Preparation Of Wogonin From Radix Scutellariae And Its Inhibitory Effects On The Growth Of Tumor Cells

Radix Scutellariae is the dry root of the medical plant Scutellaria baicalensis Georgi and has been used widely in traditional Chinese medicine to treat a variety of diseases. It has anti-oxidation, anti-inflammatory,neuroprotective,anti-tumor and antiviral activity. Baicalein, Wogonin, oroxylin A and baicalin are the main bioactive components of radix Scutellariae. Especially, Wogonin has been shown to exert cytostatic and proapoptotic effects on several tumor cell lines. It is a very promising and potential antitumor drug with mutiple targets.The present study was aimed to develop an efficient method for preparative separation of high-purity Wogonin from Radix Scutellariae and its inhibitory effects on the growth of HepG2cells.In the present study, we used endogenous baicalinase-catalysed hydrolysis to transform wogonoside into Wogonin to increase the yield of Wogonin. Radix Scutellariae was hydrolyzed in water (pH5) for12h and then extracted with95%ethanol which make Wogonin dissolve out more completely and obtain higher content crude extract. The crude extract was purified with polyamide resin (30-60mesh), which is suitable for separation of the polyhydric phenol compounds such as flavonoids, quinones, phenolic acids, Carbonyl compounds, and carboxyl compounds.Tannins in the crude extract was also removed. Using0,30%,60%,95%ethanol eluate sequentially, the eluent was detected by thin layer chromatography (TLC; chloroform-methanol (97:3)). The crude extract was21.6g and the residue of60%ethanol eluant fraction was5.1g. HPLC showed that the contents of wogonin were3.267%and13.57%respectively.Then Wogonin in the60%ethanol eluent was prepared by high-speed countercurrent chromatography (HSCCC). According to Wogonin nature, we employ n-hexane-ethyl acetate methanol-water (7:3:5:5) as high-speed countercurrent solvent system, upper phase as stationary phase and lower phase as the mobile phase. After HSCCC separation,525mg Wogonin was yielded from50g of crude sample and the purity of Wogonin was98.7%, as determined by the peak area percent of HPLC. We got two monomer substances at the same time. Because we want to prepare Wogonin in this experiment, it was not necessary to analyze this two monomers substances. It illustrated that this approach could prepare the high content, high purity Wogonin. And after a single injection finishing, the upper and lower phase almost run out, it would not cause reagents waste.The prepared Wogonin was evaluated in HepG2cells. We found that Wogonin at the doses of (12.5-200μmol/L) effectively inhibited the proliferation of hepatoma carcinoma cell HepG2by MTT assay. The effects of Wogonin and5-fluorouracil (5-FU) together on growth of HepG2cells were evaluated by MTT. Results showed that Wogonin(100umol/L)and5-FU (384μmol/L) synergically inhibited growth of HepG2cells.