Research Of Anatomy And Chemical Component Of Aquilaria Sinensis Induced By Plant Hormones

Aquilaria sinensis(Lour.)Spreng,a rare tree and source of agarwood,is widely planted in the subtropical zone of China.However,the quantity and quality of agarwood induced are unsatisfactory,so the development of agarwood industry is limited.As wild agarwood resources decrease and market demands increase,agarwood plantation and agarwood induced technology are rapidly developed,but lower quality and yield of agarwood induced are still great development challenges in the agarwood development.Agarwood induced technology by plant hormones is a new induction method that is first put forward in the wood industry but highly concenred rather than a mechnical and microorganism-inducing method.This study analyzes the distribution and form of dark secodary metabolites of aquilaria sinensis induced by plant hormones from the view of wood anatomy,explores the accumulation process of main compounds of agarwood induced,analyzes compound composition of agarwood induced by plant hormones and focuses on the critical agarwood essential oil extraction process via agarwood induction of Aquilaria Sinensis by plant hormones.(1)For the physiological process of agarwood induced and development of agarwood-induced technology,this study dissects and observes agarwood-induced samples by pouring and burning method,peeling method,worm bite method and chemical agent A method and indicates there are significant differences in the distribution and form of dark secondary metabolites,tissue regeneration,callose and crystals.Of all methods,only secondary metabolite deposition by chemical agent A is in lump,while the deposition region by other methods are in thin layer or point-like or bunchy.Preliminary statistics indicates that agarwood induced by chemical agent A method has the largest quantity because the agarwood region by chemical agent A method is the largest,followed by peeling method/poring and burning method that damages xylem,by fresh worm bite and by ring peeling method.However,dark secondary metabolites have different adherence amounts in the cell cavity,further adjudgment will be made combining with content determination and chemical analysis.Among all samples,no dark secondary metabolite deposition is found in 1-2 layers of parenchymal cells at the edge of interxylary phloem.Poring and burning method and the above-mentioned peeling methods stimulate the formation of tissue regeneration;No tissue regeneration is observed in aquilaria sinensis by chemical agent A,nor obvious lignification.Obvious lignification of cells is observed in the interxylary phloem of agarwood layer,although worm bite samples have no tissue regeneration due to short trauma duration.Calloses are deposited in new worm-bite places,indicating that callose acts in trauma defensive reaction.(2)Jasmonic acid methyl ester,ethephon and mixed agent in three concentrations were used to induce the generation of dark secondary metabolites.This study explores the distribution and area of those metabolites and makes GC-MS analysis for samples from the large induced area.The anatomical analysis indicates dark secondary metabolites contain the following cells in 180 days after jasmonic acid methyl ester,ethephon and mixed agent were applied to the surface of aquilaria sinensis peels:cork layer cells,phloem ray cells,phloem parenchyma cells,cambial cell,wood ray cells,phloem cells and ducts.It can be seen that plant hormones penetrate into standing aquilaria sinensis trees from peels and affect its cells to generate dark secondary metabolites.GC-MS analysis was performed for characteristic compounds of agarwood induced samples by jasmonic acid methyl ester and ethephon,which indicates that total concent and type of sesquiterpenes induced by mixed agent are more than those by the respective method.RNA detection indicates the induction rate of jasmonic acid methyl ester and ethephon was 52.8%and 37.34%respectively,decreased compared with RNA purity extracted from agarwood induced by mixed agent.(3)Silica gel column chromatography was carried out to separate aparwood abstracts.By Silica Gel Column Chromatography(hex-ane-acetone=3:2)and Gel Permeation Chromatography(GPC),organic solvent(CHC13)is dissolved into three ketones:Oxidoagarochromone A(1),brown glue-shaped,molecular formula C17H1404;Oxidoagarochromone B(2),light yellow crystalline solid(mp 142-144?),molecular formula C18H1605[m/z 313.1068[m+h]+(calcd for C18H1705:313.1076)];Oxidoagarochromone C(3),light yellow crystalline solid(mp 79-82?),molecular formula C18H1606[m/z 329.1031[m+h]+(calcd for C18H17O6:329.1025)].Compounds 1-3 have the same relative stereochemistry.HPLC analyzes AcOEt extractions.In the whole wood,no diepoxides were detected,nor from natural aparwoods.Silica Gel Column Chromatography and HPLC were carried out,and 2-(3-phenethyl)-chromone was separated from dark secondary metabolites by organic solvent CDC13 into 4 chromones:2-(3-phenethyl)-chromone,7,8-dyhydroxy-2-(3-phenethyl)-chromone,6-methoxy-2-[3-(4-methoxy-3-hydroxyphenylethyl)]chromone,7-hydroxy-2-[3-(2-hydroxyphenylethyl)]chromone.(4)Xylem and peels were quenced and extracted by methyl alcohol.Concentrating filtrate was decompressed to remove the solvent,compounds were purified by TLC and purfied compounds were identified by NMR.A purified compound was extracted from the xylem while two ones were obtained from peels.NMR analysis was performed for these compounds.The compounds from the xylem are 6-hydroxy-2-(3-phenethyl)chromone upon structure analysis.Two purified compounds were extracted from peels,one of which cannot be analyzed,while the other is(-)-Medioresinol.(5)Bath ultra-critical fluid equipment is used for aparwood essential oil extraction.The best process parameters of ultra-critical extraction are as follows:pressure 24Mpa,temperature 35?,flow 33 L/hr.The yield of aparwood essential oil by ultra-critical carbon extraction changes as pressure and flow,positively related with the pressure and flow,negatively related to the temperature.