DNA Barcoding Identification Of Hemsleya Macrosperma And Its Adulterants

Hemsleya macrosperma is one kind of perennial climbing herb from the Cucurbitaceae Hemsleya macrosperma. Its tuber can be used as traditional Chinese medicine,it mainly produced in Yunnan province. Hemsleya macrosperma used to extract Hemslein and be as APIs for “hemsleyadine tablet”and ethnodrug. We identify 13 samples of Hemsleya macrosperma, its adulterants, and 54 samples of medicinal materials through the methods of morphological taxonomy, DNA barcoding, chemical composition analysising. In this study, the new method was constructed which to achieve the goal of identifying Hemsleya macrosperma quickly and accuratly, provide more basis and references for molecular identification. It also lays the foundation for the development and utilization of Hemsleya macrosperma. The main findings are as follows:(1)Combining the main morphological indicators of color, shap, surface characteristics of Hemsleya macrosperma for classification. Their shape and size are disparate which can be divided into five categories.The first category: similar sphere, darker color and the verrucose surface has cracks. Due the si gnificant depression of stem, the shape is very similar to a bowl, representative samples are 1,2,34, representative sample of 30 without exception of stem.The second category: flat round, lighter in color, verrucose surface without obvious cracks, stem is not protruding or depressing. Representative samples are 6,10.The third category: flat round, deeper color, surface with cracks, sunken stem. 17, 20 are representative samples.The fourth category: oblate, the bottom is relatively flat, brown, surface with cracks distributes verrucae, depressing stem, representative sample are 3, 28, 41.The fifth category: irregular, verrucose surface with cracks, stem is depression, representative sample are 36, 38. According to the gathering place, property characteristics, select representative samples for subsequent molecular experiments.(2)To establish a new method for identifying the original species and its adulterants by DNA barcoding, the psbA-trnH, ITS1- ITS4, ITS2 sequences were analyzed. The results showed the amplificated efficiency of the three sequences are 100%, and they can distinguish Hemsleya macrosperma and its adulterants successfully. Combing with the sequences which from the GenBank database and experimental data, we can verify the medicinal materials of Hemsleya macrosperma. The amplificated efficiency of the three sequences(psb A-trn H, ITS1- ITS4) were 88%, 64%, 42%. 13 samples are adulterants from 21 samples of medicinal materials and 4 samples are adulterants from 13 samples of pharmaceutical companies by calculating of BLAST software. On the basis of the result, specific primers(6-R, 6-F) were designed for the psb A-trn H sequence, it can distinguish the Hemsleya macrosperma and its adulterants, achieve the purpose of efficient, convenient and fast identification. The Hemslein A were tested from the medicinal materials of Hemsleya macrosperma by using the HPLC method, it ranges from 0.0013%-1.1203%, the highest content is 1.1203% from the sample of 42. In the 13 samples from the pharmaceutical companies, 9 samples were tested Hemslein A. 3 samples were tested Hemslein A in the 21 samples which identifyed by the molecular experiments. But several samples were identified as Hemsleya macrosperma without Hemslein A, because of the differences of quality.