Studies On Multi-Dimensional Fingerprints Of Dendrobium Officinale

Dendrobium officinale is a precious traditional Chinese herbal medicine which belongs to Dendrobium species, Orchidaceae. Due to its unique growth condition and excessive utilization, the wild resources of Dendrobium officinale are nearly exhausted, resulting in market confusion. Multi-dimensional fingerprints of Dendrobium officinale including high performance capillary electrophoresis fingerprints, SRAP molecular fingerprints and polysaccharide fingerprints were constructed in this paper in order to promote the healthy development of this industry(1) Construction of the fingerprints by high performance capillary electrophoresis:Though optimization of extraction and the separation conditions, 15 characteristic fingerprint peaks were identified from the ethanol extracts of Dendrobium officinales from 10 different producing areas by high performance capillary electrophoresis with 40 mM borax (pH 9.5, contains 5% methanol, v/v) as the background electrolyte and 20% ethanol as extraction solvent. Among these peaks, the ninth peak was chosen as reference peak for calculating the relative retention time and the relative peak area of the other peak. The quantitative data of the fingerprints were analyzed by similiarity analysis, principal component analysis and systematically cluster analysis. The results showed that the samples from 10 different producing areas could be clustered into three groups. The first consisted of samples from Yandangshan in Zhejiang province, Fuyang in Zhejiang province, Jiangxi province, Yunnan province, Guangxi province and Guangdo province. The second was composed of the samples from Fujian province and Anhui province. The third group contained only one sample from Hunan province. In addition, four characteristic fingerprint peaks (X₂, X₇, X₁₁, X₁₃) were selected to construct discriminant analysis model, unclassified samples could be easily classified by calculating the relative peak areas of the four characteristic fingerprint peaks.(2) Construction of DNA fingerprints by SRAP technology: SRAP reaction system was optimized and the optimal reaction system contained 30 ng DNA template, 0.25 mM dNTT, 0.1 mM primer, 2.0 mM Mg²⁺, 1.5 U Taq DNA polymerase and 2.5μL 10×PCR Buffer where the total volume was 25μL. The total of 77 bands was amplified by 4 pairs of primer and 39 of them were polymorphic accounting for 51%. Among all primers, the combination of Me5—Em6 showed the highest level of polymorphism and Rp value. The dendrogram constructed with SRAP molecular makers revealed that Dendrobium officinale from seven different producing areas could be divided into 4 groups: Dendrobium officinales from Wenzhou in Zhejiang province, Guangxi province, Guangdong province were the first cluster; Dendrobium officinale from Hunan province was the second; Dendrobium officinale from Yan dangshan in Zhejiang province and Jiangxi province were clustered in the third. The last contained only the sampe from Yunnan province.(3) Construction of fingerprints of polysaccharides: Enzymatic fingerprint of Dendrobium officinale polysaccharides were investigated by fluorescence-assisted carbohydrate electrophoresis. Polysaccharides of Dendrobium officinale from different producing areas were hydrolyzed byβ-mannase and cellulose, respectively, and then separated by polyacrylamide gel electrophoresis. Some differences were observed in band location and fluorescence intensity of oligosaccharides with high polymerization degree. The polymorphism level produced byβ-mannase was higher than that by cellulase. Enzymatic fingerprints of polysaccharides from seven different Dendrobium species were also analyzed with this method and results indicated that there were significant differences not only in oligosaccharides with high polymerization degree but also in oligosaccharides with low polymerization degree. The polymorphism level produced by cellulose was higher than byβ-mannase.The above results will not only provide the theoretical bases to identify the species of Dendrobium officinale, but also supply the methods for the quality evaluation of traditional Chinese medicine (TCM).