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The morphological identification, chemical component analysis, and DNA barcode determination were investigated on Genus Mentha (including Mentha haplocalyx Briq., Mentha piperita Linn., Mentha spicata Linn. and Mentha cultivated) in order to reveal the origin of Herba Mentha as a drug, and ensure the accuracy in clinic application. The morphological characters, chemical composition analysis by GC-MS/MS and DNA content measure by polymerase chain reaction (PCR) were reported in this study for inter- or intra- species divergence. Based on the morphology, axillary verticillasters was recognized as the typical character for Mentha haplocalyx Briq. Carvone was used as an index component for chemical composition analysis of Mentha spicata Linn. Gene clustering analysis divided 22 batches of samples into two molecular groups. Mentha haplocalyx Briq. is distinguishably different from Mentha spicata Linn. Mentha piperita Linn. and other cultivated plants were distributed between these two species. The results obtained by morphological identification, chemical composition analysis, and DNA barcode determination show good correlations, but each identification method has its limit. In view of the fact that hybridization of the plants in Genus Mentha is common, identification relying on only one method is not recommended.
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Objective: The morphological characteristics of plants and the application of ITS2 and psbA-trnH sequences in the identification of Mentha haplocalyx and its related species were discussed in order to provide references for the identification of M. haplocalyx and its related species. Methods: The morphological characteristics such as the shape of leaf and leaf edge, villous density and glandular scale density of M. haplocalyx and its related species were observed. Alignment was performed to compare ITS2 and psbA-trnH sequences of the tested materials. The data were processed and analyzed by means of comparing the morphological characteristics of plants, analyzing the DNA barcode sequences, calculating the genetic distance, and constructing NJ trees. Results: The NJ trees were constructed according to the morphological characteristics of plants combined with ITS2 and psbA-trnH sequences, which showed that M. arvensis was closely related to Satureja Hortensis, Salvia miltiorrhiza was closely related to Perilla frutescens, Mentha piperita was closely related to the Monarda didyma and Agastache rugosa was closely related to Nepeta cataria. This method could distinguish M. haplocalyx from its related species. Conclusion: ITS2 and psbA-trnH sequences can be used as DNA barcode of M. haplocalyx and its related species, and the combination with morphological characters of leaves is better for the identification.
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Objective To explore the anti-influenza virus effect of phenolic fraction of Mentha haplocalyx in vitro, and to establish the HPLC characteristic spectrum of phenolic fraction of M. haplocalyx for its quality evaluation. Methods MTT method was used to detect the inhibitory effect of phenolic fraction of M. haplocalyx on influenza virus PR8 infecting MDCK cells in three ways, including adding drug firstly, adding drug and virus simultaneously and adding virus firstly. The characteristic spectrum of phenolic fraction samples of 12 batches of M. haplocalyx was established by HPLC method. The similarity of which was analyzed with Similarity Evaluation System for Chromatographic Fingerprint of Chinese Materia Medica (2.0 version). Results The phenolic fraction at 0.31—10 μg/mL showed antivirus PR8 activities in different degrees under three adding drug ways. Phenolic fraction of M. haplocalyx at six dilution concentrations displayed obvious antivirus PR8 effect under two adding drug ways including adding drug firstly and adding virus firstly, and virus inhibition rates of which were 14.17%—41.31% and 45.64%—87.48%, respectively. Total of 11 peaks were chosen as the common characteristic peaks of spectrum with the similarity degrees more than 0.9 of 12 batches of samples, which illustrated that different batches of phenolic fraction of M. haplocalyx were of high similarity. Conclusion Phenolic fraction of M. haplocalyx had better antiviral effect in vitro. The characteristic spectrum method established in this paper was simple, stable and reproducible, which could reflect the whole profile of phenolic fraction of M. haplocalyx and provide reference for quality control and efficacy stability of phenolic fraction of M. haplocalyx.
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Objective To investigate the prototype components and metabolites of Mentha haplocalyx in rats plasma. Methods UHPLC-Q-TOF-MS/MS method was developed and applied to identify the components and metabolites of rat plasma. The analysis was carried out on an Eclipse Plus C18 column (100 mm × 4.6 mm, 3.5 μm, Agilent) with the mobile phase of 0.1% acetic acid solution (A)-acetonitrile (B) at a flowing rate of 0.3 mL/min, and the injection volume was 5 μL. Results The developed method was applicable to the analysis and identification of metabolites of M. haplocalyx after oral administration. A total of 67 compounds, including 28 prototype components and 39 metabolites (one of which was a new metabolite of luteolin unreported), were identified by comparison of their retention time, ion fragmentation information with that of the blank biological samples, herb extract, and reference compounds. Conclusion The metabolic pathway of M. haplocalyx in rats were consisted of oxidation, reduction, methylation, sulfation, and glucuronidation, and the main metabolic pathway was phase II metabolic pathway among of them. This experimental method is simple and reliable, which could provide theoretical basis for elucidating the bioactive components of M. haplocalyx.
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OBJECTIVE: To develop a quantitative analysis of multi-components by single marker (QAMS) method for simultaneous determination of four flavonoid glycosides in Mentha haplocalyx Briq. by using one reference substance. METHODS: Four main effective components (hesperidin, diosmin, didymin, and buddleoside) were selected as analytes to evaluate the quality of Mentha haplocalyx Briq.. With hesperidin as internal reference standard, the relative correction factors (RCF) of the other three components to hesperidin were calculated within certain ranges. The contents of hesperidin in the samples of Mentha haplocalyx Briq. were determined by using the external standard method, and the contents of the three other ingredients were calculated by their RCFs. The method was evaluated by comparison of the quantitative results between external standard method and QAMS method by SPSS 17.0. RESULTS: No significant differences were observed between the quantitative results of these two methods (RSD 0.05). The validated HPLC method had the advantages of precision, reproducibility, and reliability. CONCLUSION: The QAMS method we established is suitable and feasible for the quality control of Mentha haplocalyx Briq.
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Objective: To study the chemical constituents from Mentha haplocalyx. Methods: Compounds were isolated and purified by various chromatographic techniques, and their structures were identified by spectral analysis. Results: Eleven compounds were isolated and identified, including menthalignin (1), 5-hydroxy-6, 7, 8, 4'-tetramethoxyflavone (2), betulinic acid (3), hesperetin 7-O-β-D-glucopyranoside (4), gentisic acid 5-O-β-D-(6'-salicylyl)-glucopyranoside (5), linarin (6), acatin (7), 5-hydroxy-6, 7, 8, 3', 4'-pentamethoxyflavone (8), 5, 6, 4'-trihydroxy-7, 8-dimethoxyflavone (9), tilianin (10), and agastachoside (11). Conclusion: Compound 1 is a new lignan named menthalignan; compound 2 is a new natural product; compound 4 is isolated from the plants in Labiatae for the first time, and compounds 3 and 5 are firstly reported in the plants of Mentha L.