Species Identification Of Chinese Patent Medicines-Ertong Qingfei Wan And Zhisou Huatan Wan

Species identification is one of the key issues in the quality evaluation system of Chinese patent medicine(CPM).Traditional CPM quality evaluation methods include characteristic identification,microscopic identification,physicochemical techniques,etc.However,they are unable to trace some species information,such as similarly morphologically related species,species with the same chemical composition,unknown toxic or endangered rare species.Therefore,it is necessary to combine molecular biology methods to identify complex species composition information of CPM.Aristolochic acids(AAs),listed in class I carcinogen due to their irreversible renal toxicity,are mainly detected in Aristolochiaceae plants such as Aristolochia and Asarum.Up to now,some herbs containing AAs are still used as medicines,so the assessment of benefit and risk for the public health deserves attention.Firstly,Ertong Qingfei Wan and Zhisou Huatan Wan contaning Aristolochiaceae plants were selected to establish a more systematic method for CPM identification by combining molecular biology with chemical methods.Then,the related real-time fluorescent PCR with TaqMan probe was used to verify the presence of important species of CPM.Meanwhile,this method was tested using Ophiocordyceps sinensis and its adulterants,Alisma plantago-aquatica and its related species A.orientale.The main results are as follows:1.The species of Ertong Qingfei Wan was identified using next generation sequencing(NGS),the real-time fluorescent PCR with TaqMan probe and liquid chromatography-mass spectrometry(LC-MS)technology.The NGS results showed that 16 prescription medicines were detected in two commercially available batches of Ertong Qingfei Wan ET01 and ET02,except for Pinelliae Rhizoma Praeparatum,Citri Exocarpium Rubrum,Gypsum Fibrosum and Chloriti Lapis.The results of real-time fluorescent PCR with TaqMan probe showed that Asari Radix et Rhizoma,ET01 and ET02 all had typical S-type amplification curves.The results of LC-MS showed that AAI was detected below 10 ?g/g in both batches of ET01 and ET02.And there is no AAI limit for Ertong Qingfei Wan in the latest Chinese Pharmacopoeia.For the safety of consumers,it is recommended that AAI content be used as one of the main evaluation indicators of Ertong Qingfei Wan in the Chinese Pharmacopoeia.This study proved that the combination methods of NGS,real-time fluorescent PCR with TaqMan probe and LC-MS were applicable to the species identification of Ertong Qingfei Wan.2.The species identification of Zhisou Huatan Wan was carried out using NGS,the real-time fluorescent PCR with TaqMan probe and LC-MS technology.The NGS results showed that 692 and 868 ITS2 sequences,287177 and 280041 Clean reads were obtained from two commercially available batches of Zhisou Huatan Wan ZS01 and ZS02,respectively.In addition,only seven prescription medicines were detected in ZS01 and ZS02,respectively.The results of real-time fluorescent PCR with TaqMan probe showed that Aristolochiae Fractus,ZS01 and ZS02 all had typical S-type amplification curves.The results of LC-MS showed that AAI was detected in both batches of ZS01 and ZS02.However,AAI content of Zhisou Huatan Wan has no limitation standard in the latest Chinese Pharmacopoeia.For safety reasons,it is recommended that the Chinese Pharmacopoeia strictly limit the AAI content in Aristolochiae Fructus and Zhisou Huatan Wan.This study showed that the combination methods of NGS,real-time fluorescent PCR with TaqMan probe and LC-MS can identify some species of Zhisou Huatan Wan.3.Identification of Ophiocordyceps sinensis and its adulterants based on the real-time fluorescent PCR with TaqMan probe.A set of specific primers and TaqMan designed using Primer Premier 6.0 Software and genomic DNA from 100 samples of O.sinensis and its adulterants were used to perform sensitivity and specificity studies on two different real-time fluorescent PCR systems(Genesig q16 and Bio-Rad CFX96).The sensitivity study showed that the detectable DNA template concentration of O.sinensis was 0.016 ng/?L in the Bio-Rad CFX96 system,which is 1000 times higher than the Genesig q16 system.Meanwhile,this method had good specificity for O.sinensis on Genesig q16 and Bio-Rad CFX96 systems,so O.sinensis could be clearly distinguished from Ophiocordyceps nutans,Cordyceps gunnii,Cordyceps militaris,Cordyceps cicadae,Cordyceps liangshanensis,Cordyceps gracilis.In conclusion,the real-time fluorescent PCR with TaqMan probe can be used to accurately identify different species with more variable sites.4.Identification of Chinese medicinal herbs Alisma plantago-aquatica and A orientale based on DNA barcoding method and the real-time fluorescent PCR with TaqMan probe.Identification results showed that the ITS2 sequence can successfully distinguish A.plantago-aquatica from A.orientale.The results of cluster analysis based on NJ tree showed that A.plantago-aquatica and A.orientale were each grouped together and had a distinct monophylaxis.The results of real-time fluorescent PCR with TaqMan probe showed that the two sets of primers and probes were not specific for A.orientale,indicating its limitation in identification of congeneric species with only one variable site.