| Callicarpa kwangtungensis Chun (CK), with phenylethanoid glycosides (PG) and flavanoids as main bioactive constituents, is the major material of Traditional Chinese Medicine (TCM) preparations "kang-gong-yan". The preparations of it is widely used for the treatment of gynecologic inflammation disease. It is called for an efficient and reliable method for the quality control of CK because of its increasing clinical application and quality problems. However, there was no any suitable and selective method reported for the quality control of CK, so far, which limits the further research and utilization of CK.ObjectiveTo make a series of studies about compounds analysis, qualitative and quantitative analysis and pharmacokinetics study of three phenylethanoid glycosides (verbascoside, forsythoside B and poliumoside) of CK for providing scientific basis for the further research and utilization of CK.Methods1. Compounds analysisUsing the column chromatography technology of macroporous resin and C18, separated and purified the components of ethanol extract of CK. Structures of them were identified using NMR (1H,13C), MS and UV technolegies.2. Qualitative and quantitative analysisThe rapidly profiling, identifying and determining of chemical constituents in CK was achieved by using a high performance liquid chromatography-electrospray ionization-iontrap-time-of-flight-tandem mass spectrometry (HPLC-ESI-IT-TOF-MS/MS) coupled with high performance liquid chromatography-photo-diode array (HPLC-PDA) method, for the qualitity control of this plant. Qualitative analysis of chemical constituents in CK was performed on HPLC-ESI-IT-TOF-MS/MS while a HPLC-PDA method was developed to simultaneous determine six index compounds (verbascoside, forsythoside B, poliumoside, luteolin-7-O-glycoside,2’-acetyl acteoside and brandioside) in CK.3. Pharmacokinetics study of three phenylethanoid glycosides (verbascoside, forsythoside B and poliumoside)In this study, a novel LC-MS/MS method was developed and validated for the simultaneous quantifcation of three phenylethanoid glycosides in rat plasma (verbascoside, forsythoside B and poliumoside), which are the major bioactive compounds of CK; MS was operated in negative mode. And the method was applied in a PK study of three phenylethanoid glycosides after a single oral administration of CK extract to rats. Results1. Compounds analysis 12 compounds were isolated from the ethanol extract of CK, and 9 of them were identified as poliumoside (Cl), forsythoside B (C2), verbascoside (C3),2’-acetyl poliumoside (C4), 2’-acetyl acteoside (C5),2’-acetyl forsythoside B (C6), diosmetin-7-O-diglycuronides (C7), luteolin-7-O-diglucuronide (C8) and luteolin-diglucuro nide-glucuronide (C9). C1-C6 were phenylethanoid glycosides, C7-C9 were flavonoids and C7～C9 were isolated from CK for the first time.2. Qualitative and quantitative analysisA total of 34 compounds including 13 phenylethanoid glycosides and 17 flavanoids were identified or tentatively characterized by LC-MS-IT-TOF based on standard components comparison, UV absorption curve and MS spectra data in both negative and positive ion modes,14 of which were firstly reported from CK. The HPLC-PDA method was fully validated and subsequently applied to simultaneous determine six index compounds (verbascoside, forsythoside B, poliumoside, luteolin-7-0-glycoside,2’-acetyl forsythoside B and brandioside) in CK of 11 batches collected from different districts in china in different harvest seasons for the first time.3. Pharmacokinetics (PK) study of three phenylethanoid glycosides (verbascoside, forsythoside B and poliumoside)This method was linear between 5.2 and 1010 ng/ml for poliumoside,7.0 and 420 ng/ml for forsythoside B and 2.60 and 260.0 ng/ml for verbascoside. The MS/MS ion transitions monitored were m/z 769.4-+160.5, m/z 755.3→593.3, m/z 623.1→160.5 and m/z 179.0→133.6 for poliumoside, forsythoside B, verbascoside and caffeic acid (IS), respectively. Linearity, accuracy, precision and extraction recovery of three analytes were all satisfactory. The PK study indicated that the PK characteristics of poliumoside, forsythoside B and verbascoside have been proposed to be similar to each other and they absorbed quickly and eliminated very slowly after administration. ConclusionIn this study, the compounds analysis of CK active part provides chemical material basis for the further study of CK in this study; the newly developed LC-ESI-MS-IT-TOF coupled with HPLC-PDA method was effective and comprehensive and could act as an useful tool for quantitative assessment and quality control of both of CK and its related medicinal products in terms of selectivity, detection levels and structural information; the PK study of three phenylethanoid glycosides (verbascoside, forsythoside B and poliumoside) has filled the blank of PK study of CK and it will be helpful to better understand the pharmacological and clinical effects of CK. |
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