| ObjectiveAquilari asinensis (Lour.) Gilg is a precious medicinal herbs, to form heartwood with resin is a long time, then creating a high-quality incense identity of Aquilari asinensis (Lour.) Gilg. By now, there not enough research on Aquilari asinensis (Lour.) Gilg, Chemical constituents of Aquilari asinensis (Lour.) Gilg are more but its chemical composition is not very clear, few study on pharmacological activity. Quality standards of Aquilati asinensis (Lour.) Gilg is a bit simple. There is only one quality control standard of Aquilari asinensis (Lour.) Gilg in the 2010 edition of Chinese Pharmacopoeia. That is ethanol extract. So it is not enough to scientifically evaluate the quality of this precious medicine.A large number of fake and shoddy Aquilari asinensis (Lour.) Gilg appeared on the market, so, a systematic research on chemical composition, pharmacological activity and quality standards of Aquilari asinensis (Lour.) Gilg is needed. Find out the relationship between the content of essential oil, ethanol soluble extractive, total chromone and 5 chromone monomer by statistical method. Establish a scientific Quality Evaluation System of Aquilari asinensis (Lour.) Gilg.MethodUse heating reflux method to get the alcohol extract (AE) of Aquilari asinensis (Lour.) Gilg, and then extracted with petroleum ether, dichloromethane, EtOAc and n-BuOH to get petroleum ether extract(PE), dichloromethane extract(DE), EtOAc extract(EAE) and n-BuOH extract (BE). The writhing responses induced by acetic acid and the hot-plate test were used to evaluate the analgesic activity of different extracts in mice.Components in essential oil of Aquilari asinensis (Lour.) Gilg were extracted by cold-maceration with ether and perform GC-MS analysis. The extract was purified by column chromatography with silica gel,ODS, Sephadex LH-20, and Polyacylamine chromatography and identified by chemical and spectral technology (IR, MS,1H-NMR,13C-NMR,2DNMR).In situ single pass perfusion model was used and the concentrations of Aquilarone A, Aquilarone B, Aquilarone D, Aquilarone E and (5S,6R,7R,8S)-2-[2-(4-methoxy-phenyl) et-hyl]-5,6,7,8-tetraydroxy-5,6,7,8-tet-ra-hydrochromone derivatives were determined by HPLC.PC12 cells was cultured in vitro, MTT method was performed for cells viability, the content of malondialadehyde (MDA) and the enzyme activity of superoxide dismutase (SOD) and glutathione reductase (GSH-PX) were determinate by kit-microplate reader methods.Reference to the 2010 edition of Chinese Pharmacopoeia with characters, microscopic, physical and chemical properties, determination of ethanol extract, make the preliminary evaluation and identification of the Aquilari asinensis (Lour.) Gilg. Determine the content of total chromone in Aquilari asinensis (Lour.) Gilg by the chromatopto-metry of molybdenum phospho tungstate-lithium sulfate. Aquilarone E was taken as the reference substance. Essential oil of Aquilari asinensis (Lour.) Gilg were extracted by cold-maceration with ether. HPLC with Waters Symmetry Shield C18 column(4.6 mm×250 mm,5μm) to determie, gradient elution with acetonitrile and 0.1% phosphoric acid at the flow rate of 0.3 mL·min-1;The detection wavelength was 234 nm with column temperature at 30℃.Perform correlation analysis between the content of the essential oil, the ethanol extract, the total chromone, Aquilarone B、Aquilarone C、Aquil arone D、Aquilarone E and (5S,6R,7R,8S)-2-[2-(4-methoxy-phenyl)et-hyl]-5,6, 7,8-tetraydroxy-5,6,7,8-tet-ra-hydrochromone with SPSS19.0. Get the inte rnal relations between the different indexes.ResultsThe AE and the PE revealed analgesic activity in the hot-plate test. The BE revealed analgesic activity in writhing response induced by acetic acid. PE and BE are the effective parts of Aquilaria sinensis (Lour.) Gilg with analgesic activity.17 compounds were identified by GC-MS from the essential oil of Aquil ari asinensis (Lour.) Gilg, includ benzyl acetone and agarospirol.8 chro mones were separated and identified from the n-BuOH extract, named Aquila rone B, Aquilarone B, Aquilarone C, Aquilarone D,Aquilarone E, (5S,6R,7R,8S)-2-[2-(4-methoxy-phenyl)et-hyl]-5,6,7,8-tetraydroxy-5,6,7,8-tet-ra-hydro chromone and CX14(a new compound, but the absolute configuration uncertai n now). Aquilarone A, Aquilarone D, Aquilarone E and CX14 are isomers;Aquila rone C and (5S,6R,7R,8S)-2-[2-(4-methoxy-phenyl)et-hyl]-5,6,7,8-tetraydro xy-5,6,7,8-tet-ra-hydrochromone are isomers.In the experiment of in situ single pass perfusion, the Ka and Papp of all five componds had no significant difference at different concentration of drugs (Except in colon). The Ka and Papp of all 5chromone deriveatives showed duodenum and colon<jejunum and ileum.In the experiment of protective effects of agarwood essential oil on the H2O2-induced PC12 cells, cell growth inhibition rate of PC12 was increased by the effect of H2O2 in 24 h. Each concentration of H2O2 was significantly inhibited to PC12 cell,200 μmol·L-1 was the maximum inhibitory concentration. The cell viability of the H2O2-induced group was lower than that of the control group, while the content of ROS and MDA were higher than that of the control group (p<0.01), and the activity of SOD and GSH-Px were sharply reduced. After being treated with the agarwood essential oil, the oxidative damage degree of the PC12 cells was significantly reduce (p<0.01, compared with the model group). Agarwood essential oil has a protective effect on PC12 cells from oxidative damage in effective dose range.In the experiment of protective effects 8 chromones in agarwood on the H2O2-induced PC12 cells, cell growth inhibition rate of PC12 was increased by the effect of H2O2 in 24 h. Each concentration of H2O2 was significantly inhibited to PC12 cell,100 μmol·L-1 was the maximum inhibitory concentration; 8 chromones showed no toxicity to PC12 cells in 0-625 μg·mL-1, so we select 1,10,100 μg·mL-1 as low, middle, high drug concentration. The result shows that Aquilarone F has a dose-dependent protective effect on PC12 cells from oxidative damage in effective dose range. Aquilarone A, Aquilarone C, CX14, CX16 has a protective effect on PC12 cells from oxidative damage with no dose-dependent characteristic. Aquilarone B, Aquilarone D, Aquilarone E showed no protective effect on PC12 cells from oxidative damage.Resin clumps are characteristic microstructure of Aguilari asinensis (Lour.) Gilg, ban be found it in every Aquilari asinensis (Lour.) Gilg. Co ntent of ethanol extract is significantly different in Aquilari asinensis (Lour.) Gilg. Zhejiang 2 Aquilari asinensis (Lour.) Gilg has the highest content of ethanol extract, is about 34.31%, and the least is Hainan 1 med icinal material, is 1.51%. Content level of ethanol extract can not explai n the quality of Aquilari asinensis (Lour.) Gilg. The highest content of e thanol extract is not the best quality of Aquilari asinensis (Lour.) Gilg. Content of essential oil is significantly different in Aquilari asinensi s (Lour.) Gilg, the highest content of essential oil is Zhejiang 2 medici nal materia, is about 41.5%, and the least is Changsha medicinal materia with a content of 0.075%. Content level of essential oil can not explain the quality of Aquilari asinensis (Lour.) Gilg at the same time. The cont ent of total chromone is stable in Aquilari asinensis (Lour.) Gilg, conte nt level can explain the quality of Aquilari asinensis (Lour.) Gilg. The content of Aquilarone B, Aquilarone C, Aquilarone D, Aquilarone E and (5S, 6R,7R,8S)-2-[2-(4-methoxy-phenyl)et-hyl]-5,6,7,8-tetraydroxy-5,6,7,8-tet-ra-hydrochromone are significantly different in Aquilari asinensis (Lou r.) Gilg too.Correlation analysis between the content of essential oil, the ethano 1 extract, the total chromone, Aquilarone B, Aquilarone C、Aquilarone D、Aq uilarone E and (5S,6R,7R,8S)-2-[2-(4-methoxy-phenyl)et-hyl]-5,6,7,8-tetra ydroxy-5,6,7,8-tet-ra-hydrochromone, we find that the content of ethanol extract has a significant correlation with the content of total chromone and Aquilarone E; The content of total chromone has a significant correla tion with each chromone; There are significant correlation between chromo nes, especially the correlation between the two isomers-Aquilarone C and (5S,6R,7R,8S)-2-[2-(4-methoxy-phenyl)et-hyl]-5,6,7,8-tetraydroxy-5,6,7,8-tet-ra-hydrochromone, the correlation coefficient is 0.932.ConelusionThe AE and the PE revealed analgesic activity in the hot-plate test. The BE revealed analgesic activity in writhing response induced by acetic acid.PE and BE are the effective parts of Aquilaria sinensis (Lour.) Gi lg with analgesic activity. From this study, we find that the previous id entification methods of Aquilari asinensis (Lour.) Gilg are not the best ones.Perform correlation analysis between the content of the essential o il, the ethanol extract,the total chromone, Aquilarone B, Aquilarone C, Aq uilarone D, Aquilarone E and (5S,6R,7R,8S)-2-[2-(4-methoxy-phenyl)et-hyl]-5,6,7,8-tetraydroxy-5,6,7,8-tet-ra-hydrochromone with SPSS19.0, we find that that the content of ethanol extract has a significant correlation w ith the content of total chromone and Aquilarone E; The content of total chromone has a significant correlation with each chromone; There are sign ificant correlation between chromones. It reveals that the significant co rrelation between the content of essential oil, the ethanol extract, the t otal chromone, Aquilarone B, Aquilarone C, Aquilarone D, Aquilarone E and (5S,6R,7R,8S)-2-[2-(4-methoxy-phenyl) et-hyl]-5,6,7,8-tetraydroxy-5,6,7,8-tet-ra-hydrochromone are useful in measuring the quality standards of Aq uilari asinensis (Lour.) Gilg. From this study, we establish method for t he determination of Aquilarone B, Aquilarone C, Aquilarone D, Aquilarone E and (5S,6R,7R,8S)-2-[2-(4-methoxy-phenyl)et-hyl]-5,6,7,8-tetraydroxy-5,6, 7,8-tet-ra-hydrochromone for the first time; Establish method for qualit y control of Aquilaria sinensis (Lour.) Gilg by the content of total chro mone,ethanol extract and chromone compounds. |
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