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Objective::To investigate the effect of betulic acid(BA) on steatosis LO2 cells. Method::LO2 cells were intervened with BA at different gradient concentrations (0, 10, 20, 40, 80, 160, 250 μmol·L-1) for 24 hours. methyl thiazolyl tetrazolium(MTT) staining was used to observed cell viability to determine the final concentration of BA. The cells were divided into control, model, dimethylsulfoxide (DMSO) and BA groups, as well as BA groups intervened with low, middle and high concentrations. First, model, DMSO and BA group's cells were cultured in 10% Lipid Mix 1 medium for 24 hours to establish a nonalcoholic fatty liver model. Then, DMSO group and low, medium and high-concentration groups were separately cultured with 0.1%DMSO medium and 20, 40, 80 μmol·L-1 BA medium for 24 hours. And control and model groups were cultured in drug-free medium for 24 hours. Oil red O staining and Nile red staining were used to observe the intracellular lipid droplets. Immunofluorescence was used to detect the protein expression of inducible nitric oxide synthase (iNOS). Western blot was used to detect the protein expression levels of receptor for advanced glycation end-products (RAGE), nuclear factor κB p65 (NF-κB p55) and iNOS. Result::BA within the concentration of 80 μmol·L-1 had no significant toxicity on LO2 cells. Compared with control group, the intracellular lipid droplets were significantly increased in the model group, and the expressions of oxidative stress-related proteins RAGE, NF-κB p65 and iNOS also increased significantly(P<0.05). Compared with model group, the intracellular lipid droplets in DMSO group were similar to those in model group, with no significant difference in the three protein expressions between the two groups. However, the intracellular lipid droplets deposition in the BA group was significantly decreased. And the expressions of RAGE, NF-κB p65 and iNOS proteins in high-concentration BA group were significantly decreased(P<0.05, P<0.01). Conclusion::BA can significantly improve the intracellular fat deposition in LO2 cells, which was probably related to the inhibition of the expressions of oxidative stress-related proteins RAGE, NF-κB p65 and iNOS.
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OBJECTIVE:To establish the method for the content determination of 4 active components in Compound xiaosuanzao chewable tablets.METHODS:HPLC-ELSD method was adopted.The determination was performed on a Grace Brava C18-BDS column with mobile phase consisted of acetonitrile-water (gradient elution) at the flow rate of 1.0 mL/min.The column temperature was 25 ℃,and sample size was 20 μL.The drift tube temperature is 100 ℃,and the carrier gas flow rate is 2.9 L/min.RESULTS:The linear ranges of betulic acid,betulinol,pachymic acid and glycyrrhizic acid were 44.50-890.0 μg/mL (r=0.999 3),20.28-405.6 μg/mL (r=0.999 7),20.50-656.0 μg/mL(r=0.999 7) and 10.50-336.0 μg/mL(r=0.999 6),respectively.RSDs of precision,stability and reproducibility were all lower than 3.0％.The recoveries were 99.44％-101.12％ (RSD=0.57％,n=6),99.41％-100.39％ (RSD=0.34％,n=6),99.31％-100.46％ (RSD=0.51％,n=6),98.96％-101.19％ (RSD=0.84％,n=6),respectively.CONCLUSIONS:The method is simple,precise,stable and reproducible,and can be used for simultaneous determination of 4 active components in Compound xiaosuanzao chewable tablets.
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Objective: To study the chemical constituents from Prunella vulgaris and their antitumor activities. Methods: Silica gel, reverse-phase octadecylsilyl (ODS), Sephadex LH-20 chromatographic methods, and HPLC were applied to isolating and purifying compounds. MS and NMR spectroscopic methods were used to determine their structures. Furthermore, the cytotoxicity of these chemical components for MCF-7, MDA-MB-231, and MCF-10A cell lines was measured by MTT method. Results: Forteen compounds were isolated from the fruits of P. vulgaris and their structures were identified as: autantiamide acetate (1), 5α,8α-epidioxy-(22E,24R)-ergosta-6,22-dien-3β-ol (2), β-amyrin (3), betulic acid (4), 3-hydroxy-11-en-11,12-dehydrogenation-28,13-oic acid lactone (5), eburicol (6), 2α,3α,24-trihydroxyolean-12-en-28-oic acid (7), candelabrone 12-methyl ether (8), cyclopentaneacetic acid (9), 2α,3β-dihydroxyursa-12-en-28-oic acid (10), α-spinasterol (11), oleanolic acid (12), ursolic acid (13), and β-sitosterol (14). Conclusion: Compounds 2, 5, 6, 8, and 9 are isolated from the genus of Prunella L. for the first time. The results of cytotoxic assay indicate that compounds 10 and 13 can obviously inhibit the activity of MCF-7, MDA-MB-231, and the normal cell lines MCF-10A. Compound 4 can selectly inhibit the activity of MCF-7 and MDA-MB-231 while show no effect on the normal cell lines MCF-10A.