| The leaf of Isatis indigotica Fort.is a traditional Chinese medicine recorded in Chinese Pharmacopoeia,2005 as Folium Isatidis.The chemical constituents,quality control methods of Folium Isatidis and pharmacokinetics of indirubin were investigated in this study.The chemical constituents of 70%ethanol extract of Folium Isatidis were isolated by using multiple column chromatographic techniques,and 18 compounds were obtained.The structures of 15 compounds were fully elucidated by chemical and spectroscopic methods （MS,UV,NMR）.Among them,one is a new compound,isoorientin 3"-O-glucopyranoside; six compounds were isolated from Isatis for the first time,isoorientin,isovitexin 6"-O-glucopyranoside,saponarin,loliolide,ethyl pyrophaeophorbideα,and methyl pyrophaeophorbideα;three compounds were isolated from Isatis indigotica Fort.for the first time,isoscoparin,isoscoparin 3"-O-glucopyranoside and isovitexin 3"-O-glucopyranoside.The MS/MS spectroscopy in negative ESI mode was applied to study the fragmentation patterns of the flavonoids isolated from Folium Isatidis.For the 3 flavone 6-C-diglucosides（isoscoparin 3"-O-glucopyranoside,isovitexin 3"-O-glucopyranoside and isoorientin 3"-O-glucopyranoside）with the rare 1→3 interglycosidic linkage,the occurrence of ion[M-H-90]-,[M-H-180]- and[M-H-270]- was observed.In the MS/MS spectra of 3 flavone C-monoglucosides（isoorientin,isovitexin,and isoscoparin）,the fragment ions [M-H-120]-（[Ag+41]-）and[M-H-90]-（[Ag+71]-）were observed which were characteristic ions of flavone 6-C glucoside.For isovitexin 6"-O-glucopyranoside which is a flavone 6-C-diglucoside with 1→6 interglycosidic linkage,ions;[Ag+71]- and[Ag+41]- with minor ions[M-H-90]- and[M-H-120]- were observed.Saponarin is O-glycosyl-C-glycosyl flavone and its main fragment ions were[M-H-90]-和[M-H-120]-.The chromatographic fingerprints（CFPs）of Folilum Isatidis from different resources were established by ultra-performance liquid chromatography with photo diode array and electrospray ionization tandem mass spectrometry detection（UPLC-PDA-MS）.The separation was conducted on an Acquity UPLC BEH C18column with 0.1%forbic acid-methanol as the mobile phase.With the reference substances and literatures,21 peaks were identified according to their quasi-molecular ion peaks and on-line UV spectra.The similarities of Folium Isatidis samples were evaluated with angle cosine and correlation coefficient as the measure.The similarity results indicate the CFPs of Folium Isatidis from different resources varied significantly.The constituents in leaves and root,i.e.Folium Isatidis and Radix Isatidis of one I.indigodica plant were compared based on their UV and MS CFPs.The constituents of flavonoids and alkaloids in leaf and root of Isatis indigodica Fort.are different.The CFPs of leaves of I.indigodica,Polygonum tinctorium Ait and Clerodendrun cyrtophyllum Turcz.were compared,and only a few common peaks were obtained from the three kinds of leaves,indicating that although they were used as Folium Isatidis in history,their constituents are totally different.The antibiotic activities of Folium Isatidis of water extract and extracts with ethanol of various concentrations against Staphylococcus aureus,Escherichia coli,Pseudomonas aeruginosa and Candida albicans were investigated by liquid dilution method.The results indicated that Folium Isatidis extract showed excellent anti-S,aureus activities and weak antibiotic activities against E.coli and C.albicans.The values of minimal inhibitory concentration（MIC）of 50%ethanol extract of Folium Isatidis samples from different resourses were determined.Taking the MIC values as the activity index,the correlationship between CFPs and MICs was studied.Sixteen very significant and thirteen significant correlation peaks were obtained,and most of those peaks were flavonoids.The results indicated that flavonoids might be one of the antibiotic active constituent groups in Folium Isatidis.Flavonoids and nucleosides are two major kinds of constituents in Folium Isatidis.A UPLC-MS/MS method was developed for simultaneous quantification determination of 6 flavonoids in Folium Isatidis.An HPLC-UV method was developed for simultaneous determination of 5 nucleosides in Folium Isatidis.The contents of those compounds in samples from different resources varied significantly.The two methods were specific, accurate,sensitive and were suitable for the quality control of Folium Isatidis.The chromatographic fingerprints of essential oils in Folilum Isatidis by gas chromatography（GC）were established.The fingerprint was performed by GC on DB-1 column with programmed column temperature and FID as the detector.The GCFPs of samples from different resources were analyzed.Forty eight common peaks were identified by GC/MS.The relative content of common peaks was more than 95%of the total content of the essential oils in Folium Isatidis by area normalization.The results of the Hierarchical Cluster Analysis showed the samples were clustered into different categories accoding to their resourses by using the GC fingerprints of essential oils.Indirubin is an active compound in Folium Isatidis with various pharmacological effects such as anti-tumor and anti-inflammatory effects.A specific,simple and sensitive HPLC method with UV detection was developed and validated for the pharmacokinetic study of indirubin in rat plasma.Indirubin,with osthole as the internal standard,was extracted from plasma samples by liquid-liquid extraction.Chromatographic separation was conducted on a reverse-phase ODS column（200mm×4.6mm,i.d.,5μm）,using a mixture of methanol-water （75:25,v/v）as the mobile phase with UV detection at 289 nm.The calibration curve of indirubin was linear over the range of 6.5-1950 ng/mL in rat plasma.The lower limit of quantification（LLOQ）was 6.5ng/mL.The intra-and inter-day precisions（RSDs）were not more than 4.1 and 9.1%,respectively.The intra-and inter-day accuracies（REs）were within -2.9 to 4.4%.This developed method was applied to the pharmacokinetic study of indirubin in rats after intravenous and intraperitoneal administration.The pharmacokinetic parameters were determined by noncompartmental analysis.And the parameters for the two dosage groups after i.v.were as follows:Cmax201ng·mL-1and 155ng·mL-1,AUC0-∞308 ng·h·mL-1 and 130 ng·h·mL-1,Ke 0.670 h-1and 0.683 h-1,t1/21.03 and 1.02.The pharmacokinetic parameters for i.p.group were:Cmax20.8ng·mL-1,Tmax0.010h,AUC0-∞25.9 ng·h·mL-1,Ke 0.640 h-1,t1/21.08.The absolute bioavailability of indirubin after intraperitoneal administration was 8.40%.A UPLC-MS/MS method with ESI negative mode was developed and validated for the pharmacokinetic studies of indirubin in rat plasma,and was applied to the pharmacokinetic study of indirubin in rats after oral administration.After liquid-liquid extraction,the plasma samples were analysied under ESI negative mode with MRM 261 →157and 217 ions.The calibration curve of indirubin was linear over the range of 1.25-200 ng/mL in rat plasma.The LLOQ was 1.25ng/mL.The intra-and inter-day precisions（RSDs） were not more than 4.8 and 10.8%,respectively.The intra-and inter-day accuracy（REs）was within-4.2 to 5.6%.The pharmacokinetic parameters were as follows Cmax4.69 ng·mL-1, Tmax0.333h,Ke 0.425h-1,t1/21.63h,AUC0-∞7.30ng·h·mL-1.The pharmacokinetic studies on three administration methods of indirubin indicated that the bioavailability of indirubin via extravascular administration was very low and the elimination.In conclusion,by utilizing various professional knowledge and analytical techniques,the constituents in Folium Isatidis were investigated,quality control methods based on UPLC-PDA-MS,HPLC and GC were established,the antibiotic activity of Folium Isatidis was determined and the pharmacokinetic study on indirubin in Folium Isatidis was conducted. This study provided useful information for the exploration of active constituents and utilization of Folium Isatidis. |
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