| The plants of the genus Siegesbeckiae (Compositae) are annual herbs, Three species of Siegesbeckiae(Siegesbeckiae orientalis L., Siegesbeckiae pubescens M., Siegesbeckiae glabrescens M.) grow in China, and their aerial parts have been used as a traditional Chinese medicine, "Xi Xian Cao" for the treatment of rheumatic arthritis, In this paper, the chemical constituents, pharmacological activity in vitro, quality control methods of Siegesbeckiae were investigated by means described as follows.The chemical constituents of the extract with 70% ethanol from Siegesbeckiae pubescens M. were studied,13 compounds were isolated by using multiple column chromatographic techniques, including silica gel column chromatography, Sephadex LH-20 column chromatography and ODS column chromatography. On the basis of analysis with 1H-NMR、13C-NMR、HMBC、MS, the chemical structures of 11 compounds were clucidated as Kirenol, Darutigenol, Hythiemoside B, ent-16aH,17,18-dihydroxy-kauran-19-oic acid, ent-16β,17,18-trihydroxy-kauran-19-oic acid, ent-16β,17-dihydroxy-kauran-19-oic acid, ent-16αH-kauran-17,19-dioic acid,β-sitosterol, Rhamnetin,3’,4’-dimethoxy quercetin, Fumaric acid.3’,4’-dimethoxy quercetin, Rhamnetin, ent-16αH-kauran-17,19-dioic acid were isolated from Siegesbeckiae for the first time.An HPLC-ESI-TOF-MS method in negative mode was developed and applied to study the chemical constituents of extract of Siegesbeckiae pubescens M.. According to the retention time, precise molecular weight and fragmentation patterns of the eight standard compounds, eight compounds were accurately identified. The chemical names of the identified compounds are Kirenol, Hythiemoside B, ent-16aH,17,18-dihydroxy-kauran-19-oic acid, ent-16/3,17,18-trihydroxy-kauran-19-oic acid, ent-16β,17-dihydroxy-kauran-19-oic acid, ent-16αH-kauran-17,19-dioic acid, Rhamnetin,3’,4’-dimethoxy quercetin. As well as on the basis of analysis precise molecular weight, fragmentation patterns and literatures,4 Kauranoids,1 Kaurane glycoside compound,2 Pimarene glycoside compound,3 flavonoids,22 fatty acids were tentatively identified.Five fractions with macroporous resin column and five compounds isolated from Siegesbeckiae pubescens M. were tested for their anti-inflammatory activity in vitro. by NO production in LPS-activated RAW264.7 Cell lines. the production of NO is induced by LPS and determined by Griess reaction, the inhibitory rate on the NO production is calculated and the cell cytotoxicity is evaluated by MTT method.The fractionⅢ,ⅣandⅤof Siegesbeckiae pubescens M showed stronger inhibitory activity on NO production thanⅠandⅡ, inhibition rate> 62%. Five compounds also showed strong inhibitory activity on NO production, inhibition rate>62%, and higher than the positive control KirenolUsing three universal detectors (MS, RID, ELSD), methods were developed for simultaneous determination of Siegesbeckiae①An HPLC-ESI/TOF-MS method in negative mode was established for the simultaneous determination of eight compounds, Kirenol, Hythiemoside B, ent-16αH,17,18-dihydroxy-kauran-19-oic acid, ent-16β,17,18-trihydroxy-kauran-19-oic acid, ent-16β,17-dihydroxy-kauran-19-oic acid, ent-16αH-kauran-17,19-dioic acid, Rhamnetin,3’,4’-dimethoxy quercetin, using a mobile phase consisting of 0.1% aqueous formic acid and acetonitrile containing 0.1% formic acid in selected ion monitoring (SIM) at m/z 373.21,561.28,351.21,335.22, 333.21,315.05 and 329.06. The precision was evaluated by intra-and inter-day tests, which revealed relative standard deviation (RSD) values less than 3.68%. The recoveries for the quantified compounds were between 97.4 and 101.2%. LOQ were between 0.10 and 8.0μg/mL. All calibration curves showed good linearity within the test ranges. This method was applied to determine the amounts of the four compounds in Siegesbeckiae from five sources.②Two HPLC-RID methods were established for simultaneous determination, Under chromatographic condition 1, mobile phase 0.3% formic acid-acetonitrile (72:28), Kirenol, Hythiemoside B, ent-16aH,17,18-dihydroxy-kauran-19-oic acid, ent-16β,17,18-trihydroxy-kauran-19-oic acid, ent-16β,17-dihydroxy-kauran-19-oic acid, were determined. The RSDs of repeatability were less than 3.97%. The recoveries for the quantified compounds were between 97.8 and 100.7%. LOQ were between 2.0 and 8.0μg. All calibration curves showed good linearity within the test ranges. This method was applied to determine the amounts of the five compounds in Siegesbeckiae from two sources. Under chromatographic condition 2, mobile phase 0.3% formic acid-acetonitrile (42:45), Darutigenol, ent-16β,17-dihydroxy-kauran-19-oic acid, ent-16αH-kauran-17,19-dioic acid,3’,4’-dimethoxy quercetin were determined. The RSDs of repeatability were less than 3.66%. The recoveries for the quantified compounds were between 97.4 and 99.0%. All calibration curves showed good linearity within the test ranges. This method was applied to determine the amounts of the four compounds in Siegesbeckiae from two sources.③An HPLC-ELSD method was established for the simultaneous determination of eight compounds, Kirenol, Hythiemoside B, Darutigenol, ent-16aH,17,18-dihydroxy-kauran-19-oic acid, ent-16β,17,18-trihydroxy-kauran-19-oic acid, ent-16β,17-dihydroxy-kauran-19-oic acid, ent-16αH-kauran-17,19-dioic acid, 3’,4’-dimethoxy quercetin using a mobile phase consisting of 0.1%aqueous formic acid and acetonitrile. The RSDs of repeatability were less than 3.93%. The recoveries for the quantified compounds were between 97.8 and 101.0%. All calibration curves showed good linearity within the test ranges. This method was applied to determine the amounts of the eight compounds in Siegesbeckiae from two sources. The results show that the HPLC-ESI/TOF-MS method was sensitive and specific, and suitable for qualitative and quantitative analysis of complex systems like traditional Chinese medicine. The HPLC-RID method which can not use the gradient elution, was suitable for single component analysis or multi-component analysis with similar polarity, or for the preparation of single compounds. The HPLC-ELSD method which can use the gradient elution, was suitable for multi-component analysis with no UV absorption determination, and its sensitive was higher than HPLC-RID for one to two orders of magnitude. In conclusion, by utilizing various professional knowledge and analytical techniques, the chemical constituents in Siegesbeckiae pubescens M.were investigated, the extracts of five parts from Siegesbeckiae pubescens and five compounds were tested for their anti-inflammatory activity in vitro. Methods were developed for multi-component quantitative determination using three universal detectors. This study provided useful information for the interpretation of activity-related substance, quality control, exploration and utilization of Siegesbeckiae. |
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