(19)
(11)EP 3 705 130 A1

(12)EUROPEAN PATENT APPLICATION
published in accordance with Art. 153(4) EPC

(43)Date of publication:
09.09.2020 Bulletin 2020/37

(21)Application number: 18877230.5

(22)Date of filing:  25.10.2018
(51)International Patent Classification (IPC): 
A61K 36/78(2006.01)
A61K 31/045(2006.01)
A61K 33/06(2006.01)
C07H 17/04(2006.01)
(86)International application number:
PCT/CN2018/111846
(87)International publication number:
WO 2019/091287 (16.05.2019 Gazette  2019/20)
(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
Designated Extension States:
BA ME
Designated Validation States:
KH MA MD TN

(30)Priority: 10.11.2017 CN 201711103420

(71)Applicant: Shijiazhuang Yiling Pharmaceutical Co., Ltd.
Shijiazhuang, Hebei 050035 (CN)

(72)Inventors:
  • ZHANG, Chuangfeng
    Shijiazhuang Hebei 050035 (CN)
  • SHEN, Shuo
    Shijiazhuang Hebei 050035 (CN)
  • SONG, Lianqiang
    Shijiazhuang Hebei 050035 (CN)

(74)Representative: Isarpatent 
Patent- und Rechtsanwälte Behnisch Barth Charles Hassa Peckmann & Partner mbB Friedrichstrasse 31
80801 München
80801 München (DE)

  


(54)METHOD FOR SEPARATING EIGHT COMPONENTS IN CHINESE TRADITIONAL MEDICINE COMPOSITION


(57) The solution provides a method for separating a Chinese traditional medicine composition. To explain a pharmacological effect mechanism of a medicine made of two or more components and scientific content in rules of compatibilityamong components ofa compound medicine, systematic researches on the material basis is very necessary. Accordingly, deep researches are done on chemical components of the pharmaceutical composition in the solution, and eight compounds are separated, which areio-O-(p-hydroxycinnamoyl)-adoxosidic acid, aloe-emodin-8-O-β-D-glucopyranoside, quercitrin, matairesinol-4'-O-glucoside, liquiritinapioside, epi-vogeloside, vogeloside and ethyl caffeate, which provides a new quality control method for the composition in the solution.


Description

TECHNICAL FIELD



[0001] The solution relates to a method for separating multiple components in a Chinese traditional medicine composition.

BACKGROUND



[0002] The Chinese traditional medicine compound prescription is the main form of Chinese traditional medicine, after thousands of years of clinical use, the compound prescription can obtain a stronger therapeutic effect than a single medicine, which has fully proved the scientific nature of the compound prescription. The medicinal composition of the solutionis composed of 13Chinese traditional medicines such as fructusforsythiae, honeysuckle, herbaephedrae (processed) and the like, has the effects of clearing heat and detoxifying, removing lung hotness, and is used for treating epidemic influenza. Clinical studies prove that the medicinal composition of the solutionhas definite therapeutic effect and remarkable effect on treating influenza and acute upper respiratory tract infection. To explain a pharmacological effect mechanism of a medicine made of two or more components and scientific content in rules of compatibility among components ofa compound medicine, systematic researches on the material basis is very necessary. Accordingly, deep researches are done on chemical components of the pharmaceutical composition in the solution, and eight compounds are separated, which areio-O-(p-hydroxycinnamoyl)-adoxosidic acid, aloe-emodin-8-O-β-D-glucopyranoside, quercitrin, matairesinol-4'-O-glucoside, liquiritinapioside, epi-vogeloside, vogeloside and ethyl caffeate, which provides a new quality control method for the composition in the solution.

SUMMARY OF THE INVENTION


TECHNICAL PROBLEM



[0003] The solution provides a method for separating eight compounds ina Chinese traditional medicine composition.

TECHNICAL SOLUTION



[0004] The Chinese traditional medicine composition is prepared from the following raw materials in parts by weight: 200-300 parts of fructusforsythiae, 60-100 parts of ephedra, 40-60 parts of rheum officinale, 200-300 parts of houttuyniacordata, 200-300 parts of honeysuckle, 200-300 parts of isatis root, 60-100 parts of pogostemoncablin, 200-300 parts of rhizomadryopteriscrassirhizomatis, 60-100 parts of rhodiolarosea, 5-9 parts of menthol, 60-100 parts of semen armeniacaeamarum, 60-100 parts of liquorice and 200-300 parts of gypsum fibrosum.

[0005] The separation method in the solutioncomprises the following steps of:
  1. (1) adsorbing the total extract of the Chinese traditional medicine composition by AB-8 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. (2) taking the 50% ethanol elution part extract obtained in step (1), adding reverse-phase silica gel ODS-AQ-HG, naturally airing the mixed sample, loading the sample, separating by using a reverse-phase ODS-AQ-HG 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; sequentially obtaining Fr.A-Fr.E;
  3. (3) taking Fr.A sample obtained in step (2), adding a reverse-phase silica gel ODS-AQ-HG to mix sample, naturally airing the mixed sample ODS, adding the mixed sample ODS into a sample loading column, separating liquid phase prepared by an upper medium-pressure, gradiently separating liquid phase prepared by medium-pressure, wherein the volume ratio of methanol to water is 25:75-60:40, and the flow rate is 25 mL/min, receiving the fractions in equal volume of 500 mL in a conical flask, concentrating under reduced pressure, identifying the combined fractions through a thin layer chromatography plate, and concentrating under reduced pressure again to obtain Fr.A-i - Fr.A-7;
  4. (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 in equal volume of 50 mL, eluting with a volume of 800 mL, and identifying the combined fractions through thin-layer chromatography plate to obtain Fr.A-1-1 - Fr.A-1-4;
  5. (5) dissolving the Fr.A-1-2 sample obtained in the step (4) with methanol, adopting high performance liquid chromatography, wherein the volume ratio of methanol to water in the mobile phase is 50:50, the flow rate is 12 mL/min, and 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 the following separation:

    a chromatographic peak of 6-8 min, further purified by high performance liquid chromatography, wherein the volume ratio of acetonitrile to water in the mobile phase is 25:75, the flow rate is 12 mL/min, and the detection wavelength is 210 nm, chromatographic column: YMC-Pack R&D ODS-A, 250×20 mm, S-10 µm, under these conditions, collecting the chromatographic peak with retention times of 8-9 min, recovering the solvent under decompression to obtained compound 2: aloe-emodin-8-O-β-D-glucopyranoside;

    a chromatographic peak of 9-10 min, further purified by high performance liquid chromatography, wherein the volume ratio of methanol to water in the mobile phase is 45:55, the flow rate is 12 mL/min, and the detection wavelength is 210 nm, chromatographic column: YMC-Pack R&D ODS-A, 250×20 mm, S-10 µm, under these conditions, collecting the chromatographic peak with retention times of 21-23 min, recovering the solvent under decompression to obtained compound 3: quercitrin;

    a chromatographic peak of 10-12 min, further purified by high performance liquid chromatography, wherein the volume ratio of acetonitrile to water in the mobile phase is 25:75, the flow rate is 12 mL/min, and the detection wavelength is 210 nm, chromatographic column: YMC-Pack R&D ODS-A, 250×20 mm, S-10 µm, under these conditions, collecting the chromatographic peak with retention times of 9-10 min, recovering the solvent under decompression to obtained compound 4: matairesinol-4'-O-glucoside;

  6. (6) dissolving the Fr.A-1-3 sample obtained in the step (4) with methanol, adopting high performance liquid chromatography, wherein the volume ratio of methanol to water in the mobile phase is 30:70, the flow rate is12mL/min, and 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 of 17-19 min is for compound 6: epi-vogeloside, chromatographic peaks of 21-24 min is for compound 7: vogeloside, a chromatographic peak of 10-11 min, further purified by high performance liquid chromatography, wherein the volume ratio of acetonitrile to water in the mobile phase is 15:85, the flow rate is 12 mL/min, and the detection wavelength is 210 nm, chromatographic column: YMC-Pack R&D ODS-A, 250×20 mm, S-10 µm, under these conditions, collecting the chromatographic peak with retention times of 14-16 min, recovering the solvent under decompression to obtained compound 5: liquiritinapioside; and
  7. (7) dissolving the Fr. A-3 sample obtained in the step (3) with methanol, adopting high performance liquid chromatography, wherein the volume ratio of methanol to water in the mobile phase is 60:40, the flow rate is 12 mL/min, and 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 ofchromatographic peak of 19-21 min to obtain a compound 8; a chromatographic peak of 14-15 min, further purified by high performance liquid chromatography, wherein the volume ratio of acetonitrile to water in the mobile phase is 30:70, the flow rate is 12 mL/min, and the detection wavelength is 210 nm, chromatographic column: YMC-Pack R&D ODS-A, 250×20 mm, S-10 µm, under these conditions, collecting the chromatographic peak with retention times of 18-20 min, recovering the solvent under decompression to obtained compound 1:10-O-(p-hydroxycinnamoyl)-adoxosidic acid.


[0006] The preferred separation method in the solutioncomprises the following steps of:
  1. (1) adsorbing 5 kg the total extract of the Chinese traditional medicine composition by AB-8 macroporous resin, eluting with 150 L water, 87.5 L 10% ethanol, 225 L 30% ethanol and 250 L 50% ethanol in sequence, and concentrating to obtain extract of each part;
  2. (2) taking 200 g the 50% ethanol elution part extract obtained in the step (1), adding 200g reversed-phase silica gel ODS-AQ-HG S-50 µm to mix sample, naturally airing the mixed sample ODS, loading the sample, and separating by using a reversed-phase ODS-AQ-HG S-50 µm open column, wherein the sample height ratio is 1:4; eluting with 6 L methanol-water in a volume ratio of 20:80, 7L in a volume ratio 40:60, 7L in a volume ratio 60:40, 5L in a volume ratio 80:20 and 3L 100% methanol in sequence under reduced pressure to obtain Fr.A-Fr.E;
  3. (3) taking 50.0 g Fr.A sample obtained in step (2), adding 50 g reverse-phase silica gel ODS-AQ-HG S-50 µm to mix sample, naturally airing the mixed sample ODS, adding the mixed sample ODS into a sample loading column, separating liquid phase prepared by an upper medium-pressure, whereinaseparating column filler is ODS-AQ-HG S-50 µm, gradiently separating liquid phase prepared by medium-pressure, wherein the volume ratio of methanol to water is 25:75-60:40, and the flow rate is 25 mL/min, receiving the fractions in equal volume of 500 mL in a conical flask, concentrating under reduced pressure, identifying the combined fractions through a thin layer chromatography plate, and concentrating under reduced pressure again to obtain Fr.A-1 - Fr.A-7;
  4. (4) mixing 3.2 g Fr.A-2 sample obtained in the step (3) with 6.4 g silica gel of 200-300 mesh, loading onto a silica gel column, wherein the sample height ratio is 1:50, carrying out isocratic separation by using a dichloromethane-methanol volume ratio of 8:1, receiving fractions in equal volume of 50 mL, eluting with a volume of 800 mL, and identifying the combined fractions through thin-layer chromatography plate to obtain Fr.A-1-1 - Fr.A-1-4;
  5. (5) dissolving the Fr.A-1-2 sample obtained in the step (4) with methanol, the solution is filtered through a 0.45 µm microporous membrane, adopting high performance liquid chromatography, wherein the volume ratio of methanol to water in the mobile phase is 50:50, the flow rate is 12 mL/min, and 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 the following separation:

    a chromatographic peak of 6-8 min, further purified by high performance liquid chromatography, wherein the volume ratio of acetonitrile to water in the mobile phase is 25:75, the flow rate is 12 mL/min, and the detection wavelength is 210 nm, chromatographic column: YMC-Pack R&D ODS-A, 250×20 mm, S-10 µm, under these conditions, collecting the chromatographic peak with retention times of 8-9 min, recovering the solvent under decompression to obtained compound 2:aloe-emodin-8-O-β-D-glucopyranoside;

    a chromatographic peak of 9-10 min, further purified by high performance liquid chromatography, wherein the volume ratio of methanol to water in the mobile phase is 45:55, the flow rate is 12 mL/min, and the detection wavelength is 210 nm, chromatographic column: YMC-Pack R&D ODS-A, 250×20 mm, S-10 µm, under these conditions, collecting the chromatographic peak with retention times of 21-23 min, recovering the solvent under decompression to obtained compound 3: quercitrin;

    a chromatographic peak of 10-12 min, further purified by high performance liquid chromatography, wherein acetonitrile to water in the mobile phase is 25:75, the flow rate is 12 mL/min, and the detection wavelength is 210 nm, chromatographic column: YMC-Pack R&D ODS-A, 250×20 mm, S-10 µm, under these conditions, collecting the chromatographic peak with retention times of 9-10 min, recovering the solvent under decompression to obtained compound 4: matairesinol-4'-O-glucoside;

  6. (6) dissolving the Fr.A-1-3 sample obtained in the step (4) with methanol, the solution is filtered through a 0.45 µm microporous membrane, adopting high performance liquid chromatography, wherein the volume ratio of methanol to water in the mobile phase is 30:70, the flow rate is12mL/min, and detection wavelength is 210 nm, to perform primary separation and respectively collecting chromatographic peaks with retention times of 10-11min, 17-19min and 21-24min, recovering the solvent under reduced pressure, chromatographic peaks of 17-19 min is for compound 6: epi-vogeloside, chromatographic peaks of 21-24 min is for compound 7: vogeloside, a chromatographic peak of 10-11 min, further purified by high performance liquid chromatography, wherein the volume ratio of acetonitrile to water in the mobile phase is 15:85, the flow rate is 12 mL/min, and the detection wavelength is 210 nm, chromatographic column: YMC-Pack R&D ODS-A, 250×20 mm, S-10 µm, under these conditions, collecting the chromatographic peak with retention times of 14-16 min, recovering the solvent under decompression to obtained compound 5: liquiritinapioside; and
  7. (7) dissolving the Fr.A-1-3 sample obtained in the step (3) with methanol, the solution is filtered through a 0.45 µm microporous membrane, adopting high performance liquid chromatography, wherein the volume ratio of methanol to water in the mobile phase is 60:40, the flow rate is 12 mL/min, and detection wavelength is 210 nm, to perform primary separation and respectively collecting chromatographic peaks with retention times of 14-15 min and 19-21min, 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 ofchromatographic peak of 19-21 min to obtain a compound 8; a chromatographic peak of 14-15 min, further purified by high performance liquid chromatography, wherein the volume ratio of acetonitrile to water in the mobile phase is 30:70, the flow rate is 12 mL/min, and the detection wavelength is 210 nm, chromatographic column: YMC-Pack R&D ODS-A, 250×20 mm, S-10 µm, under these conditions, collecting the chromatographic peak with retention times of 18-20 min, recovering the solvent under decompression to obtained compound 1: 10-O-(p-hydroxycinnamoyl)-adoxosidic acid.


[0007] Preferably, the Chinese traditional medicine composition is prepared from the following raw materials in parts by weight:
200 parts of fructusforsythiae, 300 parts of honeysuckle, 200 parts of isatis root, 40 parts of rheum officinale, 60 parts of pogostemoncablin, 300 parts of rhizomadryopteriscrassirhizomatis, 100 parts of rhodiolarosea, 9 parts of menthol, 60 parts of ephedra, 100 parts of semen armeniacaeamarum, 200 parts of houttuyniacordata, 100 parts of liquorice and 200 parts of gypsum fibrosum.

[0008] Preferably, the Chinese traditional medicine composition is prepared from the following raw materials in parts by weight:
300 parts of fructusforsythiae, 200 parts of honeysuckle, 300 parts of isatis root, 60 parts of rheum officinale, 100 parts of pogostemoncablin, 200 parts of rhizomadryopteriscrassirhizomatis, 60 parts of rhodiolarosea, 5 parts of menthol, 100 parts of ephedra, 60 parts of semen armeniacaeamarum, 300 parts of houttuyniacordata, 60 parts of liquorice and 300 parts of gypsum fibrosum.

[0009] Preferably, the Chinese traditional medicine composition is prepared from the following raw materials in parts by weight:
278 parts of fructusforsythiae, 294 parts of honeysuckle, 285 parts of isatis root, 55 parts of rheum officinale, 95 parts of pogostemoncablin, 290 parts of rhizomadryopteriscrassirhizomatis, 87 parts of rhodiolarosea, 8.5 parts of menthol, 88 parts of ephedra, 80 parts of semen armeniacaeamarum, 284 parts of houttuyniacordata, 95 parts of liquorice and 277 parts of gypsum fibrosum.

[0010] The total extract of the Chinese traditional medicine composition in the scheme is prepared from the following steps of:
  1. (1) weighing the Chinese traditional medicinal materials according to the weight ratio of the raw material medicines, cleaning and selecting, and crushing as appropriate;
  2. (2) crushing pogostemoncablin, adding 10 times of water to extract volatile oil, extracting for 8 hours, and collecting the volatile oil for later use; after filtering the extracting solution, discarding the residue, and taking the filtrate for later use;
  3. (3) extracting fructusforsythiae, ephedra, houttuyniacordata and rheum officinale three times with 12 times of 70% ethanol for 2.5 hours each time, combining and filtering the extracting solutions, recovering ethanol, and taking the filtrate for later use;
  4. (4) honeysuckle, gypsum fibrosum, isatis root, rhizomadryopteriscrassirhizomatis, liquorice and rhodiolarosea, adding 12 times of water for decocting to boil, adding semen armeniacaeamarum, decocting twice for 1 hour each time, combining and filtering the extracting solutions, combining the obtained filtrate with the filtrate after pogostemoncablin oil extraction in the step (2), concentrating to a clear pastewith a relative density of 1.10-1.15 measured at 60°C, adding ethanol to adjust the alcohol concentration to 70%, and refrigerating and standing; filtering and recovering ethanol until no alcohol smell exists to obtain clear paste for later use; and
  5. (5) combining the clear paste obtained in the step (4) with the alcohol extract obtained in the step (3), concentrating to a clear paste with a relative density of 1.15-1.20 measured at 60°C, and drying to obtain the total extract for later use.

Advantageous Effects



[0011] The separation method provided by the solutioncan separate and obtain eight compounds, namely 10-O-(p-hydroxycinnamoyl)-adoxosidic acid, aloe-emodin-8-O-β-D-glucopyranoside, quercitrin, matairesinol -4'-O-glucoside, liquiritinapioside, epi-vogeloside, vogeloside and ethyl caffeate.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS


Example 1



[0012] Weighing in proportion: 20 kg fructusforsythiae, 30 kg honeysuckle, 20 kg isatis root, 4 kg rheum officinale, 6 kg pogostemoncablin, 30 kg rhizomadryopteriscrassirhizomatis, 10 kg rhodiolarosea, 0.9 kg menthol, 6 kg ephedra, 10 kg semen armeniacaeamarum, 20 kg houttuyniacordata, 10 kg liquorice and 20 kg gypsum fibrosum, and extracting according to the following processes:
  1. (1) weighing the Chinese traditional medicinal materials according to the weight ratio of the raw material medicines, cleaning and selecting, and crushing as appropriate;
  2. (2) crushing pogostemoncablin, adding 10 times of water to extract volatile oil, extracting for 8 hours, and collecting the volatile oil for later use; after filtering the extracting solution, discarding the residue, and taking the filtrate for later use;
  3. (3) extracting fructusforsythiae, ephedra, houttuyniacordata and rheum officinale three times with 12 times of 70% ethanol for 2.5 hours each time, combining and filtering the extracting solutions, recovering ethanol, and taking the filtrate for later use;
  4. (4) honeysuckle, gypsum fibrosum, isatis root, rhizomadryopteriscrassirhizomatis, liquorice and rhodiolarosea, adding 12 times of water for decocting to boil, adding semen armeniacaeamarum, decocting twice for 1 hour each time, combining and filtering the extracting solutions, combining the obtained filtrate with the filtrate after pogostemoncablin oil extraction in the step (2), concentrating to a clear pastewith a relative density of 1.15 measured at 60°C, adding ethanol to adjust the alcohol concentration to 70%, and refrigerating and standing; filtering and recovering ethanol until no alcohol smell exists to obtain clear paste for later use; and
  5. (5) combining the clear paste obtained in the step (4) with the alcohol extract obtained in the step (3), concentrating to a clear paste with a relative density of 1.20 measured at 60°C, and drying to obtain the total extract for later use.


[0013] The separation method comprises the following steps:

1. Instruments and materials



[0014] Bruker Alpha infrared spectrometer (Bruker, Switzerland); Bruker AVIIIHD 600 nuclear magnetic resonance spectrometer (Bruker, Switzerland); Synapt G2-S Mass spectrometer (Waters, USA); Combi Flash Rf medium and low-pressure preparative liquid chromatograph (Teledvne ISCO, USA); NP7000 preparative liquid chromatograph (Jiangsu Hanbom Science and Technology Co., Ltd.) Milli-Q pure water purifier (Millipore, USA); AL204 analytical electronic balance (Mettler Toledo, USA); YMC ODS-A-HG 50 µm reverse phase silica gel (YMC Corporation, Japan); column chromatography silica gel (100-200 mesh and 200-300 mesh, Qingdao Haiyang Chemical CO., Ltd.); thin-layer chromatography silica gel plate GF254 (Qingdao Haiyang Chemical CO., Ltd); YMC-Pack R&D ODS-A (250×20mm, S-10 µm, YMC Corporation, Japan); total extract of the Chinese traditional medicine composition of the solution, (Shijiazhuang Yiling Pharmaceutical Co., Ltd., batch number: B1509001); chromatographically pure acetonitrile, methanol (Adamas Reagent Co., Shanghai); analytical pure reagents (Beijing Chemical Works).

2. Extraction and separation



[0015] 
  1. (1) adsorbing 5kg the total extract of the Chinese traditional medicine composition by AB-8 macroporous resin, eluting with 150L water, 87.5L 10% ethanol, 225L 30% ethanol and 250L 50% ethanol in sequence, and concentrating to obtain extract of each part;
  2. (2) taking 200g the 50% ethanol elution part extract obtained in the step (1), adding 200g reversed-phase silica gel ODS-AQ-HG S-50 µm to mix sample, naturally airing the mixed sample ODS, loading the sample, and separating by using a reversed-phase ODS-AQ-HG S-50 µm open column, wherein the sample height ratio is 1:4; eluting with 6 L methanol-water in a volume ratio of 20:80, 7L in a volume ratio 40:60, 7L in a volume ratio 60:40, 5L in a volume ratio 80:20 and 3L 100% methanol in sequence under reduced pressure to obtain Fr.A-Fr.E;
  3. (3) taking 50.0g Fr.A sample obtained in step (2), adding 50.og reverse-phase silica gel ODS-AQ-HG S-50 µm to mix sample, naturally airing the mixed sample ODS, adding the mixed sample ODS into a sample loading column, separating liquid phase prepared by an upper medium-pressure, whereinaseparating column filler was ODS-AQ-HG S-50 µm, gradiently separating liquid phase prepared by medium-pressure, wherein the volume ratio of methanol to water is 25:75-60:40, and the flow rate is 25 mL/min, receiving the fractions in equal volume of 500 mL in a conical flask, concentrating under reduced pressure, identifying the combined fractions through a thin layer chromatography plate, and concentrating under reduced pressure again to obtain Fr.A-1 - Fr.A-7;
  4. (4) mixing 3.2g Fr.A-2 sample obtained in the step (3) with 6.4g silica gel of 200-300 mesh, loading onto a silica gel column,whereinthe sample height ratio was 1:50, carrying out isocratic separation by using a dichloromethane-methanol volume ratio of 8:1, receiving fractions in equal volume of 50 mL, eluting with a volume of 800 mL, and identifying the combined fractions through thin-layer chromatography plate to obtain Fr.A-1-1 - Fr.A-1-4;
  5. (5) dissolving the Fr.A-1-2 sample obtained in the step (4) with methanol, the solution was filtered through a 0.45 µm microporous membrane, adopting high performance liquid chromatography, wherein the volume ratio of methanol to water in the mobile phase was 50:50, the flow rate was 12 mL/min, and detection wavelength was 210 nm, to perform primary separation and respectively collecting chromatographic peaks with retention times of 6-8min, 9-10min and 12-13min, recovering the solvent under reduced pressure, and respectively performing the following separation:

    a chromatographic peak of 6-8 min, further purified by high performance liquid chromatography, wherein the volume ratio of acetonitrile to water in the mobile phase was 25:75, the flow rate was 12 mL/min, and the detection wavelength was 210 nm, chromatographic column: YMC-Pack R&D ODS-A, 250×20 mm, S-10 µm, under these conditions, collecting the chromatographic peak with retention times of 8-9 min, recovering the solvent under decompression to obtained compound 2: aloe-emodin-8-O-β-D-glucopyranoside;

    a chromatographic peak of 9-10 min, further purified by high performance liquid chromatography, wherein the volume ratio of methanol to water in the mobile phase was 45:55, the flow rate was 12 mL/min, and the detection wavelength was 210 nm, chromatographic column: YMC-Pack R&D ODS-A, 250×20 mm, S-10 µm, under these conditions, collecting the chromatographic peak with retention times of 21-23 min, recovering the solvent under decompression to obtained compound 3: quercitrin;

    a chromatographic peak of 10-12 min, further purified by high performance liquid chromatography, wherein acetonitrile to water in the mobile phase was 25:75, the flow rate was 12 mL/min, and the detection wavelength was 210 nm, chromatographic column: YMC-Pack R&D ODS-A, 250×20 mm, S-10 µm, under these conditions, collecting the chromatographic peak with retention times of 9-10 min, recovering the solvent under decompression to obtained compound 4: matairesinol-4'-O-glucoside;

  6. (6) dissolving the Fr.A-1-3 sample obtained in the step (4) with methanol, the solution was filtered through a 0.45 µm microporous membrane, adopting high performance liquid chromatography, wherein the volume ratio of methanol to water in the mobile phase was 30:70, the flow rate was 12mL/min, and detection wavelength was 210 nm, to perform primary separation and respectively collecting chromatographic peaks with retention times of 10-11min, 17-19min and 21-24min, recovering the solvent under reduced pressure, chromatographic peaks of 17-19 min is for compound 6: epi-vogeloside, chromatographic peaks of 21-24 min is for compound 7: vogeloside, a chromatographic peak of 10-11 min, further purified by high performance liquid chromatography, wherein the volume ratio of acetonitrile to water in the mobile phase was 15:85, the flow rate was 12 mL/min, and the detection wavelength was 210 nm, chromatographic column: YMC-Pack R&D ODS-A, 250×20 mm, S-10 µm, under these conditions, collecting the chromatographic peak with retention times of 14-16 min, recovering the solvent under decompression to obtained compound 5: liquiritinapioside; and
  7. (7) dissolving the Fr.A-1-3 sample obtained in the step (3) with methanol, the solution was filtered through a 0.45 µm microporous membrane, adopting high performance liquid chromatography, wherein the volume ratio of methanol to water in the mobile phase was 60:40, the flow rate was 12 mL/min, and detection wavelength was 210 nm, to perform primary separation and respectively collecting chromatographic peaks with retention times of 14-15 min and 19-21min, 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 ofchromatographic peak of 19-21 min to obtain compound 8; a chromatographic peak of 14-15 min, further purified by high performance liquid chromatography, wherein the volume ratio of acetonitrile to water in the mobile phase was 30:70, the flow rate was 12 mL/min, and the detection wavelength was 210 nm, chromatographic column: YMC-Pack R&D ODS-A, 250×20 mm, S-10 µm, under these conditions, collecting the chromatographic peak with retention times of 18-20 min, recovering the solvent under decompression to obtained compound 1: 10-O-(p-hydroxycinnamoyl)-adoxosidic acid.

3. Structure identification


3.1 Structure identification of new compounds



[0016] Compound 1: light yellow powder, UV λmax (MeOH):228,312 nm. Infrared showed hydroxyl (3330 cm-1), α, β-unsaturated carbonyl (1680, 1630cm-1) and benzene ring (1603, 1514 cm-1). HR-ESI-MS m/z: 521.1699 [M-H]- (calculated value: 521.1659), in combination with NMR data, the molecular formula of the compound was determined to be C25H30O12. Unsaturation degree is 11.

[0017] 1H-NMR (DMSO-d6, 600MHz) spectrum (Table 1) showed that the compound contained a pair of trans double bonds δ7.56 (1H, d, J=16.2Hz), 6.39 (1H, d, J=16.2Hz) and AB system aromatic hydrogens δ7.55 (2H, d, J=8.4Hz), 6.79 (2H, d, J=8.4Hz), chemical shift of hydrogen spectrum was 4.52 (1H, d, J=7.8Hz), presumed to be the terminal hydrogen of sugar.

[0018] 13C-NMR (DMSO-d6, 150MHz) spectrum (Table 1) showed that the compound contained two conjugated carbonyl carbons (δc:168.4, 167.2) and two distinct fragments, δc: 116.2 (2C), 130.8 (2C), 160.2 should be para-hydroxyphenyl fragments, δc:99.3, 77.6, 77.1, 73.6, 70.4, 61.6 is a glucosyl group fragment.

[0019] By HMBC, double bonds δH 7.56, 6.39 are related to phenyl carbon δc 125.5, double bonds δH6.39 and -CH2-δH4.12 are related to carbonyl group δc 167.2, and the above fragments are not related to other C and H chemical shifts, and the fragments are presumed to be independent fragments of p-hydroxycinnamoyl, and the remaining part of the sugar-free fragment is presumed to be the core. The unsaturation of the parent nucleus is calculated to be 4 (containing a carbonyl group and a double bond), suggesting that the parent nucleus is a double-ring structure. Through the assignment and connection of HSQC and HMBC, it is speculated that the compound is an iridoid, and the parent nucleus of the compound is determined to be adoxosidic acid through literature search.

[0020] The remaining fragment was known to be p-hydroxycinnamic acid, esterified to the 10-position of the parent core adoxosidic acid. The compound was identified as a new compound (the configuration of which was not yet confirmed in this experiment and will be confirmed in subsequent studies) as 10-O-(p-hydroxycinnamoyl)-adoxosidic acid by searching the SciFinder and Reaxys databases.

Table 1 NMR data of compound 1



[0021] 
Table 1 NMR data of compound 1
Carbon siteδHδc
1 5.14 (1H, d, J=6.6 Hz) 96.5
3 7.40 (1H, s) 151.6
4   111.4
5 2.77 (1H, m) 35.1
6 1.42 (1H, m), 2.09 (1H, m) 32.1
7 1.35 (1H, m), 1.79 (1H, m) 27.6
8 2.25 (1H, m) 40.2
9 1.92 (1H, m) 43.1
10 4.12 (2H, m) 67.1
11   168.4
1' 4.52 (1H, d, J=7.8Hz) 99.3
2' 3.01 (1H, m) 73.6
3' 3.17 (1H, m) 77.1a
4' 3.14 (1H, m) 70.4
5' 3.17 (1H, m) 77.6a
6' 3.43 (1H, d, J=11.4 Hz), 3.68 (1H, d, J=11.4 Hz) 61.6
1"   125.5
2"6" 7.55 (2H, d, J=8.4 Hz) 130.8
3 "5" 6.79 (2H, d, J=8.4 Hz) 116.2
4"   160.2
7" 7.56 (1H, d, J=16.2Hz) 145.2
8" 6.39 (1H, d, J=16.2Hz) 114.5
9"   167.2
a Chemical shifts may need to be exchanged





3.2 Structure identification of known compounds



[0022] Compound 2:yellow powder, ESI-MS m/z: 431 [M-H]-, in combination with NMR data, the molecular formula of the compound was determined to be C21H20O10. 1H-NMR (DMSO-d6, 600MHz) δH: 12.88 (1H, s, OH), 7.89 (1H, dd, J=1.2, 8.4Hz, H-5), 7.86 (1H, t, J=7.8Hz, H-6), 7.72 (1H, dd, J=1.2, 8.4Hz, H-7), 7.66 (1H, brs, H-4), 7.28 (1H, brs, H-2), 5.17 (1H, d, J=7.8Hz, anomeric-H), 4.62 (2H, s, CH2OH), 3.72-3.23 (Glc-H). 13C-NMR (DMSO-d6, 150MHz) δH: 188.8 (C-9), 182.6 (C-10), 162.2 (C-1), 158.7 (C-8), 152.7 (C-3), 136.4 (C-6), 135.3 (C-10a), 132.7 (C-4a), 122.9 (C-7), 121.2 (C-2), 121.0 (C-5), 116.4 (C-8a, C-9a), 100.9 (C-1'), 77.7 (C-5'), 77.0 (C-3'), 73.7 (C-2'), 70.0 (C-4'), 62.5 (CH2OH), 61.0 (C-6') . The characteristics of hydrogen spectrum and data of carbon spectrum above were consistent with what reported in the literature, and the compound was identified as aloe-emodin-8-O-β-D-glucopyranoside.

[0023] Compound 3: yellow powder, ESI-MS m/z: 447 [M-H]-, in combination with NMR data, the molecular formula of the compound was determined to be C21H20O11. 1H-NMR (DMSO-d6, 600MHz) δH: 12.66 (1H, s, 5-OH), 7.31 (1H, d, J=2.4Hz, H-2'), 7.26 (1H, d, J=2.4, 8.4Hz, H-6'), 6.87 (1H, d, J=8.4Hz, H-5'), 6.39 (1H, d, J=2.4Hz, H-8), 6.21 (1H, d, J=2.4Hz, H-6), 5.26 (1H, d, J=1.8Hz, anomeric-H), 0.82 (3H, d, J=6.0Hz, CH3). 13C-NMR (DMSO-d6, 150MHz) δc: 178.2 (C-4), 164.6 (C-7), 161.7 (C-5), 157.7 (C-2), 156.9 (C-9), 148.9 (C-4'), 145.6 (C-3'), 134.7 (C-3), 121.5 (C-6'), 121.2 (C-1'), 116.1 (C-5'), 115.9 (C-2'), 104.5 (C-10), 102.3 (C-1"), 99.1 (C-6), 94.1 (C-8), 71.6 (C-4"), 71.0 (C-3"), 70.8 (C-2"), 70.5 (C-5"), 17.9 (C-6") . The characteristics of hydrogen spectrum and data of carbon spectrum above were consistent with what reported in the literature, and the compound was identified as quercitrin.

[0024] Compound 4: yellow powder, ESI-MS m/z: 519 [M-H]-, in combination with NMR data, the molecular formula of the compound was determined to be C26H32O11. 1H-NMR (DMSO-d6, 600MHz) δH: 6.99 (1H, d, J=8.4Hz, H-5), 6.78 (1H, d, J=1.8Hz, H-2'), 6.67 (2H, m, H-5, H-6'), 6.63 (1H, s, H-2), 6.50 (1H, dd, J=1.8, 8.4Hz, H-6), 4.84 (1H, d, J=7.8Hz, H-1"), 4.09 (1H, t, J=7.8Hz, H-9a), 3.86 (1H, t, J=8.4Hz, H-9b), 3.72 (6H, d, J=2.4Hz, 2×OCH3) . 13C-NMR (DMSO-d6, 150MHz) δC: 178.9 (C-9'), 149.1 (C-3'), 147.9 (C-3), 145.7 (C-4'), 145.4 (C-4), 132.2 (C-1'), 130.0 (C-1), 121.8 (C-6'), 121.2 (C-6), 115.9 (C-5'), 115.6 (C-5), 114.3 (C-2'), 113.1 (C-2), 100.6 (C-1"), 77.4 (C-5"), 77.3 (C-3"), 73.7 (C-2"), 71.1 (C-9), 70.1 (C-4"), 61.1 (C-6"), 56.1 (OCH3), 56.0 (OCH3), 46.0 (C-8'), 41.3 (C-8), 37.3 (C-7), 33.9 (C-7'). The data of carbon spectrum above were consistent with what reported in the literature, and the compound was identified as matairesinol-4'-O-glucoside.

[0025] Compound 5: white powder, ESI-MS m/z: 549 [M-H]-, in combination with NMR data, the molecular formula of the compound was determined to be C26H30O13. 1H-NMR (CD3OD, 600 MHz) δH: 7.70 (1H, d, J=9.0Hz, H-5), 7.40 (2H, d, J=8.4Hz, H-2', 6'), 7.09 (2H, d, J=9.0Hz, H-3', 5'), 6.48 (1H, dd, J=2.4, 9.0Hz, H-6), 6.34 (1H, d, J=2.4Hz, H-8), 5.46 (1H, d, J=1.2Hz, H-1'''), 5.39 (1H, dd, J=2.4, 13.2Hz, H-2), 4.98 (1H, d, J=7.2Hz, H-1"), 4.04 (1H, d, J=9.6Hz, H-5"'a), 3.89 (1H, d, J=1.2Hz, H-2'''), 3.88 (1H, dd, J=i.2, 12.0Hz, H-6"a), 3.79 (1H, d, J=9.6Hz, H-5'''b), 2.99 (1H, m, H-3a), 2.74 (1H, dd, J=2.4, 16.8Hz, H-3b) . 13C-NMR (CD3OD, 150 MHz) δC: 193.2 (C-4), 166.7 (C-7), 165.3 (C-8a), 159.0 (C-4'), 134.3 (C-1'), 129.9 (C-5), 128.8 (C-2', 6'), 117.6 (C-3', 5'), 114.9 (C-4a), 111.8 (C-6), 110.7 (C-1'''), 103.8 (C-8), 100.7 (C-1"), 80.7 (C-2), 80.6 (C-3"0, 78.8 (C-5"), 78.6 (C-2'''), 78.0 (C-2"), 77.9 (C-3"), 75.4 (C-4'''), 71.4 (C-4"), 66.0 (C-5'''), 62.4 (C-6"), 44.9 (C-3). The characteristics of hydrogen spectrum and data of carbon spectrum above were consistent with what reported in the literature, and the compound was identified as liquiritinapioside.

[0026] Compound 6: white powder, ESI-MS m/z: 387 [M-H]-, in combination with NMR data, the molecular formula of the compound was determined to be C17H24O10. 1H-NMR (CD3OD, 600 MHz) δH: 7.60 (1H, d, J=2.4Hz, H-3), 5.55 (1H, d, J=1.8Hz, H-1), 5.48 (1H, m, H-8), 5.31 (1H, s, H-7), 5.29 (1H, m, H-10a), 5.25 (1H, m, H-10b), 4.67 (1H, d, J=7.8Hz, H-1'), 3.50 (1H, s, 7-OCH3), 3.18 (1H, m, H-5), 2.63 (1H, m, H-9), 1.85 (1H, dd, J=6.0, 13.2Hz, H-6a), 1.69 (1H, td, J=3.0, 13.8Hz, H-6b) . 13C-NMR (CD3OD, 150 MHz) δC: 167.4 (C-11), 154.4 (C-4), 133.3 (C-8), 121.0 (C-10), 105.3 (C-4), 103.3 (C-7), 100.3 (C-1'), 98.5 (C-1), 78.3 (C-5'), 78.0 (C-3'), 74.6 (C-2'), 71.4 (C-4'), 62.6 (C-6'), 57.0 (7-OCH3), 43.5 (C-9), 30.2 (C-6), 22.8 (C-5). The characteristics of hydrogen spectrum and data of carbon spectrum above were consistent with what reported in the literature, and the compound was identified as epi-vogeloside.

[0027] Compound 7: white powder, ESI-MS m/z: 387 [M-H]-, in combination with NMR data, the molecular formula of the compound was determined to be C17H24O10. 1H-NMR (CD3OD, 600 MHz) δH: 7.58 (1H, d, J = 2.4Hz, H-3), 5.55 (1H, d, J = 1.2Hz, H-1), 5.47 (1H, m, H-8), 5.31 (1H, s, H-7), 5.29 (1H, m, H-1oa), 5.26 (1H, m, H-1ob), 4.66 (1H, d, J=7.8Hz, H-1'), 3.54 (1H, s, 7-OCH3), 3.16 (1H, m, H-5), 2.67 (1H, m, H-9), 1.97 (1H, m, H-6a), 1.44 (1H, m, H-6b) . 13C-NMR (CD3OD, 150 MHz) δc: 167.6 (C-11), 154.1 (C-4), 133.0 (C-8), 121.1 (C-10), 105.4 (C-4), 105.1 (C-7), 99.7 (C-1'), 97.9 (C-1), 78.4 (C-5'), 77.8 (C-3'), 74.7 (C-2'), 71.5 (C-4'), 62.6 (C-6'), 57.1 (7-OCH3), 43.7 (C-9), 31.7 (C-6), 25.3 (C-5) . The characteristics of hydrogen spectrum were consistent with what reported in the literature carbon, the carbon signal is assigned, and the compound was identified as vogeloside.

[0028] Compound 8: white powder, ESI-MS m/z: 209 [M+H]+, in combination with NMR data, the molecular formula of the compound was determined to be C11H12O4. 1H-NMR (DMSO-d6, 600MHz) δH: 7.46 (1H, d, J=15.9Hz, H-7), 7.04 (1H, brs, H-2), 6.99 (1H, d, J=8.1Hz, H-6), 6.75 (1H, d, J=8.1Hz, H-5), 6.24 (1H, d, J=15.9Hz, H-8), 4.15 (2H, q, J=7.1Hz, H-10), 1.24 (3H, t, J=7.1Hz, H-11) . 13C-NMR (DMSO-d6, 150MHz) δc: 166.5 (C-9), 148.6 (C-4), 145.0 (C-7), 145.6 (C-3), 121.3 (C-6), 125.3 (C-1), 115.7 (C-5), 114.7 (C-2), 113.9 (C-8), 59.6 (C-10), 14.3 (C-11) . The characteristics of hydrogen spectrum and data of carbon spectrum above were consistent with what reported in the with a literature, and the compound was identified as ethyl caffeate.

Example 2



[0029] Weighing in proportion: 30kg fructusforsythiae, 20kg honeysuckle, 30kg isatis root, 6kg rheum officinale, 10kg pogostemoncablin, 20kg rhizomadryopteriscrassirhizomatis, 6 kg rhodiolarosea, 0.5kg menthol, 10kg ephedra, 6 kg semen armeniacaeamarum, 30kg houttuyniacordata, 6 kg liquorice and 30kg gypsum fibrosum, and extracting according to the following processes:
  1. (1) weighing the Chinese traditional medicinal materials according to the weight ratio of the raw material medicines, cleaning and selecting, and crushing as appropriate;
  2. (2) crushing pogostemoncablin, adding 10 times of water to extract volatile oil, extracting for 8 hours, and collecting the volatile oil for later use; after filtering the extracting solution, discarding the residue, and taking the filtrate for later use;
  3. (3) extracting fructusforsythiae, ephedra, houttuyniacordata and rheum officinale three times with 12 times of 70% ethanol for 2.5 hours each time, combining and filtering the extracting solutions, recovering ethanol, and taking the filtrate for later use;
  4. (4) honeysuckle, gypsum fibrosum, isatis root, rhizomadryopteriscrassirhizomatis, liquorice and rhodiolarosea, adding 12 times of water for decocting to boil, adding semen armeniacaeamarum, decocting twice for 1 hour each time, combining and filtering the extracting solutions, combining the obtained filtrate with the filtrate after pogostemoncablin oil extraction in the step (2), concentrating to a clear pastewith a relative density of 1.10 measured at 60°C, adding ethanol to adjust the alcohol concentration to 70%, and refrigerating and standing; filtering and recovering ethanol until no alcohol smell exists to obtain clear paste for later use; and
  5. (5) combining the clear paste obtained in the step (4) with the alcohol extract obtained in the step (3), concentrating to a clear paste with a relative density of 1.15 measured at 60°C, and drying to obtain the total extract for later use.

1. The instrument and materials:the same as in Example 1.


2. Extraction and separation:



[0030] adsorbing the solution total extract 5 kg the Chinese traditional medicine composition by d-101 macroporous resin, eluting with water, 10% ethanol, 30% ethanol and 50% ethanol in sequence, and concentrating to obtain extract of each part; remaining steps were the same as in Example 1.

3. Results identification:the same as Example 1, eight compounds are obtained from the separation which are identical to those obtained in Example 1.


Example 3



[0031] The raw material medicine formula is: 27.8 kg fructusforsythiae, 29.4 kg honeysuckle, 28.5 kg isatis root, 5.5 kg rheum officinale, 9.5 kg pogostemoncablin, 29 kg rhizomadryopteriscrassirhizomatis, 8.7 kg rhodiolarosea, 0.85 kg menthol, 8.8 kg ephedra, 8 kg semen armeniacaeamarum, 28.4 kg houttuyniacordata, 9.5 kg liquorice and 27.7 kg gypsum fibrosum, and extracting according to the following processes:
  1. (1) weighing the Chinese traditional medicinal materials according to the weight ratio of the raw material medicines, cleaning and selecting, and crushing as appropriate;
  2. (2) crushing pogostemoncablin, adding 10 times of water to extract volatile oil, extracting for 8 hours, and collecting the volatile oil for later use; after filtering the extracting solution, discarding the residue, and taking the filtrate for later use;
  3. (3) extracting fructusforsythiae, ephedra, houttuyniacordata and rheum officinale three times with 12 times of 70% ethanol for 2.5 hours each time, combining and filtering the extracting solutions, recovering ethanol, and taking the filtrate for later use;
  4. (4) honeysuckle, gypsum fibrosum, isatis root, rhizomadryopteriscrassirhizomatis, liquorice and rhodiolarosea, adding 12 times of water for decocting to boil, adding semen armeniacaeamarum, decocting twice for 1 hour each time, combining and filtering the extracting solutions, combining the obtained filtrate with the filtrate after pogostemoncablin oil extraction in the step (2), concentrating to a clear pastewith a relative density of 1.13 measured at 60°C, adding ethanol to adjust the alcohol concentration to 70%, and refrigerating and standing; filtering and recovering ethanol until no alcohol smell exists to obtain clear paste for later use; and
  5. (5) combining the clear paste obtained in the step (4) with the alcohol extract obtained in the step (3), concentrating to a clear paste with a relative density of 1.18 measured at 60°C, and drying to obtain the total extract for later use.

1. The instrument and materials were the same as in Example 1.


2. Extraction and separation:



[0032] adsorbing the solution total extract 5 kg the Chinese traditional medicine composition by HPD-100 macroporous resin, eluting with water, 10% ethanol, 30% ethanol and 50% ethanol in sequence, and concentrating to obtain extract of each part; remaining steps were the same as in Example 1.

3.Results identification



[0033] The same as Example 1, eight compounds are obtained from the separation which are identical to those obtained in Example 1.

[0034] While the foregoing is directed to the preferred examples of the scheme, it is not intended to limit the scheme to the precise form disclosed, any modification, equivalent replacement or improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.


Claims

1. A method for separating eight components in a Chinese traditional medicine composition, wherein the Chinese traditional medicine composition is prepared from the following raw materials in parts by weight: 200-300 parts of fructusforsythiae, 60-100 parts of ephedra, 40-60 parts of rheum officinale, 200-300 parts of houttuyniacordata, 200-300 parts of honeysuckle, 200-300 parts of isatis root, 60-100 parts of pogostemoncablin, 200-300 parts of rhizomadryopteriscrassirhizomatis, 60-100 parts of rhodiolarosea, 5-9 parts of menthol, 60-100 parts of semen armeniacaeamarum, 60-100 parts of liquorice and 200-300 parts of gypsum fibrosum, characterized in that the separation method comprises the following steps of:

(1) adsorbing total extract of the Chinese traditional medicine composition by AB-8 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 50% ethanol elution part extract obtained in step (1), adding reverse-phase silica gel ODS-AQ-HG, naturally airing a mixed sample, loading the sample, separating by using a reverse-phase ODS-AQ-HG 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; sequentially obtaining Fr.A-Fr.E;

(3) taking Fr.A sample obtained in step (2), adding a reverse-phase silica gel ODS-AQ-HG to mix sample, naturally airing the mixed sample ODS, adding the mixed sample ODS into a sample loading column, separating liquid phase prepared by an upper medium-pressure, gradiently separating liquid phase prepared by medium-pressure, wherein the volume ratio of methanol to water is 25:75-60:40, and the flow rate is 25 mL/min, receiving the fractions in equal volume of 500 mL in a conical flask, concentrating under reduced pressure, identifying the combined fractions through a thin layer chromatography plate, and concentrating under reduced pressure again to obtain Fr.A-1 - Fr.A-7;

(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 in equal volume of 50 mL, eluting with a volume of 800 mL, and identifying the combined fractions through thin-layer chromatography plate to obtain Fr.A-1-1-Fr.A-1-4;

(5) dissolving the Fr.A-1-2 sample obtained in the step (4) with methanol, adopting high performance liquid chromatography, wherein the volume ratio of methanol to water in the mobile phase is 50:50, the flow rate is 12 mL/min, and detection wavelength is 210 nm, to perform primary separation and respectively collecting chromatographic peaks with retention times of 6-8min, 9-10min and 12-13min, recovering the solvent under reduced pressure, and respectively performing the following separation:

a chromatographic peak of 6-8 min, further purified by high performance liquid chromatography, wherein the volume ratio of acetonitrile to water in the mobile phase is 25:75, the flow rate is 12 mL/min, and the detection wavelength is 210 nm, chromatographic column: YMC-Pack R&D ODS-A, 250×20 mm, S-10 µm, under these conditions, collecting the chromatographic peak with retention times of 8-9 min, recovering the solvent under decompression to obtained compound 2: aloe-emodin-8-O-β-D-glucopyranoside;

a chromatographic peak of 9-10 min, further purified by high performance liquid chromatography, wherein the volume ratio of methanol to water in the mobile phase is 45: 55, the flow rate is 12 mL/min, and the detection wavelength is 210 nm, chromatographic column: YMC-Pack R&D ODS-A, 250×20 mm, S-10 µm, under these conditions, collecting the chromatographic peak with retention times of 21-23 min, recovering the solvent under decompression to obtained compound 3: quercitrin;

a chromatographic peak of 10-12 min, further purified by high performance liquid chromatography, wherein the volume ratio of acetonitrile to water in the mobile phase is 25:75, the flow rate is 12 mL/min, and the detection wavelength is 210 nm, chromatographic column: YMC-Pack R&D ODS-A, 250×20 mm, S-10 µm, under these conditions, collecting the chromatographic peak with retention times of 9-10 min, recovering the solvent under decompression to obtained compound 4: matairesinol-4'-O-glucoside;

(6) dissolving the Fr.A-1-3 sample obtained in the step (4) with methanol, adopting high performance liquid chromatography, wherein the volume ratio of methanol to water in the mobile phase is 30:70, the flow rate is12mL/min, and detection wavelength is 210 nm, to perform primary separation and respectively collecting chromatographic peaks with retention times of 10-11min, 17-19min and 21-24min, recovering the solvent under reduced pressure, chromatographic peaks at 17-19 min is for compound 6: epi-vogeloside, chromatographic peaks at 21-24 min is for compound 7: vogeloside, chromatographic peak of 10-11 min, further purified by high performance liquid chromatography, wherein the volume ratio of acetonitrile to water in the mobile phase is 15:85, the flow rate is 12 mL/min, and the detection wavelength is 210 nm, chromatographic column: YMC-Pack R&D ODS-A, 250×20 mm, S-10 µm, under these conditions, collecting the chromatographic peak with retention times of 14-16 min, recovering the solvent under decompression to obtained compound 5: liquiritinapioside; and

(7) dissolving the Fr. A-3 sample obtained in the step (3) with methanol, adopting high performance liquid chromatography, wherein the volume ratio of methanol to water in the mobile phase is 60:40, the flow rate is 12 mL/min, and 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 ofchromatographic peak of 19-21 min to obtain a compound 8; chromatographic peak of 14-15 min, further purified by high performance liquid chromatography, wherein the volume ratio of acetonitrile to water in the mobile phase is 30:70, the flow rate is 12 mL/min, and the detection wavelength is 210 nm, chromatographic column: YMC-Pack R&D ODS-A, 250×20 mm, S-10 µm, under these conditions, collecting the chromatographic peak with retention times of 18-20 min, recovering the solvent under decompression to obtained compound 1: 10-O-(p-hydroxycinnamoyl)-adoxosidic acid.


 
2. The method for separating eight components according to claim 1, characterized in that the method for separating comprises the following steps of:

(1) adsorbing5 kg the total extract of the Chinese traditional medicine composition by AB-8 macroporous resin, eluting with 150 L water, 87.5 L 10% ethanol, 225 L 30% ethanol and 250 L 50% ethanol in sequence, and concentrating to obtain extract of each part;

(2) taking 200 g the 50% ethanol elution part extract obtained in the step (1), adding 200g reversed-phase silica gel ODS-AQ-HG S-50 µmtomix sample, naturally airing the mixed sample ODS, loading the sample, and separating by using a reversed-phase ODS-AQ-HG S-50 µm open column, wherein the sample height ratio is 1:4; eluting with 6 L methanol-water in a volume ratio of 20:80, 7 L in a volume ratio 40:60, 7 L in a volume ratio 60:40, 5 L in a volume ratio 80:20 and 3 L 100% methanol in sequence under reduced pressure to obtain Fr.A-Fr.E;

(3) taking 50.0 g Fr.A sample obtained in step (2), adding 50 g reverse-phase silica gel ODS-AQ-HG S-50 µm to mix sample, naturally airing the mixed sample ODS, adding the mixed sample ODS into a sample loading column, separating liquid phase prepared by an upper medium-pressure, whereinaseparating column filler is ODS-AQ-HG S-50 µm, gradiently separating liquid phase prepared by medium-pressure, wherein the volume ratio of methanol to water is 25:75-60:40, and the flow rate is 25 mL/min, receiving the fractions in equal volume of 500 mL in a conical flask, concentrating under reduced pressure, identifying the combined fractions through a thin layer chromatography plate, and concentrating under reduced pressure again to obtain Fr.A-1 - Fr.A-7;

(4) mixing 3.2 g Fr.A-2 sample obtained in the step (3) with 6.4 g silica gel of 200-300 mesh, loading onto a silica gel column,whereinthe sample height ratio is 1:50, carrying out isocratic separation by using a dichloromethane-methanol volume ratio of 8:1, receiving fractions in equal volume of 50 mL, eluting with a volume of 800 mL, and identifying the combined fractions through thin-layer chromatography plate to obtain Fr.A-1-1 - Fr.A-1-4;

(5) dissolving the Fr.A-1-2 sample obtained in the step (4) with methanol, the solution is filtered through a 0.45 µm microporous membrane, adopting high performance liquid chromatography, wherein the volume ratio of methanol to water in the mobile phase is 50:50, the flow rate is 12 mL/min, and detection wavelength is 210 nm, to perform primary separation and respectively collecting chromatographic peaks with retention times of 6-8min, 9-10min and 12-13min, recovering the solvent under reduced pressure, and respectively performing the following separation:

thechromatographic peak of 6-8 min, further purified by high performance liquid chromatography, wherein the volume ratio of acetonitrile to water in the mobile phase is 25:75, the flow rate is 12 mL/min, and the detection wavelength is 210 nm, chromatographic column: YMC-Pack R&D ODS-A, 250×20 mm, S-10 µm, under these conditions, collecting the chromatographic peak with retention times of 8-9 min, recovering the solvent under decompression to obtained compound 2:aloe-emodin-8-O-β-D-glucopyranoside;

thechromatographic peak of 9-10 min, further purified by high performance liquid chromatography, wherein the volume ratio of methanol to water in the mobile phase is 45: 55, the flow rate is 12 mL/min, and the detection wavelength is 210 nm, chromatographic column: YMC-Pack R&D ODS-A, 250×20 mm, S-10 µm, under these conditions, collecting the chromatographic peak with retention times of 21-23 min, recovering the solvent under decompression to obtained compound 3: quercitrin;

thechromatographic peak of 10-12 min, further purified by high performance liquid chromatography, wherein the volume ratio of acetonitrile to water in the mobile phase is 25:75, the flow rate is 12 mL/min, and the detection wavelength is 210 nm, chromatographic column: YMC-Pack R&D ODS-A, 250×20 mm, S-10 µm, under these conditions, collecting the chromatographic peak with retention times of 9-10 min, recovering the solvent under decompression to obtained compound 4: matairesinol-4'-O-glucoside;

(6) dissolving the Fr.A-1-3 sample obtained in the step (4) with methanol, the solution is filtered through a 0.45 µm microporous membrane, adopting high performance liquid chromatography, wherein the volume ratio of methanol to water in the mobile phase is 30:70, the flow rate is12 mL/min, and detection wavelength is 210 nm, to perform primary separation and respectively collecting chromatographic peaks with retention times of 10-11min, 17-19min and 21-24min, recovering the solvent under reduced pressure, chromatographic peaks of 17-19 min is for compound 6: epi-vogeloside, chromatographic peaks of 21-24 min is for compound 7: vogeloside, a chromatographic peak of 10-11 min, further purified by high performance liquid chromatography, wherein the volume ratio of acetonitrile to water in the mobile phase is 15:85, the flow rate is 12 mL/min, and the detection wavelength is 210 nm, chromatographic column: YMC-Pack R&D ODS-A, 250×20 mm, S-10 µm, under these conditions, collecting the chromatographic peak with retention times of 14-16 min, recovering the solvent under decompression to obtained compound 5: liquiritinapioside; and

(7) dissolving the Fr.A-3 sample obtained in the step (3) with methanol, the solution is filtered through a 0.45 µm microporous membrane, adopting high performance liquid chromatography, wherein the volume ratio of methanol to water in the mobile phase is 60:40, the flow rate is 12 mL/min, and detection wavelength is 210 nm, to perform primary separation and respectively collecting chromatographic peaks with retention times of 14-15 min and 19-21min, 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 ofchromatographic peak of 19-21 min to obtain a compound 8; a chromatographic peak of 14-15 min, further purified by high performance liquid chromatography, wherein the volume ratio of acetonitrile to water in the mobile phase is 30:70, the flow rate is 12 mL/min, and the detection wavelength is 210 nm, chromatographic column: YMC-Pack R&D ODS-A, 250×20 mm, S-10 µm, under these conditions, collecting the chromatographic peak with retention times of 18-20 min, recovering the solvent under decompression to obtained compound 1: 10-O-(p-hydroxycinnamoyl)-adoxosidic acid.


 
3. The method for separating eight components according to any one of the claims 1 to 2, characterized in that the Chinese traditional medicine composition is prepared from the following raw materials in parts by weight:
200 parts of fructusforsythiae, 300 parts of honeysuckle, 200 parts of isatis root, 40 parts of rheum officinale, 60 parts of pogostemoncablin, 300 parts of rhizomadryopteriscrassirhizomatis, 100 parts of rhodiolarosea, 9 parts of menthol, 60 parts of ephedra, 100 parts of semen armeniacaeamarum, 200 parts of houttuyniacordata, 100 parts of liquorice and 200 parts of gypsum fibrosum.
 
4. The method for separating eight components according to any one of the claims 1 to 2, characterized in that the Chinese traditional medicine composition is prepared from the following raw materials in parts by weight:
300 parts of fructusforsythiae, 200 parts of honeysuckle, 300 parts of isatis root, 60 parts of rheum officinale, 100 parts of pogostemoncablin, 200 parts of rhizomadryopteriscrassirhizomatis, 60 parts of rhodiolarosea, 5 parts of menthol, 100 parts of ephedra, 60 parts of semen armeniacaeamarum, 300 parts of houttuyniacordata, 60 parts of liquorice and 300 parts of gypsum fibrosum.
 
5. The method for separating eight components according to any one of the claims 1 to 2, characterized in that the Chinese traditional medicine composition is prepared from the following raw materials in parts by weight:
278 parts of fructusforsythiae, 294 parts of honeysuckle, 285 parts of isatis root, 55 parts of rheum officinale, 95 parts of pogostemoncablin, 290 parts of rhizomadryopteriscrassirhizomatis, 87 parts of rhodiolarosea, 8.5 parts of menthol, 88 parts of ephedra, 80 parts of semen armeniacaeamarum, 284 parts of houttuyniacordata, 95 parts of liquorice and 277 parts of gypsum fibrosum.
 
6. The method for separating eight components according to any one of the claims 1 to 2, characterized in that the total extract of the Chinese traditional medicine composition is prepared by the following steps of:

(1) weighing the Chinese traditional medicinal materials according to the weight ratio of the raw material medicines, cleaning and selecting, and crushing as appropriate;

(2) crushing pogostemoncablin, adding 10 times of water to extract volatile oil, extracting for 8 hours, and collecting the volatile oil for later use; after filtering the extracting solution, discarding the residue, and taking the filtrate for later use;

(3) extracting fructusforsythiae, ephedra, houttuyniacordata and rheum officinale three times with 12 times of 70% ethanol for 2.5 hours each time, combining and filtering the extracting solutions, recovering ethanol, and taking the filtrate for later use;

(4) honeysuckle, gypsum fibrosum, isatis root, rhizomadryopteriscrassirhizomatis, liquorice and rhodiolarosea, adding 12 times of water for decocting to boil, adding semen armeniacaeamarum, decocting twice for 1 hour each time, combining and filtering the extracting solutions, combining the obtained filtrate with the filtrate after pogostemoncablin oil extraction in the step (2), concentrating to a clear pastewith a relative density of 1.10-1.15 measured at 60°C, adding ethanol to adjust the alcohol concentration to 70%, and refrigerating and standing; filtering and recovering ethanol until no alcohol smell exists to obtain clear paste for later use; and

(5) combining the clear paste obtained in the step (4) with the alcohol extract obtained in the step (3), concentrating to a clear paste with a relative density of 1.15-1.20 measured at 60°C, and drying to obtain the total extract for later use.


 





Search report