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OBJECTIVE@#To investigate whether kirenol, the major pharmacologically active compound of the Chinese medicinal herb , can protect mice from dextran sulfate sodium (DSS)-induced ulcerative colitis (UC).@*METHODS@#C57BL/6 mice with or without kirenol pretreatment were treated with DSS in drinking water for 7 days to induce UC. The symptoms of UC including weight loss, diarrhea and bloody stool were observed daily and graded using the disease activity index (DAI). Colon injury of the mice was assessed by measuring the length of the colon and HE staining of the colon tissue. The levels of inflammatory cytokines produced by the mesenteric lymph nodes (MLNs) lymphocytes were measured using enzyme-linked immunosorbent assay; the apoptosis of the lymphocytes and CD4 T cells was analyzed using flow cytometry.@*RESULTS@#The mice receiving pretreatment with kirenol showed obviously ameliorated symptoms of UC and milder pathological changes in the colon as compared with the control mice. Kirenol treatment significantly down-regulated the secretion of IFN-γ, IL-17A, IL-6 and TNF-α by the MLNs lymphocytes and increased the apoptosis of lymphocytes, especially CD4 T cells in the DSS-treated mice.@*CONCLUSIONS@#Kirenol can protect against T cell-mediated colon injury in DSS-treated mice possibly by suppressing the secretion of inflammatory mediators and inducing apoptosis of the inflammatory lymphocytes.
Subject(s)
Animals , Mice , Apoptosis , Colitis, Ulcerative , Cytokines , Dextran Sulfate , Diterpenes , Mice, Inbred C57BL , T-LymphocytesABSTRACT
Objective To establish a joint wavelength switching HPLC gradient elution method for simultaneous determination of darutoside, kirenol, darutigenol, fangchinoline and d-tetrandrine in Xixian Fengshi Wan. Methods A Kromasil C18 column (4. 6 mm × 250 mm, 5 μm) was used with a mobile phase A; acetonitrile-methano (2: 1) and mobile phase B: 0. 05% acetic acid solution with gradient elution. The flow rate was 1. 0 mL/min; the injection volume was 20 μL; darutoside, kirenol and darutigenol were detected at 215 nm, and fangchinoline and d-tetrandrine were detected at 280 nm. Results In the given concentration range, the linearity ranges of darutoside, kirenol, darutigenol, fangchinoline and d-tetrandrine were 6. 45-129. 00 μg/mL (r=0. 999 5), 5. 61-112. 20 μg/mL (r=0. 999 9), 4. 25-85. 00 μg/mL (r=0. 999 3), 9. 19-183. 80 μg/mL (r =0.999 8), and 11. 05-221. 00 μg/mL (r=0. 999 7), respectively; and their average recoveries and RSD were 98. 73% (1. 43%), 97. 63% (1. 28%), 99. 44% (1. 29%), 98. 33% (1. 38%), and 97. 36% (1. 37%), respectively. Conclusion The established joint wavelength switching HPLC gradient elution method is simple, accurate and reproducible; it can simultaneously determine the contents of darutoside, kirenol, darutigenol, fangchinoline and d-tetrandrine in Xixian Fengshi Wnn and can be used for quality control of Xixinn Fengshi Wan.
ABSTRACT
The study established a LC-MS/MS method for the simultaneous determination of two active diterpenoids:kirenol and ent-16β, 17-dihydroxy-kauran-19-oic acid (DHKA) from Herba Siegesbeckiae in rat plasma using osthole as an internal standard (IS). Plasma sample pretreatment involved a one-step liquid-liquid extraction with ethyl acetate. Chromatographic separation was performed on a Waters Symmetry C18 column (2.1 mm×100 mm, 3.5 μm) with isocratic elution using methanol-5 mmol·L-1 aqueous ammonium acetate (80:20) as mobile phase at a flow rate of 0.2 mL·min-1. The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode under positive and negative electrospray ionization. Quantification was performed using SRM of the transitions m/z 356.4→321.4 for kirenol, m/z 335.3→335.3 for DHKA, and m/z 245.1→188.9 for the IS, respectively. The calibration curves were linear over the range of 50.0-25000 ng·mL-1 for kirenol, 25.0-12500 ng·mL-1 for DHKA. The extraction recoveries of two analytes and IS were more than 85%. The intra- and inter-day precision (relative standard deviation) values were less than 13.9% and accuracy (relative error) was from -10.7% to 10.3% at four quality control levels. The pharmacokinetic parameters of different medication administration teams were analyzed with SPSS statistics 13.0 software. The validated method was successfully applied to a comparative pharmacokinetic study of the two diterpenoids in rat plasma after intragastric administration of kirenol, DHKA and Herba Siegesbeckiae extract. Kirenol appeared to be both absorbed and eliminated fast in vivo, and DHKA absorbed fast but eliminated slowly in vivo. And there were obvious differences between the pharmacokinetic behaviors after oral administration of Herba Siegesbeckiae extract compared with single substances. Compared with the value after oral administration of kirenol, the extract might inhibit the absorption and postpone the elimination of kirenol in rats after administration of the extract. For DHKA, the absorption rate of DHKA increased rapidly after administration of the extract. This work can provide some experimental basis for the clinical use of Herba Siegesbeckiae. The method is simple, rapid and sensitive, which is suitable for pharmacokinetics study of the two diterpenoids from Herba Siegesbeckiae in rats.
ABSTRACT
OBJECTIVE:To study the reasonable parts used as medicinal herbs in Herba Siegesbeckiae from the point of chemical contents.METHODS:Using the methods of Ch.P 2005 to determine the percentage contents of ethanol-soluble extractives and Kirenol(an effective antirheumatic constituent) in the tender stems and leaves and thick stems of Herba Siegesbeckiae. RESULTS:The contents of the ethanol-soluble extractives and Kirenol in the tender stems and leaves were much higher than in the thick stems.CONCLUSION:The above results suggest that tender stems and leaves of Herba Siegesbeckiae should be used as the medicinal parts.