| Lamiophlomis rotate (Benth.) Kudo is a perennial herb distributed in Bhutan, India, Nepal, and China. Moreover, the L. rotate and its preparations are officially used as hemostasis and pain alleviation and recorded in Chinese Pharmacopoeia. In Tibetan medicine volunteers, three plants are used as DABUBA, L. rotata (Benth.) Kudo of the L. genus (Lamiacea), Ajuga ovalifolia Bur. et Franch.var. calantha (Diels) C.Y.Wu et C.Chen of the Ajuga Bunge genus (Lamiacea) and Oreosolen wartii Hook. f of the Oreosolen genus (Scrophulariaceae). It is often confused in different races and different areas.Metabolomics is one omics approach that can be used to acquire comprehensive information on the composition of a metabolite pool to provide a functional screen of the cellular state. Metabolomic techniques are fast becoming a hotspot of global biological research, and increasingly playing an important role in every aspect of the biomedical and phytochemical research fields, including biomarker screening, quality control, activity and toxicity prediction, clinical chemistry, chemotaxonomy and environmental metabolism.Purposes:In this paper, to distinguish L. rotata herb from its adulterants, the study was used to verify the stability and accuracy of DNA barcodes in identification of Chinese materia medica for the first time. It is quite arduous to obtain the whole picture of chemical constituents appropriately with the existing analytical techniques. The study focused on the metabolomics of L. rotate were performed by 1H-NMR spectroscopy and HPLC-TOF/MS.Methods:1. All genomic DNAs from thirty one samples were extracted. The ITS (internal transcribed spacer) regions were amplified and sequenced bi-directionally. Obtained sequences were assembled using the CodonCode Aligner. And the sequences of the ITS regions were aligned through Clustal-W and the genetic distances were computed using MEGA 4.0 in accordance with the kimura 2-parameter (K2P) model. The neighbor-joining (NJ) phylogenetic trees were constructed. The ITS2 regions were obtained by using the hidden Markov model (HMM)-based annotation methods from the ITS sequences.2. A high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight (?)andem mass spectrometry (HPLC-ESI-TOF-MS) method was employed for the qualitative identification of constituents in L. rotata. The optimal condition of separation and detection were achieved on a Waters Symmetry C18 column with a gradient elution with methanol and 0.1% formic acid. Identification was made by elucidating the mass spectral data and further confirmed by comparing retention times and UV spectra of target peaks with those of reference compounds.3. Metabolomics of L. rotate were performed by 1H-NMR spectroscopy and multivariate statistical analysis. In NMR measurements, metabolic profile of L.rotata was prepared by methanol-d4 solvent. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were applied to analyze the’H-NMR spectroscopy of L. rotata samples.4. We investigated the ability of nine compounds to modulate platelet activation and aggregation induced by ADP.Results:1. Results indicated that the lengths of ITS2 regions of L. rotata were 219bp. The intra-specific genetic distances were smaller than inter-specific ones in ITS2 regions of L. and its adulterants.2. With this method, a total of twenty-four phytochemicals were identified including fourteen iridoids, seven phenylethanoid glycosides, and three flavonoid glycosides.3. Nine iridoid glycosides, one flavonoid, three phenylpropanoid glycosides, five sugars and one fatty acid present in L. rotata were detected by means of 1H-NMR spectroscopy. The samples of Gansu location had higher relative concentrations of iridoid glycosides, while the L. rotata samples from Tibet accumulate higher relative amounts of phenylpropanoid glycosides. The results is by means of HCA analysis.4. The results indicated that nine compounds have anti-platelet aggregation activity. The detail were as follows:Forsythoside B> Verbascoside> Alyssonoside> Luteolin-7-O-β-D-glucoside>7,8-dehydropenstemoside> Phlorigidoside C> Shanzhiside methylester>8-0-Acetyl shanzhiside methylester.Conclusions:1. ITS2 regions as DNA barcodes can stably and accurately distinguish L. from its adulterants and could provide a new technique to ensure clinical safety in utilization of traditional Chinese medicines.2. All of results indicated that the method presented here is able to classify the sample species and to reveal characteristic details of the chemical constituents of L. rotata.3.1H NMR measurements together with multivariate statistical analysis were used to successfully discriminate samples from three growth locations.4. Our studies provide basis on the endeavors of screening chemicals with that anti-platelet aggregation activity.Innovations:1.In this study, we applied DNA barcoding technology,1H-NMR metabolomic and LC-TOF-MS techniques and multivariate statistical analyses methods to study the L. rotata identification, quality evaluation.2. This paper was verify the stability and accuracy of DNA barcodes in identification of L. rotate and its adulterants for the first time. ITS/ITS2 regions as DNA barcodes can stably and accurately distinguish L. rotata from its adulterants and could provide a new technique to ensure clinical safety in utilization of traditional Chinese medicines.3. Fourteen major iridoid glycosides, seven phenylpropanoid glycosides, and three flavonoid glycosides were identified or tentatively characterized based on their retention times, UV and TOF MS data. The major fragmentation pathways of PGs in L. rotata obtained through the MS data was schemed systematically for the first time, which provides a reference for other PGs derivatives.4. The overall metabolites present in L. rotate were simultaneously detected for the first time including Nine iridoid glycosides, one flavonoid, three phenylpropanoid glycosides, five sugars and one fatty acid.5. In this paper, it indicated that nine compounds have anti-platelet aggregation-aetivity for the first time, and it may be the mechanism of L. rotate on inhibiting the platelet aggregation induced by ADP. |
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