Research On Quality Characteristics Of Agarwood Produced By Whole-tree Agarwood-inducing Technique

As a valuable fragrant medicine,agarwood is a resin-containing wood of Aquilaria spp.of Thymelaeaceae,which is widely used in medicine,perfume,religion and collection,and has a large market demand.A.sinensis(Lour.)Gilg is the only source of authentic plant for China's precious medicinal material agarwood.And because of excessive logging,wild agarwood resources have been on the verge of exhaustion.With the advent of "Whole-tree agarwood-inducing technique(Agar-Wit)" and its popularization in China and Southeast Asia,it has developed into the most widely used agarwood-inducing technology in China and the world's main agarwood region.But what is the chemical ingredient of the agarwood produced by Agar-Wit?How about its quality?And is it safe?A series of questions remain the doubts about the application of the new technology in industrialization.Therefore,this study focuses on the quality and safety of agarwood by Agar-Wit,Research and analysis the chemical composition,quality,possible exogenous pollutants and toxicity of this kind of agarwood,and the main findings are as follows:1.The quality index of agarwood produced by Agar-Wit was systematically analyzed.Its properties,microscopic identification characteristics,physicochemical identification characteristics and characteristic atlas are consistent with those of wild agarwood.They all conform to the Chinese Pharmacopoeia 2015 edition,and do not contain rosin acids.Based on the pharmaceutical standards,the properties,microscopic characteristics,TLC,physicochemical color reaction,extract,content determination,characteristic atlas and rosin acids of 20 batches of representative samples from five main producing areas,Hainan,Guangdong,Guangxi,Yunnan and Fujian,were determined and analyzed.The results showed that the properties,microscopic identification characteristics,physicochemical identification characteristics and characteristic atlas are consistent with those of wild agarwood.They all conform to the Chinese Pharmacopoeia 2015 edition,and do not contain rosin acids.The average contents of extract and agarotetrol in 20 batches of agarwood produced by Agar-Wit were 19.24%and 0.35%respectively,which were higher than the average values of wild agarwood samples.2.To make a systematic analysis of the agarwood produced by Agar-Wit,the Pb,Hg,As,Cd and Cu of 16 batches representative samples were in the reference range,and no pesticide residue and aflatoxin contamination were found from the representative samples.In order to investigate whether there are heavy metals,organochlorine pesticides,aflatoxin and other exogenous pollutants in agarwood produced by Agar-Wit,a method for the determination of Pb,Hg,As,Cd and Cu in agarwood was established.The contents of Pb,Hg,As,Cd and Cu in 16 batches of representative samples of agarwood by Agar-Wit were all within the reference limits.In addition,no pesticide residues such as DDT,HCH and PCNB were found in 20 batches of representative samples,and no aflatoxin contamination was found in 2 batches of representative samples.3.A more comprehensive assessment of the safety of the agarwood produced by Agar-Wit in the experimental animals was found,and the extracts had no obvious acute toxicity symptoms to KM mice and beagle dogs,and there was no significant long-term toxic reaction to SD rats.The acute toxicity and long-term toxicity of agarwood on KM mice and Beagle dogs after oral administration and 90 days feeding in rats were determined.It was found that the extract of agarwood produced by Agar-Wit had no obvious acute toxicity to KM mice,and the dosage was 760.5 times of the maximum dosage that clinical people intended to use.No obvious toxicity symptoms were found in the acute toxicity test of Beagle dogs by intragastric administration,and the dosage was converted to 300 times of the maximum dosage for clinical use.No obvious toxic reaction was found in the long-term toxicity test of SD rats at high,medium and low doses,and the maximum dosage was about 80 times as much as the daily dosage of human clinical use.4.A systematic study on the chemical constituents of agarwood produced by Agar-Wit showed that 2-(2-phenylethyl)chromone and sesquiterpenes were the main chemical constituents,and they all contained four characteristic sesquiterpenes,agarospirol,aristolene,guaiol and baimuxinal,with the relative content similar to that of the control agarwood sample.Twenty-one characteristic peaks identified in the UPLC fingerprint of agarwood by Agar-Wit were the skeleton types of 2-(2-phenylethyl)chromones commonly found in wild agarwood.The chemical constituents of agarwood produced by Agar-Wit were systematically studied by modern chromatographic separation and spectral analysis techniques.65 compounds were isolated and identified from it,including 41 2-(2-phenylethyl)chromone derivatives,15 sesquiterpenes,5 lignans and 4 other types of compounds.Among them,7 new compounds are common skeleton types of compounds reported in wild agarwood,and 8 compounds have significant anti-inflammatory activity.By analyzing the characteristic components of the samples produced by Agar-wit,it was found that 21 characteristic peaks were identified by UPLC fingerprint as skeleton types of 2-(2-phenylethyl)chromone derivatives compounds commonly found in wild agarwood.The agarwood by Agar-Wit all contained four characteristic sesquiterpenes,namely,agarospirol,aristolene,guaiol and baimuxinal,with the relative content similar to that of the control agarwood sample.The content of alcohol-soluble extract and agarotetrol in 135 batches of samples produced by Agar-Wit was much higher than that of reference wild agarwood,and the content of alcohol-soluble extract and agarotetrol area in different habitats and species was quite different.This study is the first time to systematically study the quality characteristics of agarwood produced by Agar-Wit,such as quality preparation,chemical composition and safety.It is found that the quality characteristics of agarwood produced by Agar-Wit are as follows.Its properties,microscopic identification characteristics,physicochemical identification characteristics and characteristic atlas are consistent with those of wild agarwood,which are in accordance with the provisions of the Chinese Pharmacopoeia 2015 edition and do not contain rosin acids.Its main chemical constituents are 2-(2-phenylethyl)chromones and sesquiterpenes,The relative contents of four characteristic sesquiterpenoids,agarospirol,aristolene,guaiol and baimuxinal,were similar to those of the control agarwood.The 21 characteristic peaks in UPLC fingerprints were the skeleton types of 2-(2-phenylethyl)chromone compounds commonly found in wild agarwood.the Pb,Hg,As,Cd and Cu of 16 batches representative samples were in the reference range,and no pesticide residue and aflatoxin contamination were found;their extracts have no obvious acute toxicity symptoms to KM mice and Beagle dogs,and no obvious long-term toxicity to SD rats.This study has guiding significance for optimizing and upgrading the Agar-Wit,and solving the problems of uncertain composition,quality and safety of agarwood in the popularization and industrialization of the Agar-Wit,then promote the industrialization and application of the new technology.It will lay a foundation for the clinical application and the development of large healthy products of agarwood produced by Agar-Wit.