Method for separating eight components in Chinese traditional medicine composition

A method for separating eight components in a composition, wherein the composition is prepared from raw materials in parts by weight comprising:

• 200-300 parts of fructus Forsythiae, 60-100 parts of ephedra, 40-60 parts of Rheum officinale, 200-300 parts of Houttuyni acordata, 200-300 parts of honeysuckle, 200-300 parts of isatis root, 60-100 parts of Pogostemon cablin, 200-300 parts of Rhizoma dryopteris crassirhizomatis, 60-100 parts of Rhodiola rosea, 5-9 parts of menthol, 60-100 parts of semen Armeniacae amarum, 60-100 parts of liquorice and 200-300 parts of Gypsum fibrosum, wherein the method comprises following steps of:
• (1) adsorbing total extract of the composition by macroporous resin, eluting with water, 10% ethanol, 30% ethanol and 50% ethanol in sequence, respectively collecting eluent of each part, and concentrating to obtain extract of each part;
• (2) taking the extract of an elution part of the 50% ethanol (50% ethanol elution part extract) obtained in step (1), adding reverse-phase octadecyl-modified silica gel to form a first mixture, air drying the first mixture, loading the first mixture, separating by using a reverse-phase octadecyl-modified silica gel open column, and sequentially eluting by using methanol with methanol-water volume ratios of 20:80, 40:60, 60:40, 80:20 and 100% methanol, and sequentially obtaining Fr.A -Fr.E samples, wherein the reverse-phase octadecyl-modified silica gel has a hydrophobic carbon loading and a hydrophilic surface;
• (3) taking the Fr.A sample obtained in step (2), adding the reverse-phase octadecyl-modified silica gel to form a second mixture, air drying the second mixture, adding the second mixture into a sample loading column, separating by medium pressure preparative liquid chromatography, wherein a volume ratio of methanol to water is 25:75-60:40, and a flow rate is 25 mL/min, receiving fractions, concentrating the fractions under reduced pressure, evaluating each fraction through thin layer chromatography, and concentrating under reduced pressure again to obtain Fr.A-1-Fr.A-7 samples;
• (4) mixing the Fr.A-2 sample obtained in the step (3) with silica gel, loading onto a silica gel column, carrying out isocratic separation by using a dichloromethane-methanol volume ratio of 8:1, receiving fractions, evaluating each fraction through thin-layer chromatography, and obtaining Fr.A-2-1-Fr.A-2-4 samples;
• (5) dissolving the Fr.A-2-2 sample obtained in the step (4) with methanol, adopting high performance liquid chromatography, wherein a volume ratio of methanol to water in a mobile phase is 50:50, a flow rate is 12 mL/min, and a detection wavelength is 210 nm, to perform primary separation, and respectively collecting chromatographic peaks with retention times of 6-8 min, 9-10 min and 12-13 min, recovering the solvent under reduced pressure, and respectively performing following separation:
• the chromatographic peak of 6-8 min, further purified by high performance liquid chromatography, wherein a volume ratio of acetonitrile to water in the mobile phase is 25:75, a flow rate is 12 mL/min, and a detection wavelength is 210 nm, chromatographic column:
• liquid chromatographic column using octadecyl-modified silica gel with fully-endcapped C18 phase, 250×20 mm, under these conditions, collecting the chromatographic peak with retention times of 8-9 min, recovering the solvent under decompression to obtained a compound 2:
• aloe-emodin-8-O-β-D-glucopyranoside;
• the chromatographic peak of 9-10 min, further purified by high performance liquid chromatography, wherein a volume ratio of methanol to water in a mobile phase is 45:55, a flow rate is 12 mL/min, and a detection wavelength is 210 nm, chromatographic column:
• liquid chromatographic column using the octadecyl-modified silica gel with fully-endcapped C18 phase, 250×20 mm, under these conditions, collecting the chromatographic peak with retention times of 21-23 min, recovering the solvent under decompression to obtained a compound 3:
• quercitrin;
• and
• the chromatographic peak of 12-13 min, further purified by high performance liquid chromatography, wherein a volume ratio of acetonitrile to water in a mobile phase is 25:75, a flow rate is 12 mL/min, and a detection wavelength is 210 nm, chromatographic column:
• liquid chromatographic column using the octadecyl-modified silica gel with fully-endcapped C18 phase, 250×20 mm, under these conditions, collecting the chromatographic peak with retention times of 9-10 min, recovering the solvent under decompression to obtained a compound 4:
• matairesinol-4′-O-glucoside;
• (6) dissolving the Fr.A-2-3 sample obtained in the step (4) with methanol, adopting high performance liquid chromatography, wherein a volume ratio of methanol to water in a mobile phase is 30:70, a flow rate is 12 mL/min, and a detection wavelength is 210 nm, to perform primary separation and respectively collecting chromatographic peaks with retention times of 10-11 min, 17-19 min and 21-24 min, recovering the solvent under reduced pressure, chromatographic peaks at 17-19 min is for a compound 6:
• epi-vogeloside, chromatographic peaks at 21-24 min is for a compound 7:
• vogeloside, chromatographic peak of 10-11 min, further purified by high performance liquid chromatography, wherein a volume ratio of acetonitrile to water in a mobile phase is 15:85, a flow rate is 12 mL/min, and a detection wavelength is 210 nm, chromatographic column:
• liquid chromatographic column using the octadecyl-modified silica gel with fully-endcapped C18 phase, 250×20 mm, under these conditions, collecting the chromatographic peak with retention times of 14-16 min, recovering the solvent under decompression to obtained a compound 5:
• liquiritin apioside;
• and
• (7) dissolving the Fr.A-3 sample obtained in the step (3) with methanol, adopting high performance liquid chromatography, wherein a volume ratio of methanol to water in a mobile phase is 60:40, a flow rate is 12 mL/min, and a detection wavelength is 210 nm, to perform primary separation and respectively collecting chromatographic peaks with retention times of 14-15 min and 19-21 min, and recovering the solvent under reduced pressure, separating out a white precipitate in a dichloromethane-methanol solution with volume ratio of 2:1 from a collecting liquid of chromatographic peak of 19-21 min to obtain a compound 8;
• and
• wherein chromatographic peak of 14-15 min is further purified by high performance liquid chromatography, wherein a volume ratio of acetonitrile to water in a mobile phase is 30:70, a flow rate is 12 mL/min, and a detection wavelength is 210 nm, chromatographic column:
• liquid chromatographic column using the octadecyl-modified silica gel with fully-endcapped C18 phase, 250×20 mm, under these conditions, collecting the chromatographic peak with retention times of 18-20 min, recovering the solvent under decompression to obtained a compound 1:
• 10-O-(p-hydroxycinnamoyl)-adoxosidic acid.