| RUBIAE RADIX ET RHIZOMA is commonly used in traditional Chinese medicine, bitter, cold, heart liver, with blood stasis, cooling blood to stop bleeding, relieving cough and eliminating phlegm, used clinically in the treatment of traumatic bleeding, hematemesis, uterine bleeding, amenorrhea and blood stasis, joint pain, swelling or flutter, which was found firstly recorded in 《shen nong’s material medica》,named "qiangen",《Chinese pharmacopoeia》 2010 edition contains the source of the RUBIAE RADIX ET RHIZOMA for rubiaceae plant Rubia cordifolia L. of dry root and rhizome.as the herbs have similar morphological characteristics, so in the market, a lot of plant root or rhizome of other genera as adulterants was often used as Rubiae Radix et Rhizoma.Therefore, this paper studies on the base of the original identification and quality control of RUBIAE RADIX ET RHIZOMA, it is important to ensure the accuracy and effectiveness of clinical medication.Objective:1. To establish identification method which can identify RUBIAE RADIX ET RHIZOMA and its adulterants by DNA barcode and provide the standard sequence for the《Chinese Pharmacopoeia》 DNA barcode database;2. Study on the fingerprint of HPLC and determination of content with RUBIAE RADIX ET RHIZOMA bought on the market.Methods:1. Using DNA barcode identify RUBIAE RADIX ET RHIZOMA medicinal herbs and its adulterants with psbA-trnH, matK and rbcL sequences and their combination sequences;2. With purpurin and rubimaillin as reference, study fingerprint of RUBIAE RADIX ET RHIZOMA and the contents determination by HPLC.Results:1. The DNAs were successfully extracted from 76 original plant samples.Fortunately, R. cordifolia could be identified from all the adulterants by psbA-trnH+matK+rbcL combination marker. The intra-and inter-specific K2P genetic distances of psbA-trnH+matK+rbcL were 0-0.0014 and 0.0007-0.6303.R. cordifolia could be identified from all the adulterants by the psbA-trnH+matK+rbcL combination using NJ tree method. Among the 30 unidentified samples of Rubiae Radix et Rhizoma which were collected from medicinal herb markets, all three markers, including psbA-trnH, matK and rbcL, could be amplified from 22 samples. The 11 samples of Rubiae Radix et Rhizoma clustered with R. cordifolia and the others were divided into three clusters by the psbA-trnH+matK+rbcL combination using NJ tree method.2. The determination results the contents of 11 batches RUBIAE RADIX ET RHIZOMA herb show that the content of purpurin is 0.14%-0.46%, rubimaillin content is 0.16%-0.44%,the content of 11 adulterants show that the content of purpurin is 0.27%-0.50%, rubimaillin content is 0.11%-0.32%.as for the content of purpurin, the content of adulterants is slightly higher than the genuine, while the content of rubimaillin is only slightly lower than the genuine.3 The HPLC fingerprint of RUBIAE RADIX ET RHIZOMA herbs was established,12 peaks have been confirmed.The similarity of 11 batch quality sample is between0.947 with 0.992.The similarity of adulterants sample is between 0.805 with 0.961.The samples of S17 and S18 were 0.961 and 0.960 which greater than the 0.947 of minimum genuine similarity.Conclusion:1. psbA-trnH+matK+rbcL combination can be used as DNA barcode to identify R. cordifolia from adulterants.A preliminary technical identification method of identify RUBIAE RADIX ET RHIZOMA herbs using DNA barcode were established.2. The quality of RUBIAE RADIX ET RHIZOMA of traditional Chinese Medicine were analysised,we found that the content of purpurin and rubimaillin and fingerprints of RUBIAE RADIX ET RHIZOMA and its adulterants are very similar. |
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