| ObjectiveThere is only one quality control standard of aquilariae lignum resinatum in the2010edition of Chinese Pharmacopoeia. That is ethanol soluble extractive. It is difficult to evaluate the quality of this precious medicinal material on the basis of ethanol soluble extractive. The content of essential oil in aquilariae lignum resinatum is very low, so it is inappropriate to be the quality control standard of aquilariae lignum resinatum. Therefore, the modern analytical technique and the statistical method are applied to research the relationship between chromones and the active principles in the essential oil of aquilariae lignum resinatum. Then study the establishment of a quality evaluation system of aquilariae lignum resinatum. Provides the scientific basis for the modernization and standardization of quality standard and the establishment of GAP base construction of aquilariae lignum resinatum.Method1. The aquilariae lignum resinatum powders were reflux extracted with70%ethanol (solid-liquid ratio is1:10), and get the extracts. Adds some water to the extracts and extract with cyclohexane, then get the extracts of cyclohexane parts. Separated by silica gel column chromatography (200mesh). Purified by the solvent method, and get the target compound1. Identify the purity of compound1by TLC and HPLC. Identify the structure of compound1by nuclear magnetic resonance (NMR) spectroscopy. After extracting with cyclohexane, the aquilariae lignum resinatum powders were reflux extracted with methanol, and get the extracts. Separated by silica gel column chromatography (200mesh). Purified by the solvent method, and get the target compound2. Identify the purity of compound2by TLC and HPLC. Identify the structure of compound2by NMR.2. Reference to the2010edition of Chinese Pharmacopoeia, make the preliminary evaluation and identification of the aquilariae lignum resinatum. Thin layer chromatography(TLC) method was used to analyze the methanol extract and ether extract of aquilariae lignum resinatum. Compare two kinds of evolutionary systems and get tow TLC fingerprints of different polar compositions of aquilariae lignum resinatum.3. To make a full spectrum scan to ether extract of aquilariae lignum resinatum by HPLC-PDA. Confirm a wavelength which can provide steady baseline and more information. The6,7-dimethoxy-2-(2-phenylethyl) chromone was used as the reference peak. Establish the fingerprint, and obtain common peaks information. Similarity analysis, cluster analysis and principal component analysis. The Chinese pharmacopoeia "Similarity Evaluation System edition A for Chromatographic Fingerprint of TCM" was used to evaluate the similarity. Then perform the hierarchical cluster analysis and principal component analysis with SPSS16.0.4.6,7-dimethoxy-2-(2-phenylethyl) chromone was determined by UPLC with ACQUITY BEH Shield RP18column(2.1mm100mm,1.7μm)by gradient elution using acetonitrile and0.4%phosphoric acid in water as the mobile phase at the flow rate of0.3mL·min-1; The detection wavelength was234nm with column temperature at35℃.5. The content of total chromone in aquilariae lignum resinatum was determined by the chromatopto-metry of molybdenum phospho tungstate-lithium sulfate.5,6,7,8-tetrahydroxy-2-(3’-hydroxy-4’-methoxyphenethyl)-5,6,7,8-tetrahydro-4H-chromone was taken as the reference substance.6. Ursolic acid was used as the reference substance. To determinate the total triterpene in vanillin-glacial acetic acid, perchloric acid system by spectrophotometry.7. Components in Volatile Oil of aquilariae lignum resinatum were extracted by cold-maceration with ether and perform GC-MS analysis.8. Perform correlation analysis between the content of6,7-dimethoxy-2-(2-phenylethyl) chromone, the total chromone, the total triterpene, ether extract and the benzylacetone with SPSS16.0. Get the internal relations between the different indexes.Results1Two chromone compounds were isolated and identified as6,7-dimethoxy-2-(2-phe-nylethyl) chromone5,6,7,8-tetrahydroxy-2-(3’-hydroxy-4’-methoxyphenethyl)-5,6,7,8-tet-rahydro-4H-chromone.2. Established the thin-layer chromatography of different polar compositions of aquilariae lignum resinatum and got the TLC fingerprints. The TLC sports developed were fairly clear and separated very well. It can provide a lot of chemical composition informations. Three batches of aquilariae lignum resinatum which collected from Qing ping Market and Viet Nam were identified as fake and shoddy products by TCL.3. Established the HPLC fingerprint of aquilariae lignum resinatum.6,7-dimethoxy-2-(2-phenylethyl) chromone was used as the reference peak. There were12common peaks in the fingerprint. By similarity calculation, nine batches of aquilariae lignum resinatum were differentiated, and their similarity was0.541-0.983. By the cluster analysis and principal component analysis, nine batches of aquilariae lignum resinatum were divided into three classes.4. Established the UPLC method for determination of6,7-dimethoxy-2-(2-phenylethyl) chromone in aquilariae lignum resinatum.6,7-dimethoxy-2-(2-phenylethyl) chromone was separated clearly and respectively in13min, resolution is larger than1.5. The linear range of6,7-dimethoxy-2-(2-phenylethyl) chromone was0.0074～-0.1235μg, and the linear regression equation was Y=15507229.64952X-20016.04908(r=0.9997). The average recovery was95.69%with RSD=1.89%(n=6). There is great difference among The contents of all batches of aquilariae lignum resinatum. The content of6,7-dimethoxy-2-(2-phenylethyl) chromone in the range of0.45to14.53mg·g-1.5. Established the best extraction technique and the optimum color conditions.5,6,7,8-tetrahydroxy-2-(3’-hydroxy-4’-methoxyphenethyl)-5,6,7,8-tetrahydro-4H-chromo-ne had a good linear relationship with the absorbance in the range of0.00886to0.04430mg, and the linear regression equation was y=4.8488x+0.0290(r=0.9994). The average recovery was95.69%with RSD=2.61%(n=6). There is great difference among the contents of all batches of aquilariae lignum resinatum. The content of total chromone in the range of15.93to65.78mg·g-1.6. Established the best extraction technique and the optimum color conditions. Ursolic acid had a good linear relationship with the absorbance in the range of0.0212to0.1696mg, and the linear regression equation was y=4.8488x+0.0290(r=0.9994). The average recovery was95.69%with RSD=2.61%(n=6). There is great difference among the contents of all batches of aquilariae lignum resinatum. The content of total triterpenoid in the range of19.46to86.19mg·g-1.7. Determined the content of ether extract of each batch of aquilariae lignum resinatum, obtained the total ion current. Some components were identified, including active component benzylacetone and the feature component agarospirol. Others components were identified, including some sesquiterpenoids, fatty acids, oleic acid, aromatic compounds. The content of common components were determined through the percentage composition.8. The statistics analysis showed that, there were high positive correlation between the content of ether extract and the content of6,7-dimethoxy-2-(2-phenylethyl) chromone(r=0.83, P<0.01), there were high positive correlation between the content of ether extract and the content of benzylacetone(r=0.864, P<0.01), There were moderate positive correlation between the content of ether extract and the content of total chromone (r=0.673, P<0.05). The correlation coefficients of the content of ether extract and the content of6,7-dimethoxy-2-(2-phenylethyl), total chromone, benzylacetone are higher than the correlation coefficients of ethanol soluble extraction and these three indexes. Conclusion:1Two chromone compounds were extracted and separated from aquilariae lignum resinatumThe main chemical compositions in aquilariae lignum resinatum include volatile oil, chromone, triterpene, fatty acids and so on. Among them, the chromone compounds are many and varied. Two chromone compounds were isolated and identified as6,7-dimethoxy-2-(2-phenylethyl) chromone. The chromone compounds of aquilariae lignum resinatum were of potential value in exploitation and utilization. It worth further research.2Preliminary establishment of quality evaluation system of aquilariae lignum resinatumThe modern analytical technique is applied to establish TLC, HPLC fingerprint, content determination and GC-MS analysis on the ether extract. Quality evaluation system of aquilariae lignum resinatum can be established and provides a scientific basis and technical reference for the establishment quality standard and GAP base construction of aquilariae lignum resinatum.3. It was found that there are correlations between the different indexes of aquilariae lignum resinatumThe statistics analysis showed that, there were high positive correlation between the content of ether extract and the content of6,7-dimethoxy-2-(2-phenylethyl) chromone, there were high positive correlation between the content of ether extract and the content of benzylacetone, There were moderate positive correlation between the content of ether extract and the content of total chromone. These results suggested that the content of ether extract is a good index to reflect the trend of changes in the content of6,7-dimethoxy-2-(2-phenylethyl) chromone, the content of benzylacet-one and the content of total chromone. So it is possible to greatly simplify the quality evaluation method. The correlation coefficients of the content of ether extract and the content of6,7-dimethoxy-2-(2-phenylethyl), total chromone, benzylacetone are higher than the correlation coefficients of ethanol soluble extraction and these three indexes.These results suggested that the content of ether extract can reflect more the quality of aquilariae lignum resinatum than ethanol soluble extraction. |
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