T/GDP 010-2020 Preparation technique standards for high purity blisterworm-based compounds

Scope

Main technical content: Using the multi-stage liquid chromatography separation of compounds based on the overload critical point of the column, and adopting three-stage high-performance liquid chromatography separation. (I) Drying and grinding the fresh bitter melon stems and leaves into powder; (II) Extracting the dried bitter melon stems and leaves with 100% methanol for 24 hours, with a ratio of methanol to dried bitter melon stems and leaves of 1:5–10 (g:mL). Filtering and concentrating the extract to obtain a crude extract; (III) Conducting first-stage high-performance liquid chromatography separation of the crude extract, collecting the effluents from the time periods of 18–26 minutes and 30–42 minutes, and concentrating them to obtain first-stage fractions 1 and 2; (IV) Conducting second-stage high-performance liquid chromatography separation of the first-stage fractions 1 and 2. For first-stage fraction 1, collecting the effluents from the time periods of 18–22 minutes and 24–26 minutes, drying and concentrating them to obtain second-stage fractions 1-1 and 1-2. For first-stage fraction 2, collecting the effluents from the time period of 33–37 minutes, drying and concentrating them to obtain second-stage fraction 2-1; (V) Conducting third-stage high-performance liquid chromatography separation of the second-stage fractions 1-1, 1-2, and 2-1, collecting the effluents from the time periods of 20–24 minutes, 23–26 minutes, and 34–37 minutes, respectively, drying and concentrating them to obtain bitter melon glycosides II, bitter melon glycoside L, and bitter melon glycoside I. (VI) The parameters of high-performance liquid chromatography separation are: mobile phase of 80% methanol, column temperature of 28°C, flow rate of 2.5mL/min, and separation column of Agilent XB-C18. The detection wavelength is 210nm. Pure bitter melon glycoside compounds with a purity of over 99% were obtained, with a yield 100% higher than previous extraction methods and a time reducti

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