ABSTRACT
OBJECTIVE:To establish a method for content determination of thymosin α1 in Thymopolypeptides enteric-coated tablets.METHODS:HPLC-MS/MS method was adopted.The determination was performed on Luna C18(2) with mobile phase consisted of 0.1％ formic acid-acetonitrile (gradient elution) at the flow rate of 0.7 mL/min.The column temperature was set at 30 ℃,and sample size was 20 μL.ESI was used with ion spray voltage of 4.5 kV,sheath gas flow rate of 60 arb,aux gas flow rate of 30 arb,sweep gas flow rate of 10 arb and capillary temperature at 320 ℃.The working mode was positive ion monitoring mode.RESULTS:The linear range for thymosin α1 was 1-1 000 ng/mL (r=0.999 9).The limit of quantitation was 1 ng/mL.The limit of detection was 0.1 ng/mL.RSDs of precision,stability and reproducibility tests were all lower than 2.0％.The recoveries were 95.0％-98.0％ (RSD=1.2％,n=6),respectively.CONCLUSIONS:The method is simple,rapid,sensitive and accurate,and can be suitable for simultaneous determination of thymosin α1 in Thymopolypeptides enteric-coated tablets.
ABSTRACT
To investigate the antiviral effect of thymopolypeptides combined with 4 kinds of matrine type alkaloids on HepG2.2.15 cells, oxymatrine, sophocarpidine, sophocarpine, and sophoridine (at concentration of 0.2 mmol•L⁻¹ respectively) were respectively combined with thymopolypeptides (0.025, 0.1 g•L⁻¹), and after 48 h and 72 h treatment on HepG2.2.15 cells, the cells and supernatants were collected. The cells activity in various groups was determined by CCK-8 method to evaluate the toxic effects of the drugs on HepG2.2.15 cells. Enzyme linked immunosorbent assay (ELISA) was used to determine HBeAg and HBsAg levels in cellular supernatants. HBV DNA levels in cellular supernatants andcells were quantified with fluorogenic quantitative PCR method; and the expression level of IFN-α in supernatants was detected with CBA method. The results indicated that single thymopolypeptides at 0.025-0.4 g•L⁻¹ had no toxicity to cells. Thymopolypeptides in this concentration range combined with 0.2 mmol•L⁻¹ matrine type alkaloids also had no toxicity to cells. Anti-HBV activity of drug combination was better than that of alkali or thymopolypeptides alone. Thymopolypeptides at 0.025 g•L⁻¹ had better inhibitory effect than thymopolypeptides at 0.1 g•L⁻¹ on intracellular HBV DNA expression, but the inhibitory effect on supernatant HBeAg level was on the contrary. Anti-HBV activity was similar between alkaloids combined with 0.1 g•L⁻¹ and alkaloids combined with 0.025 g•L⁻¹. There was no statistical difference in anti-HBV effect between various combined groups (P<0.05). In general, 72 h anti-HBV effect was better than 48 h anti-HBV effect (P<0.05). The expression of IFN-α was increased after drug combination, with positive correlation to the changes of other four indicators (P<0.05). In conclusion, oxymatrine, sophocarpidine, sophocarpine and sophoridine combined with thymopolypeptides could inhibit HBsAg and HBeAg secretion in HepG2.2.15 cells and HBV DNA replication, and further promote the antiviral effect by promoting the expression of IFN-α.