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Objective: To analyze six components in Bletillae Rhizoma using high-performance liquid chromatography. Through the linear calibration using two reference substances method, the retention time of the components was predicted, and the feasibility and accuracy of this technique in the qualitative identification of chromatographic peaks were explored. This method was also compared with the relative retention time (RRT) method. Methods: High-performance liquid chromatography was employed using an Ultimate XB-C18 column (250 mm×4.6 mm, 5 μm). The mobile phase consisted of water (A) and acetonitrile (B), with a gradient elution as follows: 0-11 min,12% B; 11-58 min, 12% B to 68% B; 58-65 min, 68% B to 12% B. The flow rate was 0.9 mL · min-1, the injection volume was 5 μL, the column temperature was maintained at 30 °C, and the detection wavelength was set at 280 nm. By measuring the actual retention time of the six components on 16 different C18 chromatographic columns, the standard retention time was calculated. Gastrodin (Peak 1) and gymnadenin Ⅲ (Peak 4) were used as the double-standard compounds.The linear calibration using two reference substances method was used to predict the retention time of other components,and the prediction was verified on another three unknown C18 chromatographic columns. With the relative retention time(RRT) method, using bletilloside (Peak 5) as the reference compound, the retention times of the other five components were predicted. Results: A qualitative analysis method for the six components in Bletillae Rhizoma was established,which could accurately predict the retention times of each component in Bletillae Rhizoma. The standard retention times of the six components as as follows: gastrodin 4.279 min, gymnadenin Ⅲ 32.145 min, pleione bulbocodioides factor Ⅰ27.220 min, 2-O-glucosylbletilloside 28.603 min, bletilloside 33.509 min, and batatasin Ⅲ 45.504 min. Conclusion: The linear calibration using two reference substances method can improve the prediction ability of the retention time of each component in Bletillae Rhizoma, thereby enhancing the accuracy and reliability of the analysis results.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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