| Dan Shen is the root or rhizome of Salvia miltiorrhiza Bge and one of the most widely used drugs in traditional Chinese medicine.The first record of Salvia Miltiorrhiza was in “Shen Nong's Herbal”,and was commonly used for increasing blood circulation.Acording to the color of its flower,Dan Shen was divided into Salvia miltiorrhiza Bunge.f.alba.and Salvia miltiorrhiza Bunge.s.Tanshinones is its main chemical composition.There are researchs show that tanshinones from Salvia Miltiorrhiza have good anti-inflammatory effect.The research of our study was to investigate the difference of tanshinones from Salvia miltiorrhiza Bunge.f.alba.and Salvia miltiorrhiza Bunge.s for isolate,extract and identify the tanshinones from Salvia miltiorrhiza.The anti-inflammatory activity of Tanshinone IIA,Tanshinone I and Methyl tanshinone were studied to investigate the anti-inflammatory mechanism of Tanshinones.To investigate the difference of tanshinones from Salvia miltiorrhiza Bunge.f.alba.and Salvia miltiorrhiza Bunge.s,an accurate and reliable analytical method was investigated for simultaneous determination of ten active components(Tanshinone IIA,Tanshinone I,Sugiol,ferruginol,cryptotanshinone,Danshenxinkun B,Tanshinonal,Salviolone,dihydrotanshinone I,Methyl tanshinonate)in plants of Salvia Miltiorrhiza.The major components of fractions were studied by HPLC.A Agilent XDB-C18 column(4.6 mm × 250 mm,5 ?m)was used at a flow rate of 1 ml/min.Mobile phase A was water-formic acid(1000: 1,v/v)and mobile phase B was methanol.Using ten tanshinones as control,the tanshinones content of Salvia Miltiorrhiza of different origins.The detection wavelength was set at 280 nm.The results showed that the calibration curve of Tanshinone IIA was linear in the range of 2.00?g-100.00?g,r=0.9979.The linear equation is Y=40.601X+159.68.RSD of the average recovery is 0.46%.The calibration curve of Tanshinone I was linear in the range of 2.00?g-100.00?g,r=0.9975.The linear equation is Y=22.335X+72.957.RSD of the average recovery is 1.76%.The calibration curve of Sugiol was linear in the range of 2.00?g-100.00?g,r=0.9984.The linear equation is Y=30.296X+98.144.RSD of the average recovery is 2.28%.The calibration curve of ferruginol was linear in the range of 2.00?g-100.00?g,r=0.9982.The linear equation is Y=6.7841X+27.737.RSD of the average recovery is 1.37%.The calibration curve of cryptotanshinone was linear in the range of 2.00?g-100.00?g,r=0.9981.The linear equation is Y=19.936X+72.423.RSD of the average recovery is 2.09%.The calibration curve of Danshenxinkun B was linear in the range of 2.00?g-100.00?g,r=0.9978.The linear equation is Y=41.554X+161.46.RSD of the average recovery is 1.27%.The calibration curve of Tanshinonal was linear in the range of 2.00?g-100.00?g,r=0.9984.The linear equation is Y=23.688X+64.779.RSD of the average recovery is 1.98%.The calibration curve of Salviolone was linear in the range of 2.00?g-100.00?g,r=0.9999.The linear equation is Y=79.323X-66.428.RSD of the average recovery is 1.93%.The calibration curve of dihydrotanshinone I was linear in the range of 2.00?g-100.00?g,r=0.9980.The linear equation is Y=52.933X+194.45.RSD of the average recovery is 1.86%.The calibration curve of Methyl tanshinone was linear in the range of 2.00?g-100.00?g,r=0.9999.The linear equation is Y=27.794X+0.392.RSD of the average recovery is 1.72%.The method was simple,accurate and reproducible.The result shows that there were significant differences in the contents of the compounds of tanshinones in Salvia Miltiorrhiza from different origins.Sugiol,Danshenxinkun B and Tanshinonal only could be detected in Salvia miltiorrhiza Bunge.f.alba.The compounds of Tanshinones were decocted in dichloromethane and methanol.The extract was subject to silica gel and gel chromatography eluted at a flow rate of 1BV/h with different proportions of solvents according to the ascending polarity of cyclohexane: dichloromethane(10:1,v/v)and dichloromethane system.Each gradient elute 5-6 column volume.The fractions obtained from cyclohexane-dichloromethane(1: 2)was eluted with different gradient of dichloromethane-methanol.The extracts were filtered and concentrated on a rotary evaporator under reduced pressure followed by drying in a freeze-dryer.Seven fractions(FrA-FrG)and four compounds were acquired.Two compounds obtained from the FrE fraction were separated by gradient elution of different proportions of cyclohexane-dichloromethane.Two compounds obtained from the FrF fraction were separated by gradient elution of different proportions of cyclohexane-dichloromethane and HPLC semi-preparative.Two compounds obtained from FrG were separated by gradient elution with different proportions of cyclohexane-dichloromethane.The structures of ten compounds of tanshinones were analyzed by 1HNMR and 13 CNMR,determined by comparing with literature and reference.The structures of new compounds of tanshinones were analyzed by LC-MS/MS and determined by comparing with literature and reference.The aim of the study was to investigate the anti-inflammatory activities of Tanshinone IIA,Tanshinone I and Methyl tanshinone obtained from the extract of Salvia Miltiorrhiza in complete freund's adjuvant induced knee arthritis mice.The serum IFN-?,IL-4,LTB4 and NO levels of mice which were induced by complete freund's adjuvant were tested and determined.The results showed that there was significant difference of the paw swelling of the mice in model control group compare with mice in normal control group the mice after modeling.After administration,the paw swelling of the mice in the group of positive,Tanshinone IIA,Tanshinone I and Methyl tanshinone were significantly reduced,compared to the model group.The serum level of IFN-?of the model group was significantly lower than the serum level of IFN-? of normal control group.The serum levels of IFN-?o in the group of positive,Tanshinone IIA,Tanshinone I and Methyl tanshinone were higher than those in the model group(P <0.05)after administration.The serum levels of IL-4,LTB4 and NO of the model group were significantly higher than those in the normal control group.The serum levels of IL-4,LTB4 and NO of the group of positive,Tanshinone IIA,Tanshinone I and Methyl tanshinone were lower than those in the model group(P <0.05)after administration.The pathologicals results showed that there was a lot of macrophages and T cells in synovial tissue.There was pannus formattedin synvial tissue.The remarks of model control group was higher than that in normal control group.In this study,eleven kinds of tanshinones were isolated and purified by silica gel column chromatography and gel column chromatography.The structures of these 10 compounds were determined by 1HNMR and 13 CNMR.The structures of new compounds of tanshinones were analyzed by LC-MS/MS and determined by comparing with literature and reference.Inflammatory activity of the study,to provide a scientific basis for further research. |
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