| Objective:Agarwood,found throughout Guangdong,Guangxi Hainan in China,and Vietnam,Indonesia,Kampuchea,Malaysia,and other Southeast Asian countries,is a fragrant resinous heartwood from species of Aquilaria and Gyrinops trees.Agarwood divided into domestic and imported products,domestic agarwood is mainly harvested from species of Aquilaria sinensis,,imported agarwood is mainly from Aquilaria malaccensi and Aquilaria crassna.The formed way and species are the main factors for the quality and price of Agarwood.Although DNA identification technology can identify the species of agarwood,different formed ways of the same species agarwood can not be distinguished by this technology.The specific species and the specific formed ways will produce a specific chemical composition.So,it is a good way to identify the species,quality control and quality classification through the analysis of those specific chemical composition.At present,it is not clear whether there are some differences between different kinds of agarwood,and there is no method to screen and identify these different marker.This paper was mainly focus on distinguishing and assorting the different types of agarwood through the multivariate statistical methods and unidimensional analytical methods by gas chromatography-mass spectrometry(GCMS)and liquid chromatography coupled with mass spectrometry(LCMS),and found out the markers between the different group of agarwood,established the method for identification of the intrinsic quality of agarwood.These studies can provide some references for screening and identification of different marker in agarwood and provide the basis for species identification,quality control and quality classification of agarwood.Methods:The 2015 edition of the Chinese Pharmacopoeia method was used in alcohol soluble extractives.HPLC analysis was performed to determine of agarotetrol on a Altima C18(150 mm×4.6 mm,5 μm),eluted with a mobile phase of water with 0.1%formic acid and acetonitrile in gradient mode with the flow rate of 0.7 mL ·min-1,the detection wavelength was set at 252 nm,the column temperature was 30℃.Exploring the quality classification of agarwood combinated with the content of the alcohol soluble extractives and agarotetrol.GC-MS analysis were performed using a angilent 7890BGC-5977A GCMS and equipped with a HP-5 MS capillary fused silica column(30m×0.25mm×0.25μm)(The oven temperature program initiated at 90℃,held for 3 min then rose at 10 ℃/·min-lto 180 ℃,held for 3 minand then rose at 3 ℃/min to 280 ℃,held for 10 min then rose at 5 ℃/min to 300 ℃,held for 8 min;carrier gas:He(99.999%),with a flow rate of 1 mL/min;injector temperature,250℃,splitless.A sample of 1 μL was injected in the split mode injection.Mass spectra were taken at 70 eV.The m/z valueswere recorded in the range of 50-500 amu.).The data are preprocessed and imported into Simca-P software for data processing and group analysis.UPLC condition(Shimadzu Nexera LC-30A(Shimadzu),InertSustainSwiftTM C18 column(2.1 mm × 150 mm,1.9 μm,Shimadzu-GL).The mobile phases were acetonitrile(A)and 0.1%formic acid-water(B)(10%A at 0-3 min,10%-30%A at 3-8 min,30%-50%A at 8-25 min,50%-100%A at 25-32 min,100%A at 32-35 min,flow rate:0.3 mL · min-1,the injection volume:10 μL).AB-Sciex 5600 Triple TOFTM mass spectrometer(ESI voltage,4500 V;nebulizer gas,55;auxiliary gas,55;curtain gas,35;turbo gas temperature,500 ℃;declustering potential,100 V;collision energy,10 eV;The TOF mass scan range:m/z 100-2000,the product ion scan range:m/z 50-2000(CE=45)).The data are preprocessed and imported into Simca-P software for data processing and group analysis.Results:Agarotetrol in fifty-two kinds of agarwood is 0.10-6.60%,the alcohol soluble extractives is 6.07-57.06%,the alcohol soluble extractives is have no relevant between formed ways and species,but the content of agarotetrol is have some relevant between formed ways and species.Nineteen differential markers,based on GCMS technology,were found out thro-ugh the multivariate statistical methods and unidimensional analytical methods,of which 5 2-(2-phenylethyl)chromones,5 sesquiterpenes and other compouds.13 marke-rs were checked between cultivated and wild A.sinensis,four of which,2-(2-phene-thyl)chromone,6,7-dimethoxy-2-(2-phenethyl)chromone,5,8-dihydroxy-4a-methyl-4,4a,-4b,5,6,7,8,8a,9,10-decahydro-2(3H)-phenanthrenone(isomer 1),hentriacontane(isomer 3),were significant difference between these two groups.2 sesquiterpenes markers between cultivated and wild A.crassna.A sesquiterpene and a 2-(2-phenylethyl)chr-omone markers between cultivated A.sinensis and cultivated A.crassna,5,8-dihydr-oxy-4a-methyl-4,4a,4b,5,6,7,8,8a,9,10-decahydro-2(3H)-phenanthrenone(isomerl)was significant difference between these two groups.Seven markers were checked in bo-th two groups(wild A.sinensis with wild A.crassna agarwood and wild A.sinens-is with wild A.malaccensis agarwood),hentriacontane(isomer 3)was the significant marker.The wild A.crassna and A.malaccensis have more sesquiterpenes and 2-(2-phenylethyl)chromones by comparing of the peak areas of fifty-two kind of samples.At the same time,cultivated A.sinensis have more sesquiterpenes and 2-(2-phenylethyl)chromones than wild A.sinensis and have more sesquiterpenes than cultivated A.crassna,wild A.sinensis have the most alkanes.The total peak areas of different components in those five different agarwood groups,sesquiterpenes and 2-(2-phenylethyl)chromones had some difference between cultivated and wild A.sinensis,alkanes and sesquiterpenes had some difference between wild A.sinensis and wild A.malaccens,alkanes had some difference between wild A.sinensis and wild A.crassna,other groups and components had not any difference.The cluster classification showed that the cultivated agarwood could be clustered into one group,and most natural agarwood could be clustered into other group.One hundred and twenty-three differential markers,based on LCMS technology,were found out through the multivariate statistical methods and unidimensional analytical methods,sixty-two of which were identified by MS,MS2 and references,and twenty-three of them were potential new compounds.Forty-five markers were checked between cultivated and wild A.sinensis,fifteen of which were significant difference between these two groups,2-(2-phenylethyl)chromone,7-hydroxy-2-(2-phenylethyl)chromone,6-methoxy-2-(2-phenylethyl)chromone(isomer 2),6,8-dihydroxy-2-(2-phenylethyl)chromone(isomer 1),6-methoxy-2-[2-(4-methoxyphenyl)ethyl]chromone,dehydroxy AH21 were the main markers.Twenty-nine markers were checked between cultivated and wild A.crassna,seven of which were significant difference between these two groups,6,8-dihydroxy-2-(2-phenylethyl)chromone(isomer 1),AH21(isomer 1),methoxy AH21(isomer 1),2,3-dihydroxy-5-phenethyl-2,3-dihydro-lah-oxireno[2,3-f]chromen-7(7bh)-one(isomer 2),dehydroxy AH21(isomer 2)were the main markers.Thirty-six markers were checked between cultivated A.sinensis and cultivated A.crassna,three of which were significant difference between these two groups,AH 12(isomer 3)were the main markers.Thirty-five markers were checked between wild A.sinensis and wild A.malaccensis,thirteen of which were significant difference between these two groups,6-methoxy-2-[2-(4’-hydroxy-3’-methoxyphenyl)ethyll]chromone(isomer 2),6,8-dihydroxy-2-[2-(3’-hydroxy-4’-methoxyphenyl)ethyl]chromone(isomer 2),agarotetrol,6-hydro xy-7-methoxy-2-(4’-hydroxy-3’-methoxyphenethyl)chromone(isomer 2),2,3-dihydroxy-5-phenethyl-2,3-dihydro1-lah-oxireno[2,3-f]chromen-7(7bh)-one(isomer 1)were the main markers.Twenty markers were checked between wild A.sinensis and wild A.malaccensis,two of which were significant difference between these two groups,6-nethoxy-2-[2-(4’-hydroxy-3’-methoxyphenyl)ethyl]chromone(isomer 2),6,8-dihydroxy-2-[2-(3’-hydroxy-4’-methoxyphenyl)ethyl]chromone(isomer 1)were the main markers.Six markers were checked between wild A.sinensis and wild A.malaccensis,there was no significant difference between these two groups.The results also showed significant characteristics of hydroxyl and methoxy substituted(phenyl)2-(2-phenylethyl)chromones may be effective for identification of cultivated and wild agarwood,5,6,7,8-tetrahydro-2-(2-phenylethyl)chromones and bi-2-(2-phenylethyl)chromones may be the key components to distinguish between different species of agarwood.The total peak areas of four different 2-(2-phenylethyl)chromones were sorted,5,6,7,8-tetrahydro-2-(2-phenylethyl)chromones,bi-2-(2-phenylethyl)chromones and tri-2-(2-phenylethyl)chromones had some difference between five groups agarwood.The cluster classification showed that the cultivated and wild agarwood could be clustered into different group.Conclusion:This paper established an analytical method based GCMS and LCMS combined with multivariate statistical analysis and unidimensional statistical analysis methods for screening and identification of different marker in agarwood.Ninteen markes,based on GCMS and NIST 14,were identified and four of which were significant difference.Sixty-two markes,based on LC-ESI-QTOF and references,were firstly identified and twenty-seven of which were significant difference,twenty-three of which were potential new compounds.The difference between cultivated and wild agarwood may be closely related to the component of hydroxyl and methoxy substituted(phenyl)2-(2-phenylethyl)chromones,5,6,7,8-tetrahydro-2-(2-phenylethyl)chromones and bi-2-(2-phenylethyl)chromones may be the key components to distinguish between different species of agarwood.To a certain extent,Alkanes,sesquiterpenes and 2-(2-phenylethyl)chromones can represent the quality of agarwood,alkanes content were lower,sesquiterpenes and 2-(2-phenylethyl)chromones content were higher,the quality may be better,otherwise the opposite.The cluster classification showed that the cultivated and wild agarwood could be clustered into different group,this method can be used to cluster and distinguish different quality agarwood.This papers provide new idea and method for the identification,quality control,quality classification of agarwood. |
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