,compound 72 had distinct cytotoxic activity on HeLa,HepG2 and MCF-7 cells,and compound 66 had remarkable cytotoxic activity on MCF-7 cell at the concentration of 30 μM,respectively.Subsequently,Flow cytometry was applied to evaluate the apoptosis effect of isolates on tumor cells.The results showed that compound 31 induced the apoptosis of HeLa cells at the concentration of 40μM,compound 65 induced the apoptosis of of HepG2 cells at the concentration of 30 μM,and compound 72 induced the apoptosis of HeLa cells at the concentration of 30μM.Finally,the key proteins of PARP,caspase 3,JNK,ERK1/2,p38 and AKT were tested by western blotting.The result showed that compound 31 could significantly up-regulate the expression of PARP,p-p38,and p-JNK and down-regulate the expression of caspase 3,p-AKT and p-ERK in HeLa cells,compound 65 significantly increased the expression of p-p38 and p-JNK in HepG2 cells at the concentration of 30 μM,and compound 72 significantly increased the expression of cleaved-PARP,Bax,p-p38,and p-JNK proteins and inhibited the expression of p-AKT and p-ERK proteins in HeLa cells at the concentration of 30 μM.In the part of study on RXRα transcriptional activities of isolates,a dual-luciferase reporter gene assay were used for evaluating the 9-cis-RA induced RXRαtranscription.The result showed that compounds 28,29,30,31,32,33,36,38,41,44,46,49,50,56,58,62,63,98 and 99 could reverse the 9-cis-RA induced RXRαtranscription.Furthermore,the fluorescence quenching technology was conducted to test the binding capacity of compound 31 to RXRa-LBD protein,as a result,the binding constant(Kd)of compound 31 to RXRα-LBD was 3.78 μM.The antitumor effect of compound 31 related to nuclear receptor RXRa remains to be further explored.In this article,a comprehensive study on the constituent into blood,chemical components and biological activities of F.hirta has been carried out,which clarifies the efficacy material basis and provides a scientific basis for the further development and utilization of F.hirta.