Agarwood Chemical Composition Analysis And Anti-atherosclerosis Mechanism Research

Lignum Aquilariae Resinatum?Chinese agarwood?is the resin lingum of Aquilaria sinensis?Lour?.Gilg,possessing the actions of promoting qi to relieve pain,arrestting vomiting by warming the stomach,and promoting inspiration to relieve asthma.It is used for the treatment of distension and pain in the chest and abdomen,vomiting or hiccupping due to cold in the stomach,qi and kidney-deficiency syndrome.Chinese agarwood is the main component of more than 160 kinds of Chinese traditional patent medicines,such as Bawei Chenxiang Powder,Bawei Qingxin Chenxiang Powder,Chenxiang Huaqi Pill,Zaizao Pill,and so on.Therefore,a comprehensive chemical analysis of Chinese agarwood was carried out by extensive methods,including chiral-phase HPLC,computational ECD,off-line two-dimensional liquid chromatography,serially coupled RPLC-HILIC,and quantitative NMR.Furthermore,the animal models of high-fat diet?HFD?-induced ApoE-/-mice and HFD-induced hamster were established to evaluate the anti-atherosclerosis effect of the extract of Chinese agarwood,and the differential metabolites were clarified by metabolomics and lipidomics approaches.The results include:1.Literature reviewThe research progress of 2-?2-phenylethyl?chromones,pharmacological activities,and the quality evaluation methods of agarwood have been reviewed.2-?2-Phenylethyl?chromones are mainly classified into simple 2-?2-phenethyl?chromones,5,6,7,8-tetrahydro-2-?2-phenylethyl?chromones,5,6:7,8-diepoxy-2-?2-phenylethyl?chromones,2-?2-phenylethyl?chromones dimers,and others.Pharmacological studies suggested that the crude extracts and pure compounds from agarwood have antispasmodic,antimicrobial,anti-inflammatory,neuroprotective,and cytotoxic activities.The determination of multi-component content and fingerprint of agarwood based on high performance liquid chromatography and liquid chromatography-tandam mass spectrometry were mainly used to evaluate the quality of agarwood.However,the chemical components of agarwood have not been clearly revealed.The quality markers and effective quality evaluation methods have not been established.The mechanism of pharmacological activity of agarwood,especially lipid-lowering,has not yet been elucidated.It is difficult to ensure the safety and effectiveness of clinical medication of agarwood,which hinders the healthy development of agarwood industry.2.Study on the chemical structures of 2-?2-phenylethyl?chromone dimers in Chinese agarwoodOn the basis of previous chemical studies on Chinese agarwood,the absolute configurations of sixteen new 2-?2-phenylethyl?chromone dimers,including four pairs of enantiomers,were confirmed through comparison of their experimental and calculated electronic circular dichroism spectra,chiral-phase HPLC,and single-crystal X-ray diffraction analyses.A putative biosynthetic pathway of the representative structures via a diepoxy derivative of a chromone with a nonoxygenated A-ring is also proposed.Twelve compounds were also evaluated for their inhibitory effects on NO production in LPS-stimulated RAW264.7 cells.All these tested compounds showed inhibition against NO production.3.Chemical clarification of Chinese agarwoodThe mass fragmentation behaviors of 2-?2-phenylethyl?chromones were proposed using LCMS-IT-TOF,such as simple 2-?2-phenethyl?chromones,5,6,7,8-tetrahydroxy-2-?2-phenylethyl?-5,6,7,8-tetrahydrochromones,5,6:7,8-diepoxy-2-?2-phenylethyl?chromones,2-?2-phenylethyl?chromone dimers,etc.Off-line two-dimensional liquid chromatography-tandam mass spectrometry method was developed for separation and identification of the chemical components of the Chinese agarwood.Nine 2-?2-phenylethyl?chromones were identified from one-dimensional chromatography,and their structures were further confirmed by 1H NMR.A total of 187 compounds were identified from the two-dimensional chromatography based on comparing mass spectral profiles with reference compounds as well as literature data,including forty-seven simple 2-?2-phenethyl?chromones,seven 2-?2-phenylethyl?chromone glycosides,twenty-five 5,6,7,8-tetrahydroxy-2-?2-phenylethyl?-5,6,7,8-tetrahydrochromones,six chlorinated-tetrahydroxy-2_{2-phenylethyl}-5,6,7,8-tetrahydrochromones,nine trihydroxy₂??2-phenylethyl?-5,6,7,8-tetrahydrochromones,four dihydroxy-2-?2-phenylethyl?-5,6,7,8-tetrahydrochromones,four 5,6:7,8-diepoxy-2-?2-phenylethyl?chromones,nine 5,6/7,8-epoxy-2-?2-phenylethyl?chromones,seventy 2-?2-phenylethyl?chromones dimers,four 2-?2-phenylethyl?chromones trimers,and two other compounds.Among them,sixty-four compounds were firstly identified in the resin lingum of Aquilaria sinensis?Lour?.Gilg,in particular 37 potential new compounds.4.Quantitative analysis of chemical compounds of Chinese agarwoodIn this study,a work-flow that integrates serially coupled RPLC-HILIC,q1H NMR,online parameter optimization,RRCEC matching is proposed to totally fulfill the requirements of precise quality assessment for Chinese agarwood.Simultaneous determination of 26 compounds was conducted,in total,in sixteen batches of Chinese agarwood,four batches of adulterants,and several relevant materials of Aquilaria sinensis?such as fruits,stems,leaves,callus,and NaCl stimulated callus,one batch for each?after method validation.In particular,authentic compound-free quantification was achieved for eight 2-?2-phenylethyl?chromone derivatives?PCDs?.Moreover,PCDs should be able to act as the Q-makers for Chinese agarwood,whereas the minor distributions of those primary metabolites should also be taken into account as the negative factors for the quality.5.Effects of agarwood extract on blood lipids disorder and atherosclerosisTwo kinds of hyperlipidemic animal models induced by high fat diet?HFD?,ApoE-/-mice and hamsters,were employed for evaluation of the effects of agarwood extract on blood lipids disorder and atherosclerosis.Based on the study of ApoE-/-mice,agarwood extract significantly reduced plasma levels of total cholesterol?TC?,low density lipoprotein?LDL-c?,and plaque area of the aortic root in ApoE-/-mice.It was confirmed that the agarwood extract inhibited the development of atherosclerosis by improving the disorder of lipid metabolism.As for the study of hamsters,agarwood extract also remarkably reduced the levels of TC,triglyceride?TG?and LDL-c,increased the content of high-density lipoprotein?HDL-c?in the hamsters plasma.These results proved that the agarwood extract decreased the atherosclerosic index AI?AI=LDL-c/HDL-c?of HFD-induced hamsters.In addition,agarwood extract also obviously reduced the epididymal fat index of hamsters.This confirmed that agarwood extract restricted body fat rate caused by hyperlipidemia-induced obesity.Moreover,agarwood extract effectively inhibited the accumulation of neutral fat in the liver tissue of hamsters,indicating that the agarwood extract inhibited the occurrence of non-alcoholic fatty liver.6.Metabolomics and lipidomics studies of agarwood extract on HFD-induced hamstersThe hamsters were randomly divided into five groups:control group?CG?,control+agarwood extract?CAg?,the HFD-induced group?HG?,Ezetimibe group?EG?,HG+agarwood extract?Ag?.The hamster plasma samples from five groups were carried out using hybrid quadrupole Orbitrap mass spectrometer?Q Exactive,ThermoFisher Scientific?.In principal component analysis?PCA?model,CG and HG groups were clearly distinguishable,while Ag group was between the two groups,indicating that the metabolites of hamsters with hyperlipidemia had a trend to become normal after agarwood extract treatment.In the orthogonal partial least squares discrivation analysis?OPLS-DA?model,HG group was completely separated from CG group,for the considerable change of PCs,LysoPCs,SMs,fatty acid and other dendogenous metabolites during the hyperlipidemia injury.After administration of agarwood extract,the PCs,LysoPCs,SMs,fatty acid exhibited significant changes,suggested that it is likely that agarwood extract alleviates hyperlipidemia through regulating the disturbed glycerophospholipid metabolism pathway.Furthermore,a hybrid quadrupole Orbitrap mass spectrometer?Q Exactive,ThermoFisher Scientific?was used to perform a comprehensive lipid metabolism profiling analysis on hamster plasma samples.The results showed that eighteen lipids including PCs,SMs,and fatty acids in plasma changed significantly after feeding high-fat diet to hamsters.After administering agarwood extract,fourteen lipids had a trend to become normal.Therefore,these compounds PC?18:0/18:2?,PC?16:0/18:1?,PC?18:2/16:0?,PC?22:6/16:0?,PC?20:3/16:0?,PC?18:0/18:1?,PC?18:0/0:0?,PC?20:4/18:0?,PC?16:0/0:0?,PC?16:0/20:4?,SM?d18:1/16:0?,FA?20:4?,and FA?18:2?might be recognized as potential biomarkers for predicting atherosclerosis.In summary,we conducted a comprehensive chemical analysis of Chinese agarwood,clarified the efficacy of agarwood against atherosclerosis,the metabolic pathways that may be affected by agarwood extract,and figured out the potential biomarkers for predicting atherosclerosis.This study will offer scientific evidence for expanding the clinical application and rational use of agarwood,enhancing the medicinal value of precious Chinese medicines,thus promoting industrial development of Chinese agarwood.