ABSTRACT
OBJECTIVE To explore the difference in th e mechanis m of baicalein and wogonin inhibiting the energy metabolism of hepatoma cells. METHODS Human hepatoma HepG 2 cells were divided into blank control group （without medicine），different dose groups of baicalein and wogonin （1.25，2.5，5，10 and 20 μmol/L）. The effects of baicalein and wogonin on the viability of HepG 2 cells were detected by MTT assay. HepG 2 cells were divided into blank control group （without medicine），baicalein group and wogonin group. After administration ，the concentration of ATP in cell was detected by enhanced ATP kit. The levels of cell glycolysis and mitochondrial energy metabolism were evaluated by glycolysis and mitochondrial pressure test kit ；the affinity of baicalein and wogonin with key enzymes of energy metabolism was predicted by molecular docking ，and the key enzymes of energy metabolism with high affinity were screened ；the expression of key enzymes of energy metabolism was detected by Western blot. RESULTS Within the dose range of 2.5-20 μmol/L，the half inhibitory concentrations of baicalein and wogonin were 12.84 and 24.09 μmol/L；baicalein 1.25 μmol/L and wogonin 2.5 μmol/L had no effect on cell viability ，so it was selected as the dosage for subsequent experiments. Compared with blank control group ，the concentration of ATP in HepG 2 cells decreased significantly in baicalein group and wogonin group （P＜0.05）；the inhibitory effects on basic acidification rate of HepG 2 cells in wogonin group were significantly stronger than those of baicalein group （P＜0.05），but there was no significant difference between them on the basic oxygen consumption rate （P＞0.05）；baicalein had strong binding to pyruvate kinase M 2 and mitochondrial enzyme complexes Ⅰ（CⅠ），C Ⅱ and C Ⅳ，while wogonin only had strong binding to pyruvate kinase M 2； wogonin could significantly down-regulate the protein expressions of hexokinase ，phosphofructokinase，pyruvate kinase M 2，CⅠ， C Ⅱ and C Ⅳ（P＜0.05），but there was no statistical significance in the effect of baicalein on the regulation of these enzymes （P＞ 0.05）. CONCLUSIONS Both baicalein and wogonin can inhibit the energy metabolism of hepatoma HepG 2 cells，but the mechanism is different ：the effect of baicalein is related to the activity of key enzymes ，while the effect of wogonin is related to the inhibition of the expression of key enzymes of energy metabolism.
ABSTRACT
OBJECTIVE：To establish the method for the content de termination of 17 quality markers in Dahuang zhechong pills（DHZCP）. METHODS ：HPLC method was adopted to determine the contents of 17 quality markers in 10 batches of DHZCP ， such as allantoin ，hypoxanthine，salidroside，hydroxypaeoniflorin，glycyrrhizin，isoglycyrrhizin，baicalin，p-methoxyphenylacetic acid，wogonin，cinnamic acid ，apigenin，naringin，norwogonin，aloe emodin ，rhein，chrysin，emodin. The determination was performed on Kromasil 100-5-C18（250 mm × 4.6 mm，5 μm）column with mobile phase consisted of 0.1％ phosphoric acid solution-acetonitrile （gradient elution ） at the flow rate of 1.0 mL/min. The column temperature was 30 ℃ ，the detection wavelength was 210 nm and the sample size was 20 μ L. RESULTS：The linear range of above 17 quality markers were 5.74-183.53，6.51-208.24，4.30-137.65，4.60-147.06，4.12-131.76，4.25-135.88，6.31-201.76，4.60-147.06，1.94-62.06，4.47- 142.94，0.69-22.06，2.29-73.24，2.33-74.41，1.42-45.29，6.65-212.94，1.11-35.44 and 1.47-47.06 μg/mL，respectively（all R2≥ 0.999 0）. RSDs of precision ，repeatability，stability and durability tests were all less than 2％（n＝6）；average recovery of 17 quality markers ranged from 96.31％ to 101.73％，and the RSDs were less than 3％（n＝6）. CONCLUSIONS ：The method is simple， rapid，speific，specise，reproducible，stable，accurate and durable ，and can be used for improving the quality standard of DHZCP.