| Agarwood is resinous wood formed from Aquilaria spp.or Gyrinops spp.of the family Thymelaeaceae in response to injury-induced defence.In China,agarwood is mainly derived from Aquilaria sinensis(Lour.)Gilg,and is a traditional and valuable herb commonly used in clinical medicine,with the effects of promoting Qi flow,relieving pain,warming the middle Jiao to arrest vomiting and promoting inspiration to relieve asthma,and is widely used in Traditional Chinese Medicine,Tibetan medicine and Japanese Kampo medicines.Kynam agarwood has been used in many medical cases as the highest quality agarwood,but for a long time it was scarce due to its resource rarity and its historic nature.In recent years,owing to a number of factors,including the technical support of artificial cultivation,the industry has taken shape and grown dynamically.However,the industry currently suffers from a lack of quality standards,an unclear number of generations of seedlings produced,confusion over varieties,varying levels of production management,and a weak awareness of the medicinal properties of Kynam agarwood.In order to effectively promote the qualitative development of the industry,this study explores the material basis of the chemical composition and active constituents of Kynam agarwood,elaborates on its scientific connotations as a high-quality agarwood,and provides a basis for the development of quality standards for Kynam agarwood.1.Through the analysis of pharmacopoeia test items related indexes of several batches of collected samples of agarwood,it was found that the high-quality herb Kynam agarwood recorded in the herbal literature did not meet the requirements of agarotetrol content and HPLC specific chromatogram added after the revision of 2015 edition of Chinese Pharmacopoeia.To solve this problem,the basic physicochemical properties and principal components of Kynam agarwood were further analyzed.The results showed that Kynam agarwood has the characteristics of high quality.This experiment was conducted based on the 2020 edition of the Chinese Pharmacopoeia to examine and analyze the ethanol extract content,microscopic characteristics,agarotetrol,and characteristic spectra of different sources of agarwood herbs.The results showed that all collected agarwood samples were in line with the characteristics of the pharmacopoeia in terms of morphology and microscopic characteristics.Among the 10 batches of imported agarwood samples and 17 batches of domestic agarwood samples collected(15 batches of normal agarwood and 2 batches of Kynam agarwood),there were 2 batches and 3 batches of unqualified samples for ethanol extract content and agarotetrol content,respectively.Among them,the collected two batches of Kynam agarwood had extremely low agarotetrol content,but their ethanol extract content was over 40%,which was far higher than the standards of the pharmacopoeia(therefore,before the changes in the detection items of the pharmacopoeia in 2015,Kynam agarwood was a high-quality medicinal herb).In our improved HPLC specific chromatogram,it can be found that there are multiple high response value chromatographic peaks in the low polarity part of Kynam agarwood.In response to these issues,we collected 12 batches of Kynam agarwwood and 12 batches of normal agarwood for ethanol extract content and total phenylethyl chromone content determination.The results showed that the ethanol extract content and total phenylethyl chromones(PECs)content of Kynam agarwood were significantly higher than those of normal agarwood(P-Value<0.01).In the correlation analysis,we found a significant correlation between the ethanol extract content and total PECs content in the collected samples,which also confirms the traditional view on the quality of agarwood,that is,the higher the content of alcohol extract(resin content),the better the quality of agarwood may be.These results indicate that Kynam agarwood has high-quality characteristics.2.A study was conducted using liquid chromatography-mass spectrometry(LC-MS)combined with untargeted metabolomics techniques to investigate the 2-(2-phenylethyl)chromones(PECs)components of Kynam agarwood and normal agarwood.The specific differences in PECs between Kynam agarwood and normal agarwood were characterized,and the common characteristic components of multiple batches of Kynam agarwood were also obtained,providing reference for the development of Kynam quality markers and quality standards,Provide scientific basis for further research and development and utilization of Kynam agarwood.In this study,a full-spectrum chemical composition analysis was performed using UPLC-Q-TOF-MS untargeted metabolomics technique to obtain statistically significant differential compositions of the two types of agarwood using 12 batches of Kynam agarwood and normal agarwood.The selection conditions were based on Pvalue<0.05,VIP value>1,FC value>2 or<0.5 to obtain the differential compositions,which were extracted and validated and identified one by one.44 and 11 differential compositions were obtained from the positive and negative ion modes,respectively,in which the contents of 2-(2-phenylethyl)chromones with different parent structures of FTPECs and THPECs were significantly different in the two types of agarwood.The semi-quantitative analysis of 18 FTPECs and 9 THPECs,which were obtained from two of the major parent nucleus structure groups after removal of the differential components of the pattern repeats,revealed that several FTPECs components(2-(2phenylethyl)chromone,2-[2-(4-methoxyphenyl)ethyl]chromone,qinanone c,6Hydroxy-2-[2-(4-methoxyphenyl)ethyl]chromone,6-Methoxy-2-[2-(4methoxyphenyl)eyhyl]chromone,6-Methoxy-2-(2-phenylethyl)chromone,2-[2-(4Hydroxyphenyl)ethyl]chromone,qinanone g,qinanone d,qinanone a,2-[2-(3Methoxy-4-hydroxyphenyl)ethyl]chromone)in the Kynam agarwood was significantly higher than that of normal agarwood(P value<0.01).Thirteen 2-(2phenylethyl)chromones were identified from the fingerprinting of the Kynam agarwood samples,of which five FTPECs were characteristic of Kynam agarwood.These components can subsequently be used to differentiate the quality criteria of Kynam agarwood.It also reveals the compositional connotations of Kynam agarwood as a high-quality agarwood.3.By using GC-MS analysis to compensate for the shortcomings of liquid quality data,we further obtained the differences in volatile chemical components between Kynam agarwood and normal agarwood.At the same time,we also obtained the common volatile components of Kynam agarwood through fingerprint analysis,providing data support for comprehensively revealing the material basis of Kynam agarwood.The 2-(2-phenylethyl)chromones are the most significant differential component between Kynam agarwood and normal agarwood,and in the analysis of the differences between 24 batches of Kynam agarwood and normal agarwood by GCMS demonstrated that all 12 volatile 2-(2-phenylethyl)chromones were high in Kynam agarwood,and that the abundance of 2-(2-phenyl)chromones and 2-[2-(4-methoxyphenyl)ethyl]chromones in Kynam agarwood,when The cleavage products upon heating may be responsible for the odour of Kynam agarwood and its more aromatic nature as incense.In this experiment,the volatile components of 12 batches of Kynam agarwood and 12 batches of normal agarwood were determined using the GC-Q-TOF-MS technique.Combined with the NIST 17 database(reverse match score>800)and relevant literature,a total of 81 volatile components of agarwood were identified,including 19 2-(2phenylethyl)chromones,36 sesquiterpenoids,9 organic acid compounds,8 aromatic compounds,5 alkanes,3 lactones and 1 aldehyde compound.The volatile components of the two types of agarwood were then obtained by non-targeted analysis and multivariate statistical analysis,with P-value<0.05,VIP value>1,FC value>2 or<0.5 used to screen for differences and identify 18 volatile components associated with differences in quality,including 12 2-(2-phenylethyl)chromones and 6 sesquiterpenes,which were present in higher concentrations in Kynam agarwood.These components are present in higher concentrations in Kynam agarwood than in normal agarwood and may be contributing to the more intensive aroma of Kynam agarwood than normal agarwood.4.In this study,virtual screening technology combined with in vitro activity screening was used to obtain the active ingredients of agarwood.Based on selfestablished small molecule database of agarwood and potential targets of agarwood,the reverse molecular docking technology was used to explore the effective ingredients and potential target of agarwood.The key targets in the docking results were studied using agarwood extract and monomer components for in vitro activity to verify the accuracy of the docking results and provide a basis for the selection of quality standards for Kynam agarwood.Molecular docking combined with network analysis revealed that the main active ingredients group in agarwood is 2-(2-phenylethyl)chromones,and that the FTPECs have a stronger binding activity to several targets including AMPK than the THPECs.In vitro cells models combined with western blot analysis of the AMPK activation capacity of Kynam agarwood and normal agarwood showed that both types of agarwood significantly activated AMPK in a dose-activity dependent relationship,and that the activation capacity was higher in the high,medium and low concentrations of Kynam agarwood than in normal agarwood.Therefore,it is possible that the Kynam agarwood has stronger activity at least in diseases involving AMPK-related targets.We further validated and tested the AMPK agonist activity of several FTPECs and THPECs in HEK 293T cells and found that 6,7-Dimethoxy-2-phenethylchromone,2-(2Phenylethyl)chromone,2-[2-(4’-Methoxyphenyl)ethyl]chromone and several other FTPECs were found to significantly activate AMPK in a dose-dependent relationship,and their activation abilities were all greater than those of the THPECs such as agarotetrol.Although further in vitro and in vivo activity studies are needed to confirm and reveal this,the present preliminary in vitro activity study of Kynam agarwood has demonstrated the excellent properties of Kynam agarwood as a high-quality agarwood.In general,this study reveals the scientific connotation of the high-quality of Kynam agarwood from multiple aspects such as total content of characteristic components,differential compositions,common compositions and the activity effect of the ingredients.It was found that the content of characteristic components PECs and ethanolic extracts in Kynam agarwood are significantly higher than that of normal agarwood,among which 13 FTPECs components of Kynam agarwood are significantly higher than that of normal agarwood,and these compositions can be used as marker ingredients to distinguish Kynam agarwood.A number of common FTPECs components were obtained from different sources of Kynam agarwood,including 2-(2phenylethyl)chromone,2-[2-(4-methoxyphenyl)ethyl]chromone,6-methoxy-2-[2-(4methoxyphenyl)ethyl]chromone,and 6-methoxy-2-[2-(4-methoxyphenyl)ethyl]chromone.methoxyphenyl)ethyl]chromone,6-Methoxy-2-[2-(2-phenylethyl)chromone have the potential to serve as signature ingredients for the establishment of quality standards for Kynam agarwood and also reveal the compositional connotations of Kynam agarwood as a high-quality agarwood.Kynam agarwood showed stronger activity in diseases involving AMPK related targets,and the activation ability of FTPECs is stronger than that of THPECs,such as agarotetrol,the differences in bioactivity between Kynam agarwood and normal agarwood may be related to the differences in the content of the different parent structural component groups of the 2-(2-phenylethyl)chromone component,and these results may provide a basis for the subsequent development of quality standards for Kynam agarwood and normal agarwood.These results provide some experimental basis for the subsequent establishment of quality standards and the development of new drugs,and partly reveal the scientific connotations of Kynam agarwood as a high-quality incense. |
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