The Preliminary Construction And Analysis On The Metabolic Profiling Of Three Medicinal Dendrobes

This study established the extraction method of three species of medicinal dendrobium GC-MS samples. This kind of method was used for the separation and identification of medicinal dendrobium samples including tube seedlings, planting seedlings, and the producing dendrobium Fengdou in Anhui Huoshan, The results were manipulated and analysed by method of multivariate statistical analysis. The aims were that dendrobium metabolic profiling was preliminary constructed through the research of the composition and content changes of metabolites in medicinal dendrobium such as sugar, alcohols, organic acids, amino acids and others.Meanwhile this study was to obtain the variable importance in the projection(VIP) to the cluster pattern. According to test P value(P<0.05), we found the biomarkers which could be used as one of the evidences for the species identification and quality control of dendrobium. The study established a analysis technology platform of medicinal dendrobium metabolic profiling of methanol and water phase metabolites. It is of great technical significance and provides a new basis for further studying on metabolism of caulis dendrobii medicinal ingredients.The main results were as follows:1. The derivatization method for the pretreatment of dendrobium samples was optimized by GC-MS. In this study, oximation conditions of the samples were that the oxime time was 120 min, temperature was 37?, and the dosage of reagent( hydroxylamine hydrochloride) was 60 ?L. While derivatization conditions were that the derivatization time was 90 min, temperature 25?, and the dosage of reagent(99%BSTFA+1%TMCS)was 60?L.2. The methanol and water phase extract of dendrobium samples was mainly sugar and glucoside, amino acids, fatty acids, organic acids, sugar alcohols, etc, the chloroform phase extract was mainly alkanes, fatty acid, alkylalcohols, etc.3. The metabolic profile analysis results of methanol and water phase extract from Huoshan three species of medicinal dendrobium stems were as follows:(1) The total sugar content of different speices of dendrobium tube seedlings stems showed that Dendrobium huoshanense was more than Dendrobium Officinale,however Dendrobium moniliforme was less than Dendrobium Officinale, and the total amino acid content showed that Dendrobium Officinale was more than Dendrobium moniliforme, while Dendrobium moniliforme was more than Dendrobium huoshanense.(2) The total amino acid content of samples from one to there years stems of cultural Dendrobium Officinale had obvious variation. The samples of annual stems were more than two years, meanwhile the annual and the two years samples of stems were far more than three years. During the development of cultural Dendrobium huoshanense, the total amino acid content was the highest in three years stems.Comparing different speices of cultural debdrobium, the total sugar content was obviously different, the results showed that cultural Dendrobium huoshanense was far more than cultural Dendrobium Officinale.(3) The total sugar content of Dendrobium huoshanense among three species of dendrobium producing Fengdou was highest, the results showed that Dendrobium huoshanense was more than Dendrobium moniliforme, whlie Dendrobium moniliforme was far more than Dendrobium Officinale,and the content of total amino acid showed that Dendrobium huoshanense was more than Dendrobium moniliforme,while Dendrobium huoshanense was simlar with Dendrobium Officinal.(4) The content of total sugar was obviously different among growing stages of Dendrobium huoshanense.The result showed that planting seedlings of Dendrobium huoshanense was more than the producing Dendrobium huoshanense Fengdou, whlie the latter was far more than tube seedlings of Dendrobium huoshanense. Among growing stages of Dendrobium Officinale, the content of proline showed that tube seedlings of Dendrobium Officinale was far more than the producing Dendrobium Officinale Fengdou,and the latter was more than planting seedlings of Dendrobium Officinal.4. The biomarkers of the methanol and water phase extract from three species of medicinal dendrobium stems were as follows:(1) The metabolites from different species of dendrobium tube seedlings stems were studied to find biomarkers, these biomarkers were urea and citric acid.(2) The metabolites from dendrobium planting seedling stems were studied, the results were that six biomarkers were found during the different growth periods of Dendrobium Officinale, respectively mannose, galactose, carbamate, butanedioic acid,inositol and oleanane nitrile. In different species of dendrobium planting seedlings stems, biomarkers were sucrose, D- mannose, D-glucose, D- fructose, D- sorbose.(3) The metabolites from different species of dendrobium were studied to find biomarkers, these were sucrose, D- mannose, D-glucose, D- fructose, D- arloketone sugar, D- tager sugar, succinic acid, inositol and acrylic acid, glycerin.(4) The metabolites from different production stages(tube seedlings, planting seedlings,dendrobium producing Fengdou) were studied, the biomarkers from different production stages of Dendrobium Officinale stems were stearic acid, palmitic acid, oleic acid, acrylic, succinic acid, carbamate, and L- proline. And the biomarkers from different production stages of Dendrobium huoshanense stems were sucrose, D-(+)- mannose, 1- cyclohexene- 1- carboxylic acid, D-(+)- galactose.5. The use of cluster analysis and multivariate statistical analysis method was able to better distinguish different species of dendrobium, therefore, the two methods can be used to classified dendrobium.6. The three species of medical dendrobium metabolism profile was preliminarily constructed which were conducive to the in-depth study of the metabolic pathway and metabolic network of dendrobium.