WO2012019373A1 - 一种制备芍药内酯苷和芍药苷的方法 - Google Patents

一种制备芍药内酯苷和芍药苷的方法 Download PDF

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WO2012019373A1
WO2012019373A1 PCT/CN2010/076636 CN2010076636W WO2012019373A1 WO 2012019373 A1 WO2012019373 A1 WO 2012019373A1 CN 2010076636 W CN2010076636 W CN 2010076636W WO 2012019373 A1 WO2012019373 A1 WO 2012019373A1
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Prior art keywords
paeoniflorin
silica gel
eluate
ethyl acetate
ratio
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PCT/CN2010/076636
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English (en)
French (fr)
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顾正兵
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张作光
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00Processes for the preparation of sugar derivatives
    • C07H1/06Separation; Purification
    • C07H1/08Separation; Purification from natural products
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18Magnoliophyta (angiosperms)
    • A61K36/185Magnoliopsida (dicotyledons)
    • A61K36/65Paeoniaceae (Peony family), e.g. Chinese peony
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00Drugs for disorders of the cardiovascular system
    • A61P9/10Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20Carbocyclic rings

Definitions

  • the invention belongs to the technical field of medicine, and relates to a method for separating and extracting compounds in the chemical field, and particularly relates to a preparation process of plant natural physiologically active substances paeoniflorin and paeoniflorin.
  • Peony is a perennial herb of the genus Paeonia, which is commonly used as a dry root.
  • the Chinese Pharmacopoeia 2010 edition contains two kinds of peony. One is Radix Paeoniae Alba, which is the dried root of Paeonia lactiflora Pall.; the other is Radix Paeoniae Rubra, which is the Ranunculaceae plant 3 ⁇ 4eo « « ve fc w Lynch Dry the roots.
  • traditional Chinese medicine preparations containing peony such as cerebral thrombosis tablets, Yinaofujian capsules, partial sputum restoration tablets, Huisheng reconstituted pills, and Dahuo Luo pills.
  • Peony mainly contains paeoniflorin, albiflorin, hydroxy peonidin, benzoyl guanosine and other ingredients. Because paeoniflorin is high in medicinal materials and pure products are easy to obtain, there are many reports on paeoniflorin. Paeoniflorin has the functions of analgesia, sedation, liver protection, anti-inflammatory, immune regulation, expansion of blood vessels, improvement of learning and memory behavior, and can be used for the treatment of rheumatoid arthritis, hepatitis and senile related diseases.
  • paeoniflorin is the main active ingredient of paeoniflorin, so that paeoniflorin is used as an indicator to measure the drug containing paeoniflorin, and the quality and performance of the drug are evaluated by the content of the paeoniflorin.
  • paeoniflorin has analgesic, sedative, anticonvulsant effects, effects on the immune system, effects on smooth muscle, anti-inflammatory effects, anti-pathogenic microorganisms, and liver protection.
  • the invention patent application with the application number 200510045840.0 discloses a kind of peony from the genus Paeonia lac tifora Pall) extracts and separates the composition containing the active compound paeoniflorin and paeoniflorin, the sum of the two components is between 50% and 95%, and paeoniflorin and paeoniflorin in the combination
  • the ratio in the mixture is 1:10 to 10: 1, and the composition is used for the preparation of a medicament for treating various causes of leukopenia, thrombocytopenia, and hemoglobin-lowering conditions.
  • Another example of the invention patent application No. 200710132810.2 discloses a pharmaceutical composition containing paeoniflorin and paeoniflorin in which the weight ratio of paeoniflorin to paeoniflorin is 10:1-50:1
  • the pharmaceutical composition can be used for preventing and treating cardiovascular and cerebrovascular diseases.
  • the above studies have only studied the pharmacological uses of paeoniflorin and paeoniflorin, and prepared a combination of paeoniflorin and paeoniflorin.
  • ester glycosides Due to the chemical characteristics of paeoniflorin, it is difficult to prepare a high-purity paeoniflorin. There is no simple and convenient process in the existing research, and high-purity paeoniflorin can be obtained at low cost and on a large scale.
  • Ester glycosides For example, the invention patent application No. 200910100680.3 discloses a method for the separation and preparation of paeoniflorin by simulated moving bed chromatography. The method was applied to the separation and preparation of paeoniflorin by moving bed chromatography. The extract of paeony glycosides was used as raw material to separate the preparation of paeoniflorin by simulated moving bed chromatography.
  • the stationary phase of simulated moving bed chromatography was C18 silica gel, mobile phase.
  • RP-C18 reversed phase silica gel
  • the object of the present invention is to provide a method for simultaneously preparing high-purity paeoniflorin and paeoniflorin.
  • the method of the invention has simple operation process and short production cycle.
  • the kilograms of paeoniflorin and paeoniflorin can be prepared in a single process, and the obtained paeoniflorin and paeoniflorin are high in content, and the purity can reach more than 90%.
  • the preparation method for extracting and purifying paeoniflorin and paeoniflorin significantly reduces the production cost, and is suitable for batch preparation and industrial production.
  • an aspect of the present invention provides a method for preparing a paeoniflorin and paeoniflorin, which comprises separating and purifying a paeoniflorin or a paeoniflorin extract by a silica gel column chromatography method.
  • Another aspect of the invention provides a method of preparing a paeoniflorin and paeoniflorin comprising the steps of:
  • the method further includes the step 4): performing the second silica gel column chromatography on the paeoniflorin or the paeoniflorin prepared in the step 3), separately collecting the eluate in stages, and drying the eluate to obtain purity respectively. Higher paeoniflorin or higher purity paeoniflorin.
  • the extraction solvent described in the step 1) is one of water, ethanol solution or methanol solution.
  • the mass percentage concentration of ethanol in the ethanol solution is 10 to 95%, preferably 10 to 70%; and the mass percentage concentration of methanol in the methanol solution is 10-70%.
  • the extraction treatment described in the step 1) is a heating reflux extraction treatment method or a percolation extraction treatment method.
  • an appropriate extraction method is adopted, and when the extraction solvent is selected from water, a mass percentage concentration of 10-50% ethanol solution or a mass percentage concentration of 10-50% methanol solution, step 1
  • the percolation extraction method is used to perform the extraction treatment on the medicinal material; and when the extraction solvent selects a methanol solution having a mass percentage concentration of 50-70% or an ethanol solution having a mass percentage concentration of 50-70%, in step 1)
  • the extraction treatment is carried out on the medicinal material by a preferred heating and reflux extraction method.
  • the ratio of the volume of the extraction solvent to the weight (dry weight) of the medicinal material is 5-8:1 during the extraction process of the medicinal material by the heating reflux extraction treatment method (ie, if the weight of the medicinal material is (dry weight) is 1 kg, the volume of the extraction solvent is 5-8 liters; if the weight (dry weight) of the medicinal material is 1 gram, the volume of the extraction solvent is 5-8 ml); the number of extractions is 2-4 times , preferably 3 times; the extraction time is 2-3 hours/time.
  • the ratio of the volume of the extraction solvent to the weight (dry weight) of the medicinal material is 10-20:1, preferably 15-18: during the extraction treatment of the medicinal material by the osmosis extraction treatment method: 1 (ie if the weight (dry weight) of the medicinal material is 1 kg, the volume of the extraction solvent is 10-20 liters; if the weight (dry weight) of the medicinal material is 1 gram, the volume of the extraction solvent is 10-20 ml).
  • the medicinal material is added to the extraction solvent for immersion for 4-6 hours, and then the percolation extraction treatment is performed.
  • the soaking time is preferably 5 hours.
  • the macroporous resin column separation treatment described in the step 2) comprises the following sequential steps: the peony extract is directly subjected to the macroporous resin column. Separation, first eluting with water as an eluent, rinsing the sugar and impurities on the resin column, and then eluting with an ethanol solution having a mass percentage of 50-60% as an eluent to collect and combine the paeoniflorin And resin column eluate of paeoniflorin.
  • the ratio of the volume of water to the macroporous resin is 2-6:1, preferably 2-3.5:1; using the ethanol solution as an eluent During the elution, the ratio of the volume of the ethanol solution to the macroporous resin is 3-6:1.
  • the macroporous resin column separation treatment described in the step 2) comprises the following steps:
  • the macroporous resin column is separated, first eluted with water as an eluent, and then eluted with an ethanol solution having a mass percentage of 50-60% as an eluent, and collected.
  • the resin column eluate containing paeoniflorin and paeoniflorin was combined.
  • the ratio of the weight of the ethanol solution to the concentrated extract added in step B) is 2.3-5:1; the volume of water and macroporous resin during the elution process using water as the eluent in step C) The ratio is 1-6:1, preferably 2-3.5:1; during the elution process using the ethanol solution as an eluent, the ratio of the volume of the ethanol solution to the macroporous resin is 3-6:1. It is preferably 4-6:1.
  • the macroporous resin described in the step 2) is selected from the group consisting of D101, AB-8, DM130H, XAD-2, D201, HP-20, X-5, H103, DM11 or ADS-17 type resin, preferably D101, AB. -8 or HP-20 type resin.
  • the ratio of the volume of the macroporous resin to the weight of the medicinal herbs (dry weight) is 0.5-2:1 (ie, when the weight of the medicinal herbs (dry weight) is 100 g, the column volume of the macroporous resin column is 50-200 ml; When the weight of the medicinal herb (dry weight) is 100 kg, the column volume of the macroporous resin column is 50-200 L), preferably 1-1.5:1.
  • the ratio of the column diameter of the macroporous resin column to the column height of the resin is 1: 5-7.5, preferably 1: 5.9-7.1.
  • the ratio of the weight of the adsorbent silica gel and the resin column eluate after drying in the silica gel column chromatography treatment in the step 3) is 5-50:1, preferably 6-30:1, further It is preferably 15-30:1.
  • one of ethyl acetate, ethyl acetate and methanol, ethyl acetate and ethanol, ethyl acetate and water or a mixed solvent of ethyl acetate and acetone is selected for elution.
  • Agent one of ethyl acetate, ethyl acetate and methanol, ethyl acetate and ethanol, ethyl acetate and water or a mixed solvent of ethyl acetate and acetone is selected for elution.
  • the ratio of the volume of ethyl acetate to methanol is 10-20:1, preferably 20:1; a mixed solvent of ethyl acetate and ethanol is selected.
  • the ratio of the volume of ethyl acetate to ethanol is 10-20:1, preferably 10-15:1, more preferably 10:1; when a mixed solvent of ethyl acetate and water is selected as an eluent , the ratio of the volume of ethyl acetate to water is 100: 2-4, preferably 100: 2.5-3.5, when the mixed solvent of ethyl acetate and acetone is used as the eluent, the ratio of the volume of ethyl acetate to acetone is 10 -20: 1, preferably 10:1.
  • the ratio of the weight of the adsorbent silica gel in the second silica gel column chromatography treatment in step 4) to the sample of paeoniflorin or paeoniflorin prepared in step 3) is 15-30:1.
  • a mixed solvent of ethyl acetate and acetone or ethyl acetate and water is selected as an eluent.
  • the eluent when the eluent is selected from a mixed solvent of ethyl acetate and acetone, the ratio of the volume of ethyl acetate to acetone is 10-20:1, preferably 10:1; the eluent is selected from the mixture of ethyl acetate and water.
  • the ratio of the volume of ethyl acetate to water is 100: 2-4, preferably 100: 2.5-3.5
  • the ratio of the column diameter of the silica gel column to the height of the silica gel in the column during the silica gel column chromatography is 1:5-10.
  • the present invention provides a method for preparing a peony lactone and paeoniflorin, comprising the steps of:
  • the method further includes the step 4): respectively, the paeoniflorin or the paeoniflorin prepared in the step 3) is subjected to a second silica gel column. After chromatographic treatment, the eluate is collected in stages, and after the eluate is dried, paeoniflorin with higher purity or paeoniflorin with higher purity is obtained.
  • the ratio of the volume of the extraction solvent to the weight (dry weight) of the medicinal material in the step 1) is 5-8:1 (ie, if the weight (dry weight) of the medicinal material is 1 kg, the volume of the extraction solvent is 5-8 liters; if the weight (dry weight) of the medicinal material is 1 gram, the volume of the extraction solvent is 5-8 ml); the number of extractions is 2-4 times, preferably 3 times; the extraction time is 2-3 hours/ Times.
  • macroporous resin column separation treatment described in the step 2) comprises the following steps:
  • the macroporous resin column is separated, first eluted with water as an eluent, and then eluted with an ethanol solution having a mass percentage of 50-60% as an eluent, and collected.
  • the resin column eluate containing paeoniflorin and paeoniflorin was combined.
  • the ratio of the weight of the ethanol solution to the concentrated extract added in step B) is 2.3-5:1; the volume of water and macroporous resin during the elution process using water as the eluent in step C) The ratio is 1-6:1, preferably 2-3.5:1; during the elution using the ethanol solution as an eluent, the ratio of the volume of the ethanol solution to the macroporous resin is 4-6:1.
  • the macroporous resin described in the step 2) is selected from the group consisting of D101, AB-8, DM130H, XAD-2, D201, HP-20, X-5, H103, DM11 or ADS-17 type resin, preferably D101, AB. -8 or HP-20 type resin.
  • the ratio of the volume of the macroporous resin to the weight of the medicinal herbs (dry weight) is 0.5-2:1 (ie, when the weight of the medicinal herbs (dry weight) is 100 g, the column volume of the macroporous resin column is 50-200 ml; When the weight of the medicinal herb (dry weight) is 100 kg, the column volume of the macroporous resin column is 50-200 L), preferably 1-1.5:1.
  • the ratio of the column diameter of the macroporous resin column to the column height of the resin is 1: 5-7.5, preferably 1: 5.9-7.1.
  • the ratio of the weight of the adsorbent silica gel and the resin column eluate after drying in the silica gel column chromatography treatment in the step 3) is 5-50:1, preferably 6-30:1, further It is preferably 15-30:1.
  • one of ethyl acetate, ethyl acetate and methanol, ethyl acetate and ethanol, ethyl acetate and water or a mixed solvent of ethyl acetate and acetone is selected for elution.
  • Agent one of ethyl acetate, ethyl acetate and methanol, ethyl acetate and ethanol, ethyl acetate and water or a mixed solvent of ethyl acetate and acetone is selected for elution.
  • the ratio of the volume of ethyl acetate to methanol is 10-20:1, preferably 20:1; a mixed solvent of ethyl acetate and ethanol is selected.
  • the ratio of the volume of ethyl acetate to ethanol is 10-20:1, preferably 10-15:1, more preferably 10:1; when a mixed solvent of ethyl acetate and water is selected as an eluent , the ratio of the volume of ethyl acetate to water is 100: 2-4, preferably 100: 2.5-3.5, when the mixed solvent of ethyl acetate and acetone is used as the eluent, the ratio of the volume of ethyl acetate to acetone is 10 -20: 1, preferably 10:1.
  • the ratio of the weight of the adsorbent silica gel in the second silica gel column chromatography treatment in step 4) to the sample of paeoniflorin or paeoniflorin prepared in step 3) is 15-30:1.
  • a mixed solvent of ethyl acetate and acetone or ethyl acetate and water is selected as an eluent.
  • the eluent when the eluent is selected from a mixed solvent of ethyl acetate and acetone, the ratio of the volume of ethyl acetate to acetone is 10-20:1, preferably 10:1; the eluent is selected from the mixture of ethyl acetate and water.
  • the ratio of the volume of ethyl acetate to water is 100: 2-4, preferably 100: 2.5-3.5
  • the ratio of the column diameter of the silica gel column to the height of the silica gel in the column during the silica gel column chromatography is 1:5-10.
  • the method further includes the step 4): performing the second silica gel column chromatography on the paeoniflorin or the paeoniflorin prepared in the step 3), separately collecting the eluate in stages, and drying the eluate to obtain a purity more. High paeoniflorin or higher purity paeoniflorin.
  • the ratio of the volume of the extraction solvent to the weight (dry weight) of the medicinal material in step 1) is 5-30:1 (ie, if the weight (dry weight) of the medicinal material is 1 kg, the volume of the extraction solvent is 5- 30 liters; if the weight (dry weight) of the medicinal material is 1 gram, the volume of the extraction solvent is 5-30 ml), preferably 10-18:1.
  • the medicinal material is added to the extraction solvent water for soaking for 4-6 hours, and then the percolation extraction treatment is performed.
  • the soaking time is preferably 5 hours.
  • the macroporous resin column separation treatment described in the step 2) comprises the following sequence of steps: the peony extract is directly subjected to separation on a macroporous resin column, first eluting with water as an eluent, and then using a mass percent concentration The 50-60% ethanol solution was eluted as an eluent, and the resin column eluate containing paeoniflorin and paeoniflorin was collected and combined.
  • the ratio of the volume of water to the macroporous resin is 2-6:1, preferably 2-3.5:1; using the ethanol solution as an eluent During the elution, the ratio of the volume of the ethanol solution to the macroporous resin is 3-6:1.
  • the macroporous resin described in the step 2) is selected from the group consisting of D101, AB-8, DM130H, XAD-2, D201, HP-20, X-5, H103, DM11 or ADS-17 type resin, preferably D101, AB. -8 or HP-20 type resin.
  • the ratio of the volume of the macroporous resin to the weight of the medicinal herbs (dry weight) is 0.5-2:1 (ie, when the weight of the medicinal herbs (dry weight) is 100 g, the column volume of the macroporous resin column is 50-200 ml; When the weight of the medicinal herb (dry weight) is 100 kg, the column volume of the macroporous resin column is 50-200 L), preferably 1-1.5:1.
  • the ratio of the column diameter of the macroporous resin column to the column height of the resin is 1: 5-7.5, preferably 1: 5.9-7.1.
  • the ratio of the weight of the adsorbent silica gel and the resin column eluate after drying in the silica gel column chromatography treatment in the step 3) is 5-50:1, preferably 6-30:1, further It is preferably 15-30:1.
  • one of ethyl acetate, ethyl acetate and methanol, ethyl acetate and ethanol, ethyl acetate and water or a mixed solvent of ethyl acetate and acetone is selected for elution.
  • Agent one of ethyl acetate, ethyl acetate and methanol, ethyl acetate and ethanol, ethyl acetate and water or a mixed solvent of ethyl acetate and acetone is selected for elution.
  • the ratio of the volume of ethyl acetate to methanol is 10-20:1, preferably 20:1; a mixed solvent of ethyl acetate and ethanol is selected.
  • the ratio of the volume of ethyl acetate to ethanol is 10-20:1, preferably 10-15:1, more preferably 10:1; when a mixed solvent of ethyl acetate and water is selected as an eluent , the ratio of the volume of ethyl acetate to water is 100: 2-4, preferably 100: 2.5-3.5, when the mixed solvent of ethyl acetate and acetone is used as the eluent, the ratio of the volume of ethyl acetate to acetone is 10 -20: 1, preferably 10:1.
  • the ratio of the weight of the adsorbent silica gel in the second silica gel column chromatography treatment in step 4) to the sample of paeoniflorin or paeoniflorin prepared in step 3) is 15-30:1.
  • a mixed solvent of ethyl acetate and acetone or ethyl acetate and water is selected as an eluent.
  • the eluent when the eluent is selected from a mixed solvent of ethyl acetate and acetone, the ratio of the volume of ethyl acetate to acetone is 10-20:1, preferably 10:1; the eluent is selected from the mixture of ethyl acetate and water.
  • the ratio of the volume of ethyl acetate to water is 100: 2-4, preferably 100: 2.5-3.5
  • the ratio of the column diameter of the silica gel column to the height of the silica gel in the column during the silica gel column chromatography is 1:5-10.
  • Another aspect of the invention provides a method of preparing a paeoniflorin and paeoniflorin comprising the steps of: 1) adding the peony medicinal material to the extraction solvent, and performing the osmotic extraction treatment on the medicinal material to obtain the peony extract, wherein the extraction solvent selects a methanol solution having a concentration of 10-50% by mass or an ethanol having a concentration of 10-50% by mass.
  • the method further includes the step 4): performing the second silica gel column chromatography on the paeoniflorin or the paeoniflorin prepared in the step 3), separately collecting the eluate in stages, and drying the eluate to obtain a purity more. High paeoniflorin or higher purity paeoniflorin.
  • the ratio of the volume of the extraction solvent to the weight (dry weight) of the medicinal material in step 1) is 5-30:1 (ie, if the weight (dry weight) of the medicinal material is 1 kg, the volume of the extraction solvent is 5- 30 liters; if the weight (dry weight) of the medicinal material is 1 gram, the volume of the extraction solvent is 5-30 ml), preferably 10-18:1.
  • the medicinal material is added to the extraction solvent for immersion for 4-6 hours, and then the percolation extraction treatment is performed.
  • the soaking time is preferably 5 hours.
  • macroporous resin column separation treatment described in the step 2) comprises the following steps:
  • the macroporous resin column is separated, first eluted with water as an eluent, and then eluted with an ethanol solution having a mass percentage of 50-60% as an eluent, and collected.
  • the resin column eluate containing paeoniflorin and paeoniflorin was combined.
  • the ratio of the weight of the ethanol solution to the concentrated extract added in step B) is 2.3-5:1; the volume of water and macroporous resin during the elution process using water as the eluent in step C) The ratio is 1-6:1, preferably 2-3.5:1; during the elution using the ethanol solution as an eluent, the ratio of the volume of the ethanol solution to the macroporous resin is 4-6:1.
  • the macroporous resin described in the step 2) is selected from the group consisting of D101, AB-8, DM130H, XAD-2, D201, HP-20, X-5, H103, DM11 or ADS-17 type resin, preferably D101, AB. -8 or HP-20 type resin.
  • the ratio of the volume of the macroporous resin to the weight of the medicinal herbs (dry weight) is 0.5-2:1 (ie, when the weight of the medicinal herbs (dry weight) is 100 g, the column volume of the macroporous resin column is 50-200 ml; When the weight of the medicinal herb (dry weight) is 100 kg, the column volume of the macroporous resin column is 50-200 L), preferably 1-1.5:1.
  • the ratio of the column diameter of the macroporous resin column to the column height of the resin is 1: 5-7.5, preferably 1: 5.9-7.1.
  • the ratio of the weight of the adsorbent silica gel and the resin column eluate after drying in the silica gel column chromatography treatment in the step 3) is 5-50:1, preferably 6-30:1, further It is preferably 15-30:1.
  • one of ethyl acetate, ethyl acetate and methanol, ethyl acetate and ethanol, ethyl acetate and water or a mixed solvent of ethyl acetate and acetone is selected for elution.
  • Agent one of ethyl acetate, ethyl acetate and methanol, ethyl acetate and ethanol, ethyl acetate and water or a mixed solvent of ethyl acetate and acetone is selected for elution.
  • the ratio of the volume of ethyl acetate to methanol is 10-20:1, preferably 20:1; a mixed solvent of ethyl acetate and ethanol is selected.
  • the ratio of the volume of ethyl acetate to ethanol is 10-20:1, preferably 10-15:1, more preferably 10:1; when a mixed solvent of ethyl acetate and water is selected as an eluent , the ratio of the volume of ethyl acetate to water is 100: 2-4, preferably 100: 2.5-3.5, when the mixed solvent of ethyl acetate and acetone is used as the eluent, the ratio of the volume of ethyl acetate to acetone is 10 -20: 1, preferably 10:1.
  • the ratio of the weight of the adsorbent silica gel in the second silica gel column chromatography treatment described in the step 4) to the sample of the paeoniflorin or paeoniflorin prepared in the step 3) is 15-30:1.
  • a mixed solvent of ethyl acetate and acetone or ethyl acetate and water is selected as an eluent.
  • the eluent when the eluent is selected from a mixed solvent of ethyl acetate and acetone, the ratio of the volume of ethyl acetate to acetone is 10-20:1, preferably 10:1; the eluent is selected from the mixture of ethyl acetate and water.
  • the ratio of the volume of ethyl acetate to water is 100: 2-4, preferably 100: 2.5-3.5
  • the ratio of the column diameter of the silica gel column to the height of the silica gel in the column during the silica gel column chromatography is 1:5-10.
  • the present invention provides a method for preparing a paeoniflorin and paeoniflorin, comprising the steps of:
  • the ratio of the volume of the extraction solvent to the weight (dry weight) of the medicinal material in the step (1) is 5-8:1 (ie, if the weight (dry weight) of the medicinal material is 1 kg, the volume of the extraction solvent is 5-8 liters; if the weight (dry weight) of the medicinal material is 1 gram, the volume of the extraction solvent is 5-8 ml); the number of extractions is 2-4 times, preferably 3 times; the extraction time is 2-3 hours/ Times.
  • the ratio of the weight of the adsorbent silica gel and the peony extract after drying in the silica gel column chromatography treatment in the step (2) is 5-50:1, preferably 6-30:1, further preferably It is 15-30:1.
  • the ratio of the weight of the adsorbent silica gel to the crude paeoniflorin or the crude paeoniflorin in the silica gel column chromatography treatment described in the step (3) is 5-50:1, preferably 6-30:1, More preferably, it is 15-30:1.
  • the ratio of the column diameter of the silica gel column to the height of the silica gel in the column during the silica gel column chromatography is 1:5-10.
  • ethyl acetate, ethyl acetate and methanol, ethyl acetate and ethanol, ethyl acetate and water or ethyl acetate and acetone are selected.
  • One of the mixed solvents is an eluent.
  • the ratio of the volume of ethyl acetate to methanol is 10-20:1, preferably 20:1; a mixed solvent of ethyl acetate and ethanol is selected.
  • the ratio of the volume of ethyl acetate to ethanol is 10-20:1, preferably 15:1; when the mixed solvent of ethyl acetate and water is selected as the eluent, the volume of ethyl acetate and water is The ratio is 100: 2-4, preferably 100: 2.5-3.5; when the mixed solvent of ethyl acetate and acetone is selected as the eluent, the ratio of the volume of ethyl acetate to acetol is 10-20:1, preferably 10:1.
  • Another aspect of the present invention provides a method for preparing paeoniflorin and paeoniflorin, which comprises subjecting a paeoniflorin extract to silica gel column chromatography, collecting the eluate in stages, and drying the eluate to obtain a single component of paeoniflorin.
  • the ratio of the weight of the chromatographically adsorbed silica gel to the sample of the paeoniflorin extract during the silica gel column chromatography is 5 to 50:1, preferably 6 to 30:1, more preferably 15 to 30:1.
  • one of ethyl acetate, ethyl acetate and methanol, ethyl acetate and ethanol, ethyl acetate and water or a mixed solvent of ethyl acetate and acetone is selected as an eluent. .
  • the ratio of the volume of ethyl acetate to methanol is 10-20:1, preferably 20:1; a mixed solvent of ethyl acetate and ethanol is selected.
  • the ratio of the volume of ethyl acetate to ethanol is from 10 to 20:1, preferably from 10 to 15:1, further preferably 10:1; when a mixed solvent of ethyl acetate and water is selected as an eluent , the ratio of the volume of ethyl acetate to water is 100: 2-4, preferably 100: 2.5-3.5; when the mixed solvent of ethyl acetate and acetone is selected as the eluent, the ratio of the volume of ethyl acetate to acetone is 10-20:1, preferably 10:1.
  • the ratio of the column diameter of the silica gel column to the height of the silica gel in the column during the silica gel column chromatography is 1:5-10.
  • the peony drug is Paeonia actiflora (Paeonialactiflora) or Chuan Chi ⁇ iPaeonia Veitchii Lynch) Any part or whole plant of rhizomes, roots, leaves, branches, stems, fruits, flowers, etc., preferably roots and rhizomes.
  • the peony extract is selected from the peony medicinal materials, and after being extracted by a conventional solvent, the mixture is obtained, and the medicinal materials of the peony are extracted by a solvent, and then the mixture obtained by the extraction method or the medicinal materials of the peony is subjected to solvent extraction, and then subjected to macroporous adsorption.
  • the mixture obtained by adsorption, elution, and concentration of the resin is selected from the peony medicinal materials, and after being extracted by a conventional solvent, the mixture is obtained, and the medicinal materials of the peony are extracted by a solvent, and then the mixture obtained by the extraction method or the medicinal materials of the peony is subjected to solvent extraction, and then subjected to macroporous adsorption.
  • One or more of the mixture obtained by adsorption, elution, and concentration of the resin are selected from the peony medicinal materials, and after being extracted by a conventional solvent, the mixture is obtained, and the medicinal materials of the peony are extracted by a
  • the silica gel particle size used in silica gel column chromatography also has a great influence on the separation effect. Theoretically, the finer the particle size of silica gel, the better the separation effect, but the column pressure will increase significantly, and the requirements for equipment will increase accordingly, and the silica gel The cost will also increase significantly.
  • the preferred particle size of the invention is from 100 to 200 mesh silica gel.
  • the raw materials used in the preparation of paeoniflorin and paeoniflorin of the invention include separation and purification from plants, especially medicinal plant paeoniflorin and Chuan Chisong, and the selected raw materials may be the rhizomes, roots, leaves, branches and stems of the herbs. Any part or all of the plants such as fruits, flowers, etc., wherein the preferred parts are roots and rhizomes.
  • paeoniflorin and paeoniflorin can be prepared in the same process flow, and the peony glucoside and the gram-class paeoniflorin can be obtained respectively, and the comprehensive utilization of the medicinal materials can be fully utilized. Value, suitable for industrial production.
  • the method of the present invention has high purity of paeoniflorin and paeoniflorin, and the content of paeoniflorin and paeoniflorin after purification by silica gel column chromatography is more than 90%.
  • the preparation method of the invention is simple, the refining efficiency is high, the energy consumption is low, the environment is environmentally friendly, the operating process conditions are easy to control, and the quality is controllable.
  • the high-purity paeoniflorin and paeoniflorin according to the present invention can be used alone for preparing medicines and functional health foods, and can also be combined with any other Chinese and Western medicines or foods, especially with some blood stasis and protection.
  • Chinese medicine for cardiovascular and cerebrovascular diseases is compatible with sedative and anti-anxiety and anti-anxiety Chinese medicines for synergistic or synergistic purposes for the preparation of drugs and functional health foods.
  • Example 1 is an HPLC detection spectrum of the obtained paeoniflorin prepared in Example 1;
  • Fig. 5 is a HPLC detection spectrum of the obtained paeoniflorin prepared in Example 7.
  • the vacuum decompression concentrate tank (produced by Wuxi Huaxin Pharmaceutical Equipment Co., Ltd.) was concentrated under reduced pressure, and concentrated to a non-alcoholic taste to obtain a paeoniflorum concentrate, wherein the temperature under reduced pressure was 70°. C, the relative vacuum is -0.09 - -0.075 MPa, and the relative density of the peony concentrate is 1.25.
  • the extraction solvent for the heating and reflux extraction treatment of the medicinal material of the invention is 70% by mass concentration.
  • other ethanol solutions having a concentration percentage ranging from 50 to 70%, and a methanol solution having a mass percentage concentration ranging from 50 to 70% are suitable for use in the present invention.
  • the supernatant is concentrated to remove ethanol (that is, concentrated to an ethanol-free taste, and the concentrated supernatant does not contain ethanol), and then separated by a macroporous adsorption resin column, wherein the macroporous adsorption resin is selected as AB-8.
  • the column volume of the macroporous adsorption resin in the macroporous adsorption resin column is 200L (the diameter of the column is 0.35 m, the height is 2.5 m, the height of the resin is 2.08 m), and the volume and resin of the resin in the resin column
  • the ratio of weight (dry weight) is 4:3 (ie if the dry weight of the medicine is 150 kg, the volume of the macroporous resin is 200 L; if the dry weight of the medicine is 150 g, the volume of the macroporous resin is 200 ml), after concentration After the supernatant completely flows into the resin column, it is washed with 2.5 column volumes (ie 500 L) of water until the effluent is clear and transparent, and then washed with 5 column volumes (ie 1000 L) of a 60% by mass ethanol solution. Take off, collect the eluent, the elution flow rate is 150L / h, and collect the first-class each 50L e
  • TLC thin layer chromatography
  • silica gel column has a particle size of 100-200 mesh, and the silica gel column has a silica gel column.
  • the ratio of the column diameter to the height of the silica gel in the column is 1:8; the weight ratio of the adsorbent silica gel to the crude product of paeoniflorin and paeoniflorin is 16:1.
  • the eluent is a mixed solvent of ethyl acetate and methanol (volume The ratio is 20: 1), the amount of eluent is 5000L, the elution flow rate is 200L/h, and the first-class fraction is collected every 50L of eluent;
  • the content of paeoniflorin and paeoniflorin in the elution fraction of silica gel column was detected by thin layer chromatography (TLC), and the eluate was combined into three parts: the first part contained only a single component of paeoniflorin.
  • the second part is a mixed part containing paeoniflorin and paeoniflorin, and the third part is a single component containing only paeoniflorin, wherein the thin layer plate used in the TLC method is a silica gel G plate, and the developing agent is chloroform or methanol.
  • the mixed solution, the volume ratio of chloroform to methanol is 4:1, after the ascending expansion, sprayed with a 5% solution of sulfuric acid in ethanol, and heated at 150 ° C for 5 minutes to develop color;
  • the three parts were separately evaporated to dryness.
  • the weight of the first part was 1.74 kg (that is, the weight of the pure paeoniflorin obtained was 1.74 kg)
  • the weight of the second part was 0.37 kg (ie, the mixture of paeoniflorin and paeoniflorin)
  • the weight is 0.37 kg
  • the weight of the third part is 0.86 kg (that is, the weight of the obtained peony lactone pure product is 0.86 kg).
  • the content of paeoniflorin and paeoniflorin was determined by HPLC.
  • the HPLC conditions were as follows: Instrument: Water 515 pump, 2487 detector; Column: Kromasil RP-C18; Mobile phase: Acetonitrile: 0.1% phosphoric acid solution ( 13:87) ; Detection wavelength: 230 nm; Flow rate: 1.0 ml/min.
  • the vacuum extractor is used to concentrate under reduced pressure, and concentrated to a non-alcoholic taste to obtain a peony concentrate, wherein the concentration under reduced pressure is 60 ° C, and the relative vacuum is -0.09- -0.08 MPa, the relative density of the paeoniflorin concentrate was 1.21.
  • the extraction solvent for the heating and reflux extraction treatment of the medicinal material is 50% to 70% ethanol solution, and the mass percentage concentration range is 50-70%, except for the ethanol solution having a mass percentage concentration of 50%.
  • the methanol solution is suitable for use in the present invention.
  • the supernatant is concentrated to an ethanol-free taste (that is, the concentrated supernatant does not contain ethanol), and then separated by a macroporous adsorption resin column, wherein the macroporous adsorption resin selects HP-20 type macroporous adsorption.
  • Resin, macroporous adsorption resin column HP-20 type macroporous adsorption resin column volume is 150L (the diameter of the column is 0.30 meters, the height is 2.5 meters, the height of the resin is 2.12 meters), the volume of resin in the resin column and the medicine
  • the ratio of weight (dry weight) is 1:1 (ie, if the medicinal material (dry weight) is 150 kg, the volume of the macroporous resin is 150 L; if the medicinal material (dry weight) is 150 g, the volume of the macroporous resin is 150 ml),
  • After the supernatant after concentration has completely flowed into the resin column it is first washed with 450 L of water until the effluent is clear and transparent, and then eluted with 750 L of a 50% ethanol solution, and the eluent is collected at an elution flow rate of 200 L/ h, every 50L eluent is collected as a first-class share;
  • TLC method thin layer chromatography (TLC method) to detect the content of paeoniflorin and paeoniflorin in the eluted fraction of the collected macroporous resin column, and eluate flow containing paeoniflorin or/and paeoniflorin The fractions are combined to obtain a resin column eluate.
  • the thin layer plate used in the TLC method is a silica gel G plate, and the developing solvent is a mixture of chloroform and methanol. The volume ratio of chloroform to methanol is 4:1. , sprayed with 5% sulfuric acid ethanol solution, heated at 150 ° C for 5 minutes and then developed;
  • the ratio of the column diameter of the silica gel column to the height of the silica gel in the column is 1:8; the ratio of the weight of the adsorbent silica gel to the crude mixture of paeoniflorin and paeoniflorin is 20: 1, ethyl acetate and ethanol in the eluent
  • the volume ratio is 10: 1, the eluent dosage is 6000L, the elution flow rate is 200L/h per hour, and each 50L eluent is collected as a first-class fraction;
  • the eluate is combined into two parts: the first part is the paeoniflorin moiety (ie including It contains only a single component part of paeoniflorin and a mixture of paeoniflorin and paeoniflorin; the second part contains only a single component of paeoniflorin, wherein the thin layer plate used in the TLC method is a silica gel G plate, and the developing agent is chloroform.
  • the first part is the paeoniflorin moiety (ie including It contains only a single component part of paeoniflorin and a mixture of paeoniflorin and paeoniflorin; the second part contains only a single component of paeoniflorin, wherein the thin layer plate used in the TLC method is a silica gel G plate, and the developing agent is chloroform.
  • a mixture of methanol the volume ratio of chloroform to methanol is 4: 1, after the upward expansion, sprayed with 5% sulfuric acid ethanol solution, heated at
  • the combined two parts were separately evaporated to dryness.
  • the weight of the first part was 2.42 kg (ie containing paeoniflorin and paeoni lactone).
  • the weight of the mixture of glycosides was 2.42 kg), and the weight of the second part was 1.16 kg (i.e., the weight of the pure paeoniflorin obtained was 1.16 kg).
  • the content of paeoniflorin and paeoniflorin was determined by HPLC.
  • the HPLC conditions were as follows: Instrument: Water 515 pump, 2487 detector; Column: Kromasil RP-C18; Mobile phase: Acetonitrile: 0.1% phosphoric acid solution ( 13:87); Detection wavelength: 230 nm; Flow rate: 1.0 ml/min.
  • the content of paeoniflorin in the first part was determined by HPLC to be 84.78%, see Figure 3; the content of paeoniflorin in the second part was 94.71%, see Figure 4.
  • Example 3 The content of paeoniflorin in the first part was determined by HPLC to be 84.78%, see Figure 3; the content of paeoniflorin in the second part was 94.71%, see Figure 4.
  • the extraction solvent may also be used in an ethanol solution having a mass concentration ranging from 20 to 50%, and a methanol solution having a mass concentration ranging from 20 to 50%.
  • the percolating solution is directly separated by a macroporous adsorption resin column, wherein the macroporous adsorption resin selects the D101 type macroporous adsorption resin, and the column volume of the D101 type macroporous adsorption resin in the macroporous adsorption resin column is 150L ( The diameter of the column is 0.30 m, the height is 2.5 m, and the height of the resin is 2.12 m.
  • the ratio of the volume of the resin to the weight of the medicinal material (dry weight) is 3:2, that is, if the medicinal material (dry weight) is 100 kg, the macroporous resin The volume is 150L; if the medicinal material (dry weight) is 100g, the volume of the macroporous resin is 150ml.
  • TLC method thin layer chromatography
  • the sample of the mixture is placed on the top of the silica gel column, and the silica gel column chromatography is performed with ethyl acetate as an eluent.
  • the particle size of the adsorbent silica gel in the silica gel column is 100-200 mesh, and the column diameter and column of the silica gel column.
  • the ratio of the inner silica gel is 1:10; the ratio of the weight of the adsorbent silica gel to the crude mixture of paeoniflorin and paeoniflorin is 20:1, the eluent is ethyl acetate, the dosage is 6000L, and the elution flow rate is 300L/h. , collect a first-class share per 50L of eluent;
  • the first part is a single component containing only paeoniflorin
  • the second part is a mixed part containing paeoniflorin and paeoniflorin
  • the third part is a single component containing only paeoniflorin
  • the thin layer plate used in the TLC method is a silica gel G plate
  • the developing agent is chloroform or methanol.
  • the mixed solution, the volume ratio of chloroform to methanol is 4:1, and after being spread upward, it is sprayed with a 5% solution of sulfuric acid in ethanol, and heated at 150 ° C for 5 minutes to develop color;
  • the first part and the third part of the eluate are separately evaporated to dryness.
  • the weight of the first part is 1.74 kg (that is, the weight of the pure paeoniflorin obtained is 1.74 kg), and the weight of the third part is 1.02 kg (ie, obtained)
  • the weight of the pure paeoniflorin was 1.02 kg).
  • the content of paeoniflorin in the first part was determined by HPLC to be 93.42%; the content of paeoniflorin in the third part was 92.77%.
  • the paeoniflorin extract (the total glycoside of paeoniflorin) was used as raw material (the paeoniflorin content was determined to be 24.2%, and the paeoniflorin was 15.9%). 1, purification treatment
  • the ratio of the column diameter to the height of the silica gel in the column is 1:6; the weight ratio of the adsorbent silica gel to the peony extract is 16:1, and the volume ratio of ethyl acetate to water in the eluent is 100: 3.5, eluent
  • the dosage is 6000L, the elution flow rate is 200L/h, and the first-class fraction is collected every 50L eluent;
  • the thin layer plate used in the TLC method is a silica gel G plate
  • the developing solvent is a mixture of chloroform and methanol, chloroform.
  • the volume ratio to methanol is 4: 1, after the upward expansion, sprayed with 5% sulfuric acid ethanol solution, heated at 150 ° C for 5 minutes and then developed;
  • the combined two parts are separately evaporated to dryness.
  • the weight of the first part is 2.53 kg (that is, the weight of the pure paeoniflorin obtained is 2.53 kg), and the weight of the second part is 1.08 kg (including the inclusion of only paeoni lactone).
  • the total weight of the single component portion of the glycoside and the mixture of the paeoniflorin and the paeoniflorin was 1.08 kg).
  • the mixture was concentrated under reduced pressure in a vacuum vacuum concentration tank, and concentrated to dryness to obtain 18 kg of peony extract, wherein the temperature under reduced pressure was 60 ° C, and the relative vacuum was -0.09 - -0.08 MPa.
  • the extraction solvent may also be selected from an ethanol solution having a mass percentage concentration of 90-100% and a methanol concentration solution having a mass percentage concentration of 95-100%.
  • the sample of the mixture is placed on top of the silica gel column, and the silica gel column chromatography is carried out by using a mixed solvent of ethyl acetate and water as an eluent.
  • the particle size of the adsorbent silica gel in the silica gel column is 100-200 mesh, and the silica gel column is used.
  • the ratio of the column diameter to the height of the silica gel in the column is 1:8; the weight ratio of the adsorbent silica gel to the extract of the paeoniflorin is 6:1, and the volume ratio of ethyl acetate to water in the eluent is 100:2.5, eluent
  • the dosage is 6000L, the elution flow rate is 200L/h per hour, and each 50L eluent is collected as a first-class fraction;
  • the thin layer plate used in the TLC method is a silica gel G plate
  • the developing solvent is a mixture of chloroform and methanol, chloroform.
  • the volume ratio to methanol is 4: 1, after the upward expansion, sprayed with 5% sulfuric acid ethanol solution, heated at 150 ° C for 5 minutes and then developed; 4)
  • the combined two parts were separately evaporated to dryness.
  • the weight of the first part was 3.61 kg (that is, the weight of the pure paeoniflorin obtained was 3.61 kg), and the weight of the second part was 2.16 kg (including the inclusion of only paeoni lactone).
  • the total weight of the mixture of the single component of the glycoside and the mixture of the paeoniflorin and the paeoniflorin was 2.16 kg).
  • the content of paeoniflorin in the first part was determined by HPLC to be 72.83%; the content of paeoniflorin in the second part was 64.59%. 3, refined treatment
  • step 2 1) Take 2 kg of the crude paeoniflorin in the second part obtained in step 2, completely dissolve it in 3.5% by mass of ethanol, and add 2.5 kg of silica gel for chromatography (100-200 mesh). Stir well, dry, and grind to obtain a sample of paeoniflorin;
  • the sample of the paeoniflorin is placed on the top of the silica gel column, and the silica gel column chromatography is carried out by using a mixed solvent of ethyl acetate and acetone as an eluent, wherein the particle size of the adsorbent silica gel in the silica gel column is 100-200 mesh.
  • the ratio of the column diameter of the silica gel column to the height of the silica gel in the column is 1:10; the weight ratio of the adsorbent silica gel to the crude paeoniflorin weight is 15:1, and the volume ratio of ethyl acetate to acetone in the eluent is 10: 1, the eluent dosage is 1400L, the elution flow rate is 200L/h per hour, and each 50L eluent is collected as a first-class fraction;
  • a mixture of 500 kg of fresh Chuanchi lychee leaves and roots with a water content of 61% was used as a raw material, and after being chopped, it was placed in a percolation tank (volume of 3 M 3 ) at a mass concentration of 30%.
  • the ethanol solution was used for percolation extraction of the percolation extract.
  • the volume of the percolation extract was 3800 L, and the flow rate of the percolation was 50 L/h .
  • the percolate was combined and concentrated under reduced pressure to concentrate and remove the ethanol to obtain a concentrated drug.
  • the content of paeoniflorin in the first part was 79.4% by HPLC; the content of paeoniflorin in the first part was 73.7%.
  • Example 7 In addition to the ethanol solution having a mass percentage concentration of 30%, an extraction solvent of water having a concentration ranging from 20 to 50% by mass and a methanol solution having a mass percentage concentration of 20 to 50% may be used.
  • an extraction solvent of water having a concentration ranging from 20 to 50% by mass and a methanol solution having a mass percentage concentration of 20 to 50% may be used.
  • Example 3 1) Take the peony lactone glycoside (purity 92.77%) obtained in Example 3 as a sample, dissolve all the paeoniflorin sample with a small amount of methanol, add 1.2 kg of silica gel powder (100-200 mesh), and mix well. , drying, preparing a sample of paeoniflorin;
  • the paeoniflorin sample is subjected to silica gel column chromatography and eluted with a mixture of ethyl acetate and water as an eluent, wherein the silica gel column has a particle size of 100-200 mesh, and the silica gel column has a silica gel column.
  • the ratio of the column diameter to the height of the silica gel in the column is 1:5; the weight ratio of the adsorbent silica gel to the paeoniflorin sample is 30:1, and the volume ratio of ethyl acetate to water in the eluent is 100:3,
  • the elution volume is 2000L, the elution flow rate is 80L/h, and the first-class fraction is collected every 50L of the eluate;
  • paeoniflorin was 99.3% as determined by HPLC, see Figure 5.
  • HPLC conditions were: Instrument: Water 515 pump, 2487 detector; Column: Kromasil RP-C18; Phase: Acetonitrile: 0.1% aqueous phosphoric acid (13:87); Detection wavelength: 230 nm ; Flow rate: 1.0 ml/min.
  • Example 9 Preparation of Paeoniflorin Compound Preparation
  • Salvianolic acid B (purity 98.1) 70 g
  • Ginsenoside Rgl (purity 99.3) 50 g
  • the above components were uniformly mixed and filled into hard gelatin capsules to prepare 4,000 capsules for the treatment of coronary heart disease and angina pectoris.

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Description

一种制备芍药内酯苷和芍药苷的方法 技术领域
本发明属医药技术领域, 涉及化学领域化合物的分离提取方法, 具体涉及植物天然生 理活性物质芍药苷和芍药内酯苷的制备工艺。
背景技术
芍药是毛茛科芍药亚科芍药属多年生草本植物, 常用其干燥根入药。 中国药典 2010年 版中收录 2种芍药。一种是白芍(Radix Paeoniae Alba) ,为毛茛科植物芍药 Paeonia lactiflora Pall. 的干燥根; 一种是赤芍 (Radix Paeoniae Rubra) , 为毛茛科植物川赤芍 ¾eo« « ve fc w Lynch 的干燥根。 目前市场上已有多种含芍药的中药制剂, 如脑血栓片、 益脑复健胶囊、 偏 瘫复原片、 回生再造丸、大活络丸等。主要用于治疗心脑血管疾病、神经痛、 高血压、流产、 痛经等症。 现代化学研究表明: 芍药主要含有芍药苷(paeoniflorin)、 芍药内酯苷 (albiflorin) , 羟基芍药苷、 苯甲酰芍药苷等成分。 由于芍药苷在药材中含量高, 纯品较易 获得, 所以对芍药苷的研究报道较多。 芍药苷具有镇痛、 镇静、 保肝、 抗炎、 免疫调节、 扩 张血管、改善学习记忆行为等活性,可用于治疗类风湿性关节炎、肝炎和老年性相关疾病等。 长期以来,大多数研究认为芍药苷是芍药的主要有效成分,从而以芍药苷作为指标来测量含 芍药药物, 并以其含量的高低来评价药物的质量性能。
近年来, 现代药理研究发现芍药内酯苷具有镇痛、 镇静、 抗惊厥作用, 对免疫系 统的作用, 对平滑肌的作用, 抗炎作用, 抗病原微生物, 护肝作用, 临床上主要用于 抗癫痫, 镇痛、 戒毒, 止眩晕, 治疗类风湿性关节炎, 治疗细菌性痢疾及肠炎, 治疗 病毒性肝炎, 老年性疾病, 抗硫酸钡絮凝和溶解粘液的作用。
因此, 对芍药内酯苷的药理研究越来越受到重视, 芍药内酯苷的研究日益增多, 例如: 申请号为 200510045840.0 的发明专利申请公开了一种从白芍(毛茛科芍药属植物芍药 { Paeonia lac tifora Pall)中提取分离得到的含有活性化合物芍药苷和芍药内酯苷的组合 物, 两组分含量之和在 50 %— 95 %之间, 并且芍药苷与芍药内酯苷在该组合物中的比例为 1: 10〜10: 1,该组合物用于制备治疗各种原因引起的白细胞减少、血小板和血红球蛋白降 低的病症的药物。 又如: 申请号为 200710132810.2的发明专利申请公开了一种含有芍药苷 和芍药内酯苷药物组合物, 该组合物中芍药苷与芍药内酯苷的重量之比为 10: 1-50: 1, 该明 药物组合物可用于预防和治疗心脑血管疾病。但是上述研究仅仅是对芍药苷、芍药内酯苷的 药理用途进行了研究, 制备的是芍药苷和芍药内酯苷的组合物。
由于芍药内酯苷的化学特性,制备纯度高的芍药内酯苷的方法困难,现有的研究中还没 有一种简便易行的工艺方法, 可以低成本、大规模制备得到高纯度的芍药内酯苷。例如申请 号为 200910100680.3 的发明专利申请公开了一种模拟移动床色谱法分离制备芍药内酯苷的 方法。该方法模拟移动床色谱法分离制备芍药内酯苷, 采用白芍总苷提取物作为原料, 应用 模拟移动床色谱法分离制备芍药内酯苷, 模拟移动床色谱的固定相采用 C18硅胶, 流动相 采用甲醇或乙腈与水、 甲酸、 异丙醇的混合溶液, 混合溶液中按体积百分计: 甲醇或乙腈 10 %〜50 %、水 50 %〜90 %、 甲酸 0〜1 %、 异丙醇 0〜2 %, 上述的各组分的总和为 100 %, 该方法制备的芍药内酯苷的纯度虽然可以达到 90 %以上, 但因为该技术需要使用大量价格 昂贵的反相硅胶(RP-C18 ), 且制备工艺流程复杂, 操作工艺控制条件苛刻, 导致产品成本 高, 不适合大规模生产。
迄今为止,尚未有一种适合产业化生产同时制备高纯度芍药内酯苷和高纯度芍药苷的工 艺路线披露。
发明内容 本发明的目的在于提供一种同时制备高纯度芍药苷和芍药内酯苷的方法, 本发明方法 操作工艺过程简单, 生产周期短。运用本发明方法, 可在一个工艺流程中, 分别制备得到公 斤级的芍药苷和芍药内酯苷, 且所得到的芍药苷和芍药内酯苷的含量高, 纯度可达到 90% 以上。采取这种制备方法提取纯化芍药苷和芍药内酯苷, 显著地降低了生产成本, 适宜批量 制备和工业化生产。
为实现本发明的目的, 本发明一方面提供一种制备芍药内酯苷和芍药苷的方法, 该方 法包括以硅胶柱层析分离方法对芍药药材或芍药提取物进行分离、 纯化。
本发明另一方面提供一种制备芍药内酯苷和芍药苷的方法,包括如下顺序进行的步骤:
1 ) 将芍药药材加入到提取溶剂中, 对药材进行提取处理, 得到芍药提取液;
2 ) 将芍药提取液进行大孔树脂柱分离处理, 收集、 合并洗脱液, 得到含有芍药苷和芍 药内酯苷的树脂柱洗脱液;
3 ) 将树脂柱洗脱液干燥后的样品进行硅胶柱层析处理, 分段收集洗脱液, 洗脱液分别 干燥后, 得到单一成分的芍药苷或单一成分的芍药内酯苷。
其中, 还包括步骤 4) : 将步骤 3 ) 制得的芍药苷或芍药内酯苷分别进行第二次硅胶柱 层析处理, 分别分段收集洗脱液, 洗脱液干燥后, 分别得到纯度更高的芍药苷或纯度更高的 芍药内酯苷。
其中, 步骤 1 ) 中所述的提取溶剂选择水、 乙醇溶液或甲醇溶液中的一种。
特别是, 所述的乙醇溶液中的乙醇的质量百分比浓度为 10-95%, 优选为 10-70%; 所 述的甲醇溶液中甲醇的质量百分比浓度为 10-70%。
其中, 步骤 1 ) 中所述的提取处理为加热回流提取处理法或渗漉提取处理法。
特别是, 根据所选提取溶剂的种类而采用恰当的提取方法, 当提取溶剂选择水、 质量 百分比浓度为 10-50%的乙醇溶液或质量百分比浓度为 10-50%的甲醇溶液时, 步骤 1 ) 中优 先选择渗漉提取法对药材进行所述提取处理; 而当提取溶剂选择质量百分比浓度为 50-70% 的甲醇溶液或质量百分比浓度为 50-70%的乙醇溶液时, 步骤 1 ) 中优先选择加热回流提取 法对药材进行所述提取处理。
特别是, 步骤 1 )中采用加热回流提取处理法进行所述的药材的提取处理过程中, 提取 溶剂的体积与药材的重量 (干重) 之比为 5-8: 1 (即如果药材的重量 (干重) 为 1公斤, 则 提取溶剂的体积为 5-8升;如果药材的重量(干重)为 1克,则提取溶剂的体积为 5-8毫升); 提取次数为 2-4次, 优选为 3次; 提取时间为 2-3小时 /次。
其中, 步骤 1 )中采用采用渗漉提取处理法进行所述药材的提取处理过程中, 提取溶剂 的体积与药材的重量 (干重) 之比为 10-20: 1, 优选为 15-18: 1 (即如果药材的重量 (干重) 为 1公斤, 则提取溶剂的体积为 10-20升; 如果药材的重量(干重) 为 1克, 则提取溶剂的 体积为 10-20毫升)。
特别是, 进行渗漉提取之前还包括将药材加入到提取溶剂中进行浸泡 4-6 小时后, 再 进行所述的渗漉提取处理。
尤其是, 浸泡时间优选为 5小时。
其中, 当使用水作为提取溶剂进行渗漉提取, 制得芍药提取液后, 步骤 2 ) 中所述的大 孔树脂柱分离处理包括以下顺序进行的步骤:将芍药提取液直接进行大孔树脂柱分离,首先 采用水为洗脱剂进行洗脱,冲洗树脂柱上的糖份和杂质,然后采用质量百分比浓度为 50-60% 的乙醇溶液为洗脱剂进行洗脱, 收集、 合并含有芍药苷和芍药内酯苷的树脂柱洗脱液。
特别是, 采用水为洗脱剂进行洗脱的过程中, 水与大孔树脂的体积之比为 2-6: 1, 优 选为 2-3.5 : 1; 采用所述乙醇溶液为洗脱剂进行洗脱的过程中, 乙醇溶液与大孔树脂的体积 之比为 3-6: 1。
其中, 当使用甲醇或乙醇溶液作为提取溶剂进行渗漉提取或加热回流提取, 制得芍药 提取液后, 步骤 2 ) 中所述的大孔树脂柱分离处理包括以下顺序进行的步骤:
A) 将芍药提取液浓缩至相对密度为 1.2-1.25, 得到浓缩提取液;
B )向浓缩提取液中加入质量百分比浓度为 90-100%的乙醇溶液, 至浓缩提取液中乙醇 浓度为 70-75%, 静置沉淀, 得到上清液;
C)将上清液浓缩去除乙醇后进行大孔树脂柱分离, 首先采用水为洗脱剂进行洗脱, 然 后采用质量百分比浓度为 50-60%的乙醇溶液为洗脱剂进行洗脱, 收集、 合并含有芍药苷和 芍药内酯苷的树脂柱洗脱液。
特别是, 步骤 B ) 中加入的乙醇溶液与浓缩提取液的重量之比为 2.3-5: 1 ; 步骤 C) 中 采用水为洗脱剂进行洗脱的过程中, 水与大孔树脂的体积之比为 1-6: 1, 优选为 2-3.5 : 1; 采用所述乙醇溶液为洗脱剂进行洗脱的过程中, 乙醇溶液与大孔树脂的体积之比为 3-6: 1, 优选为 4-6: 1。
其中, 步骤 2)中所述的大孔树脂选择 D101、 AB-8、 DM130H、 XAD-2、 D201、 HP-20、 X-5、 H103、 DM11或 ADS-17型树脂, 优选为 D101、 AB-8或 HP-20型树脂。
特别是, 大孔树脂的体积与芍药药材重量 (干重) 之比为 0.5-2: 1 (即当芍药药材重量 (干重) 为 100g, 则大孔树脂柱的柱体积为 50-200ml; 当芍药药材重量 (干重) 为 100kg, 则大孔树脂柱的柱体积为 50-200L), 优选为 1-1.5 : 1。
尤其是, 大孔树脂柱分离处理过程中, 大孔树脂柱的柱直径与树脂的柱高之比为 1 : 5-7.5, 优选为 1 : 5.9-7.1。
其中,步骤 3 )中所述的硅胶柱层析处理过程中的吸附剂硅胶与树脂柱洗脱液干燥后的 样品的重量之比为 5-50: 1, 优选为 6-30: 1, 进一步优选为 15-30: 1。
特别是, 所述硅胶柱层析处理过程中选择乙酸乙酯、 乙酸乙酯与甲醇、 乙酸乙酯与乙 醇、 乙酸乙酯与水或乙酸乙酯与丙酮的混合溶剂中的一种为洗脱剂。
尤其是, 选择乙酸乙酯与甲醇的混合溶剂为洗脱剂时, 乙酸乙酯与甲醇的体积之比为 10-20: 1 , 优选为 20: 1 ; 选择乙酸乙酯与乙醇的混合溶剂为洗脱剂时, 乙酸乙酯与乙醇的体 积之比为 10-20: 1, 优选为 10-15: 1, 进一步优选为 10:1 ; 选择乙酸乙酯与水的混合溶剂为洗 脱剂时, 乙酸乙酯与水的体积之比为 100: 2-4, 优选为 100:2.5-3.5, 选择乙酸乙酯与丙酮混 合溶剂为洗脱剂时, 乙酸乙酯与丙酮的体积之比为 10-20: 1, 优选为 10: 1。
其中, 步骤 4) 中所述的第二次硅胶柱层析处理过程中的吸附剂硅胶与步骤 3 )制得的 芍药苷或芍药内酯苷的样品的重量之比为 15-30: 1。
特别是, 所述第二次硅胶柱层析处理过程中选择乙酸乙酯与丙酮或乙酸乙酯与水的混 合溶剂为洗脱剂。
尤其是, 洗脱剂选择乙酸乙酯与丙酮的混合溶剂时, 乙酸乙酯与丙酮的体积之比为 10-20: 1 , 优选为 10: 1 ; 洗脱剂选择乙酸乙酯与水的混合溶剂时, 乙酸乙酯与水的体积之比 为 100: 2-4, 优选为 100:2.5-3.5„
特别是, 硅胶柱层析处理过程中硅胶柱的柱直径与柱内硅胶高之比为 1 :5-10。
本发明又一方面提供一种制备芍药内酯苷和芍药苷的方法,包括如下顺序进行的步骤:
1 ) 将芍药药材加入到提取溶剂中, 对药材进行加热回流提取处理, 得到芍药提取液, 其中提取溶剂选择质量百分比浓度为 50-70%的甲醇溶液或质量百分比浓度为 50-70%的乙 醇溶液;
2 ) 将芍药提取液浓缩, 去除乙醇或甲醇后, 进行大孔树脂柱分离处理, 收集、 合并洗 脱液, 得到含有芍药苷和芍药内酯苷的树脂柱洗脱液;
3 ) 将树脂柱洗脱液干燥后的样品进行硅胶柱层析处理, 分段收集洗脱液, 洗脱液分别 干燥后, 分别得到单一成分的芍药苷、 芍药内酯苷。
其中, 还包括步骤 4) : 将步骤 3 ) 制得的芍药苷或芍药内酯苷分别进行第二次硅胶柱 层析处理, 分别分段收集洗脱液, 洗脱液干燥后, 分别得到纯度更高的芍药苷或纯度更高的 芍药内酯苷。
特别是, 步骤 1 ) 中所述的提取溶剂的体积与药材的重量 (干重) 之比为 5-8: 1 (即如 果药材的重量(干重) 为 1公斤, 则提取溶剂的体积为 5-8升; 如果药材的重量(干重) 为 1克, 则提取溶剂的体积为 5-8毫升); 提取次数为 2-4次, 优选为 3次; 提取时间为 2-3小 时 /次。
其中, 步骤 2) 中所述的大孔树脂柱分离处理包括以下顺序进行的步骤:
A) 将芍药提取液浓缩至相对密度为 1.2-1.25, 得到浓缩提取液;
B )向浓缩提取液中加入质量百分比浓度为 90-100%的乙醇溶液, 使提取液中乙醇浓度 为 70-75%, 静置沉淀, 得到上清液;
C)将上清液浓缩去除乙醇后进行大孔树脂柱分离, 首先采用水为洗脱剂进行洗脱, 然 后采用质量百分比浓度为 50-60%的乙醇溶液为洗脱剂进行洗脱, 收集、 合并含有芍药苷和 芍药内酯苷的树脂柱洗脱液。
特别是, 步骤 B ) 中加入的乙醇溶液与浓缩提取液的重量之比为 2.3-5: 1 ; 步骤 C) 中 采用水为洗脱剂进行洗脱的过程中, 水与大孔树脂的体积之比为 1-6: 1, 优选为 2-3.5 : 1; 采用所述乙醇溶液为洗脱剂进行洗脱的过程中, 乙醇溶液与大孔树脂的体积之比为 4-6: 1。
其中, 步骤 2)中所述的大孔树脂选择 D101、 AB-8、 DM130H、 XAD-2、 D201、 HP-20、 X-5、 H103、 DM11或 ADS-17型树脂, 优选为 D101、 AB-8或 HP-20型树脂。
特别是, 大孔树脂的体积与芍药药材重量 (干重) 之比为 0.5-2: 1 (即当芍药药材重量 (干重) 为 100g, 则大孔树脂柱的柱体积为 50-200ml; 当芍药药材重量 (干重) 为 100kg, 则大孔树脂柱的柱体积为 50-200L), 优选为 1-1.5 : 1。
尤其是, 大孔树脂柱分离处理过程中, 大孔树脂柱的柱直径与树脂的柱高之比为 1 : 5-7.5, 优选为 1 : 5.9-7.1。
其中,步骤 3 )中所述的硅胶柱层析处理过程中的吸附剂硅胶与树脂柱洗脱液干燥后的 样品的重量之比为 5-50: 1, 优选为 6-30: 1, 进一步优选为 15-30: 1。
特别是, 所述硅胶柱层析处理过程中选择乙酸乙酯、 乙酸乙酯与甲醇、 乙酸乙酯与乙 醇、 乙酸乙酯与水或乙酸乙酯与丙酮的混合溶剂中的一种为洗脱剂。
尤其是, 选择乙酸乙酯与甲醇的混合溶剂为洗脱剂时, 乙酸乙酯与甲醇的体积之比为 10-20: 1 , 优选为 20: 1 ; 选择乙酸乙酯与乙醇的混合溶剂为洗脱剂时, 乙酸乙酯与乙醇的体 积之比为 10-20: 1, 优选为 10-15: 1, 进一步优选为 10:1 ; 选择乙酸乙酯与水的混合溶剂为洗 脱剂时, 乙酸乙酯与水的体积之比为 100: 2-4, 优选为 100:2.5-3.5, 选择乙酸乙酯与丙酮混 合溶剂为洗脱剂时, 乙酸乙酯与丙酮的体积之比为 10-20: 1, 优选为 10: 1。
其中, 步骤 4) 中所述的第二次硅胶柱层析处理过程中的吸附剂硅胶与步骤 3 )制得的 芍药苷或芍药内酯苷的样品的重量之比为 15-30: 1。
特别是, 所述第二次硅胶柱层析处理过程中选择乙酸乙酯与丙酮或乙酸乙酯与水的混 合溶剂为洗脱剂。
尤其是, 洗脱剂选择乙酸乙酯与丙酮的混合溶剂时, 乙酸乙酯与丙酮的体积之比为 10-20: 1 , 优选为 10: 1 ; 洗脱剂选择乙酸乙酯与水的混合溶剂时, 乙酸乙酯与水的体积之比 为 100: 2-4, 优选为 100:2.5-3.5„
特别是, 硅胶柱层析处理过程中硅胶柱的柱直径与柱内硅胶高之比为 1 :5-10。
本发明再一方面提供一种制备芍药内酯苷和芍药苷的方法,包括如下顺序进行的步骤:
1 ) 将芍药药材加入到提取溶剂中, 对药材进行渗漉提取处理, 得到芍药提取液, 其中 提取溶剂选择水;
2 ) 将芍药提取液进行大孔树脂柱分离处理, 收集、 合并洗脱液, 得到含有芍药苷和芍 药内酯苷的树脂柱洗脱液;
3 )将树脂柱洗脱液干燥后的样品进行硅胶柱层析处理, 分段收集洗脱液, 洗脱液分别 干燥后, 分别得到单一成分的芍药苷、 芍药内酯苷。
其中, 还包括步骤 4) : 将步骤 3 ) 制得的芍药苷或芍药内酯苷分别进行第二次硅胶柱 层析处理, 分别分段收集洗脱液, 洗脱液干燥后, 得到纯度更高的芍药苷或纯度更高的芍药 内酯苷。
其中, 步骤 1 ) 中所述提取溶剂的体积与药材的重量(干重)之比为 5-30: 1 (即如果药 材的重量 (干重) 为 1公斤, 则提取溶剂的体积为 5-30升; 如果药材的重量 (干重) 为 1 克, 则提取溶剂的体积为 5-30毫升), 优选为 10-18: 1。
特别是, 进行渗漉提取之前还包括将药材加入到提取溶剂水中进行浸泡 4-6 小时后, 再进行所述的渗漉提取处理。
尤其是, 浸泡时间优选为 5小时。
其中, 步骤 2 )中所述的大孔树脂柱分离处理包括以下顺序进行的步骤: 将芍药提取液 直接进行大孔树脂柱分离, 首先采用水为洗脱剂进行洗脱, 然后采用质量百分比浓度为 50-60%的乙醇溶液为洗脱剂进行洗脱, 收集、 合并含有芍药苷和芍药内酯苷的树脂柱洗脱 液。
特别是, 采用水为洗脱剂进行洗脱的过程中, 水与大孔树脂的体积之比为 2-6: 1, 优 选为 2-3.5 : 1; 采用所述乙醇溶液为洗脱剂进行洗脱的过程中, 乙醇溶液与大孔树脂的体积 之比为 3-6: 1。
其中, 步骤 2)中所述的大孔树脂选择 D101、 AB-8、 DM130H、 XAD-2、 D201、 HP-20、 X-5、 H103、 DM11或 ADS-17型树脂, 优选为 D101、 AB-8或 HP-20型树脂。
特别是, 大孔树脂的体积与芍药药材重量 (干重) 之比为 0.5-2: 1 (即当芍药药材重量 (干重) 为 100g, 则大孔树脂柱的柱体积为 50-200ml; 当芍药药材重量 (干重) 为 100kg, 则大孔树脂柱的柱体积为 50-200L), 优选为 1-1.5 : 1。
尤其是, 大孔树脂柱分离处理过程中, 大孔树脂柱的柱直径与树脂的柱高之比为 1 : 5-7.5, 优选为 1 : 5.9-7.1。
其中,步骤 3 )中所述的硅胶柱层析处理过程中的吸附剂硅胶与树脂柱洗脱液干燥后的 样品的重量之比为 5-50: 1, 优选为 6-30: 1, 进一步优选为 15-30: 1。
特别是, 所述硅胶柱层析处理过程中选择乙酸乙酯、 乙酸乙酯与甲醇、 乙酸乙酯与乙 醇、 乙酸乙酯与水或乙酸乙酯与丙酮的混合溶剂中的一种为洗脱剂。
尤其是, 选择乙酸乙酯与甲醇的混合溶剂为洗脱剂时, 乙酸乙酯与甲醇的体积之比为 10-20: 1 , 优选为 20: 1 ; 选择乙酸乙酯与乙醇的混合溶剂为洗脱剂时, 乙酸乙酯与乙醇的体 积之比为 10-20: 1, 优选为 10-15: 1, 进一步优选为 10:1 ; 选择乙酸乙酯与水的混合溶剂为洗 脱剂时, 乙酸乙酯与水的体积之比为 100: 2-4, 优选为 100:2.5-3.5, 选择乙酸乙酯与丙酮混 合溶剂为洗脱剂时, 乙酸乙酯与丙酮的体积之比为 10-20: 1, 优选为 10: 1。
其中, 步骤 4) 中所述的第二次硅胶柱层析处理过程中的吸附剂硅胶与步骤 3 )制得的 芍药苷或芍药内酯苷的样品的重量之比为 15-30: 1。
特别是, 所述第二次硅胶柱层析处理过程中选择乙酸乙酯与丙酮或乙酸乙酯与水的混 合溶剂为洗脱剂。
尤其是, 洗脱剂选择乙酸乙酯与丙酮的混合溶剂时, 乙酸乙酯与丙酮的体积之比为 10-20: 1 , 优选为 10: 1 ; 洗脱剂选择乙酸乙酯与水的混合溶剂时, 乙酸乙酯与水的体积之比 为 100: 2-4, 优选为 100:2.5-3.5„
特别是, 硅胶柱层析处理过程中硅胶柱的柱直径与柱内硅胶高之比为 1 :5-10。
本发明另一方面提供一种制备芍药内酯苷和芍药苷的方法,包括如下顺序进行的步骤: 1 ) 将芍药药材加入到提取溶剂中, 对药材进行渗漉提取处理, 得到芍药提取液, 其中 提取溶剂选择质量百分比浓度为 10-50%的甲醇溶液或质量百分比浓度为 10-50%的乙醇溶 液;
2 ) 将芍药提取液浓缩, 去除乙醇或甲醇后, 进行大孔树脂柱分离处理, 收集、 合并洗 脱液, 得到含有芍药苷和芍药内酯苷的树脂柱洗脱液;
3 )将树脂柱洗脱液干燥后的样品进行硅胶柱层析处理, 分段收集洗脱液, 洗脱液分别 干燥后, 分别得到单一成分的芍药苷、 芍药内酯苷。
其中, 还包括步骤 4) : 将步骤 3 ) 制得的芍药苷或芍药内酯苷分别进行第二次硅胶柱 层析处理, 分别分段收集洗脱液, 洗脱液干燥后, 得到纯度更高的芍药苷或纯度更高的芍药 内酯苷。
其中, 步骤 1 ) 中所述提取溶剂的体积与药材的重量(干重)之比为 5-30: 1 (即如果药 材的重量 (干重) 为 1公斤, 则提取溶剂的体积为 5-30升; 如果药材的重量 (干重) 为 1 克, 则提取溶剂的体积为 5-30毫升), 优选为 10-18: 1。
特别是, 进行渗漉提取之前还包括将药材加入到提取溶剂中进行浸泡 4-6 小时后, 再 进行所述的渗漉提取处理。
尤其是, 浸泡时间优选为 5小时。
其中, 步骤 2) 中所述的大孔树脂柱分离处理包括以下顺序进行的步骤:
A) 将芍药提取液浓缩至相对密度为 1.2-1.25, 得到浓缩提取液;
B )向浓缩提取液中加入质量百分比浓度为 90-100%的乙醇溶液, 至提取液中乙醇浓度 为 70-75%; 静置沉淀, 得到上清液;
C)将上清液浓缩去除乙醇后进行大孔树脂柱分离, 首先采用水为洗脱剂进行洗脱, 然 后采用质量百分比浓度为 50-60%的乙醇溶液为洗脱剂进行洗脱, 收集、 合并含有芍药苷和 芍药内酯苷的树脂柱洗脱液。
特别是, 步骤 B ) 中加入的乙醇溶液与浓缩提取液的重量之比为 2.3-5: 1 ; 步骤 C) 中 采用水为洗脱剂进行洗脱的过程中, 水与大孔树脂的体积之比为 1-6: 1, 优选为 2-3.5 : 1; 采用所述乙醇溶液为洗脱剂进行洗脱的过程中, 乙醇溶液与大孔树脂的体积之比为 4-6: 1。
其中, 步骤 2)中所述的大孔树脂选择 D101、 AB-8、 DM130H、 XAD-2、 D201、 HP-20、 X-5、 H103、 DM11或 ADS-17型树脂, 优选为 D101、 AB-8或 HP-20型树脂。
特别是, 大孔树脂的体积与芍药药材重量 (干重) 之比为 0.5-2: 1 (即当芍药药材重量 (干重) 为 100g, 则大孔树脂柱的柱体积为 50-200ml; 当芍药药材重量 (干重) 为 100kg, 则大孔树脂柱的柱体积为 50-200L), 优选为 1-1.5 : 1。
尤其是, 大孔树脂柱分离处理过程中, 大孔树脂柱的柱直径与树脂的柱高之比为 1 : 5-7.5, 优选为 1 : 5.9-7.1。
其中,步骤 3 )中所述的硅胶柱层析处理过程中的吸附剂硅胶与树脂柱洗脱液干燥后的 样品的重量之比为 5-50: 1, 优选为 6-30: 1, 进一步优选为 15-30: 1。
特别是, 所述硅胶柱层析处理过程中选择乙酸乙酯、 乙酸乙酯与甲醇、 乙酸乙酯与乙 醇、 乙酸乙酯与水或乙酸乙酯与丙酮的混合溶剂中的一种为洗脱剂。
尤其是, 选择乙酸乙酯与甲醇的混合溶剂为洗脱剂时, 乙酸乙酯与甲醇的体积之比为 10-20: 1 , 优选为 20: 1 ; 选择乙酸乙酯与乙醇的混合溶剂为洗脱剂时, 乙酸乙酯与乙醇的体 积之比为 10-20: 1, 优选为 10-15: 1, 进一步优选为 10:1 ; 选择乙酸乙酯与水的混合溶剂为洗 脱剂时, 乙酸乙酯与水的体积之比为 100: 2-4, 优选为 100:2.5-3.5, 选择乙酸乙酯与丙酮混 合溶剂为洗脱剂时, 乙酸乙酯与丙酮的体积之比为 10-20: 1, 优选为 10: 1。
其中, 步骤 4) 中所述的第二次硅胶柱层析处理过程中的吸附剂硅胶与步骤 3 )制得的 芍药苷或芍药内酯苷的样品的重量之比为 15-30: 1。 特别是, 所述第二次硅胶柱层析处理过程中选择乙酸乙酯与丙酮或乙酸乙酯与水的混 合溶剂为洗脱剂。
尤其是, 洗脱剂选择乙酸乙酯与丙酮的混合溶剂时, 乙酸乙酯与丙酮的体积之比为 10-20:1, 优选为 10:1; 洗脱剂选择乙酸乙酯与水的混合溶剂时, 乙酸乙酯与水的体积之比 为 100: 2-4, 优选为 100:2.5-3.5„
特别是, 硅胶柱层析处理过程中硅胶柱的柱直径与柱内硅胶高之比为 1:5-10。
本发明又一方面提供一种制备芍药内酯苷和芍药苷的方法, 包括如下顺序进行的步骤:
(1)将芍药药材加入到提取溶剂中, 对药材进行加热回流提取处理, 得到芍药提取液, 其中提取溶剂选择质量百分比浓度为 95-100%的甲醇溶液或质量百分比浓度为 90-100%的 乙醇溶液;
(2) 芍药提取液干燥后的样品进行第一次硅胶柱层析处理, 分段收集洗脱液, 洗脱液 分别干燥后, 得到芍药苷粗品、 芍药内酯苷粗品;
(3) 将步骤 (2) 得到的芍药苷粗品或芍药内酯苷粗品分别进行第二次硅胶柱层析, 分别分段收集洗脱液,洗脱液干燥后,分别得到单一成分的芍药苷或单一成分的芍药内酯苷。
其中, 步骤(1) 中所述的提取溶剂的体积与药材的重量(干重)之比为 5-8:1 (即如果 药材的重量 (干重) 为 1公斤, 则提取溶剂的体积为 5-8升; 如果药材的重量 (干重) 为 1 克, 则提取溶剂的体积为 5-8毫升); 提取次数为 2-4次, 优选为 3次; 提取时间为 2-3小时 /次。
其中, 步骤(2) 中所述的硅胶柱层析处理过程中的吸附剂硅胶与芍药提取液干燥后的 样品的重量之比为 5-50: 1, 优选为 6-30:1, 进一步优选为 15-30:1。
其中, 步骤(3) 中所述的硅胶柱层析处理过程中的吸附剂硅胶与芍药苷粗品或芍药内 酯苷粗品的重量之比为 5-50: 1, 优选为 6-30:1, 进一步优选为 15-30:1。
特别是, 硅胶柱层析处理过程中硅胶柱的柱直径与柱内硅胶高之比为 1:5-10。
特别是, 步骤(2)、 步骤(3) 中所述硅胶柱层析处理过程中选择乙酸乙酯、 乙酸乙酯 与甲醇、 乙酸乙酯与乙醇、 乙酸乙酯与水或乙酸乙酯与丙酮的混合溶剂的一种为洗脱剂。
尤其是, 选择乙酸乙酯与甲醇的混合溶剂为洗脱剂时, 乙酸乙酯与甲醇的体积之比为 10-20:1, 优选为 20:1; 选择乙酸乙酯与乙醇的混合溶剂为洗脱剂时, 乙酸乙酯与乙醇的体 积之比为 10-20:1, 优选为 15:1; 选择乙酸乙酯与水的混合溶剂为洗脱剂时, 乙酸乙酯与水 的体积之比为 100: 2-4, 优选为 100:2.5-3.5; 选择乙酸乙酯与丙酮的混合溶剂为洗脱剂时, 乙酸乙酯与丙酮醇的体积之比为 10-20:1, 优选为 10:1。
本发明另一方面提供一种制备芍药内酯苷和芍药苷的方法, 包括将芍药提取物进行硅 胶柱层析处理, 分段收集洗脱液, 洗脱液干燥后, 分别得到单一成分的芍药苷或单一成分的 芍药内酯苷。
其中,硅胶柱层析处理过程中的层析吸附硅胶与芍药提取物的样品的重量之比为 5-50: 1, 优选为 6-30:1, 进一步优选为 15-30:1。
特别是, 所述硅胶柱层析处理过程中选择乙酸乙酯、 乙酸乙酯与甲醇、 乙酸乙酯与乙 醇、 乙酸乙酯与水或乙酸乙酯与丙酮的混合溶剂的一种为洗脱剂。
尤其是, 选择乙酸乙酯与甲醇的混合溶剂为洗脱剂时, 乙酸乙酯与甲醇的体积之比为 10-20:1, 优选为 20:1; 选择乙酸乙酯与乙醇的混合溶剂为洗脱剂时, 乙酸乙酯与乙醇的体 积之比为 10-20:1, 优选为 10-15:1, 进一步优选为 10:1; 选择乙酸乙酯与水的混合溶剂为洗 脱剂时, 乙酸乙酯与水的体积之比为 100: 2-4, 优选为 100:2.5-3.5; 选择乙酸乙酯与丙酮的 混合溶剂为洗脱剂时, 乙酸乙酯与丙酮的体积之比为 10-20:1, 优选为 10:1。
特别是, 硅胶柱层析处理过程中硅胶柱的柱直径与柱内硅胶高之比为 1:5-10。
其中, 所述的芍药药材为毛茛科植物芍药 Paeonialactiflora )或川赤芍 iPaeonia veitchii Lynch ) 的根茎、 根、 叶、 枝、 茎、 果实、 花等任一部位或全株, 优选的部位是根 与根茎。
其中, 所述的芍药提取物选择以芍药药材为原料, 经过常规溶剂提取后浓缩得到混合 物、芍药药材经过溶剂提取后, 再采用萃取方法得到的混合物或芍药药材经过溶剂提取, 再 经过大孔吸附树脂吸附、 洗脱, 浓缩后得到的混合物中的一种或多种。
硅胶柱层析使用的硅胶粒度对分离效果亦有较大影响, 理论上, 粒度越细的硅胶, 分 离效果越好,但是柱压会明显升高,对设备的要求也相应增加,且硅胶的成本也会显著增加。 本发明优选粒度为 100-200目硅胶。
本发明制备芍药苷和芍药内酯苷所用的原料包括从植物、 特别是药用植物芍药和川赤 芍中分离提纯得到, 所选取的原料可以是这些药材的根茎、 根、 叶、 枝、 茎、 果实、 花等任 一部位或全部植株, 其中优选的部位是根与根茎。
本发明具有如下优点:
1、 应用本发明方法, 可在同一个工艺流程中, 制备得到芍药苷和芍药内酯苷, 并且可 以分别得到公斤级的芍药苷和公斤级的芍药内酯苷,充分利用药材资源的综合使用价值,适 宜工业化生产。
2、 应用本发明方法, 制备的芍药苷和芍药内酯苷纯度高, 采用硅胶柱层析法纯化处理 后的芍药苷和芍药内酯苷含量达到 90%以上。
3、 本发明制备工艺方法简单, 精制效率高, 耗能低, 环保, 操作工艺条件容易控制, 质量可控性强。
4、 本发明所述的高纯度芍药苷和芍药内酯苷, 可以单独用于制备药物和功能性保健食 品,也可以与其它任何中西药物或食物,尤其是与某些具有活血化淤和保护心脑血管疾病的 中药或是与镇静安神和抗抑郁抗焦虑的中药配伍,起到协同或者增效之目的,用于制备药物 和功能性保健食品。
附图说明
图 1为实施例 1制备所得芍药苷的 HPLC检测图谱;
图 2为实施例 1制备所得芍药内酯苷的 HPLC检测图谱;
图 3为实施例 2制备所得芍药苷的 HPLC检测图谱;
图 4为实施例 2制备所得芍药内酯苷的 HPLC检测图谱;
图 5为实施例 7制备所得芍药内酯苷的 HPLC检测图谱。
具体实施方式
下面通过实施例对本发明进行进一步说明, 本发明的优点和特点将会随着描述而更为 清楚。但这些实施例仅是范例性的, 并不对本发明的范围构成任何限制。本领域技术人员应 该理解的是,在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改 或替换, 但这些修改和替换均落入本发明的保护范围内。 实施例 1
1、 药材的提取
干燥的芍药根 150公斤, 粉碎成粗粉后, 装入不锈钢中药提取罐 (容积为 2M3 ) 中, 进行加热回流提取 3次, 其中回流提取的提取溶剂为质量百分比浓度为 70%的乙醇溶液, 提取溶剂的体积用量依次为 1200L, 900L, 900L, 提取时间依次为 3h、 2.5h、 2h;
将三次提取液合并后, 采用真空减压浓缩罐 (无锡市华新药化设备有限公司产) 进行 减压浓缩, 浓缩至无醇味, 制得芍药浓缩液, 其中减压浓缩的温度为 70°C, 相对真空度为 -0.09- -0.075MPa, 芍药浓缩液的相对密度为 1.25。
本发明对药材进行加热回流提取处理时的提取溶剂除了选用质量百分比浓度为 70%的 乙醇溶液之外, 其他质量百分比浓度范围为 50-70%的乙醇溶液、 质量百分比浓度范围为 50-70%的甲醇溶液均适用于本发明。
2、 分离处理
1 )向芍药浓缩液加入质量百分比浓度为 95%乙醇, 使浓缩液中乙醇的质量百分比含量 为 75%后, 静止放置 8-12小时, 然后取上清液, 其中乙醇溶液与芍药浓缩液的重量之比为 3.75: 1;
2 ) 将上清液浓缩去除乙醇 (即浓缩至无乙醇味, 浓缩后的上清液中不含乙醇), 然后 采用大孔吸附树脂柱进行分离处理, 其中, 大孔吸附树脂选择 AB-8型大孔吸附树脂, 大孔 吸附树脂柱内大孔吸附树脂的柱体积为 200L (层析柱的直径 0.35米, 高 2.5米, 内装树脂 高度为 2.08米), 树脂柱内树脂的体积与药材重量(干重)之比为 4:3 (即如果药材干重 150 公斤, 大孔树脂的体积是 200L; 如果药材干重 150克, 则大孔树脂的体积为 200毫升), 待 浓缩后的上清液完全流入树脂柱后, 先用 2.5倍柱体积 (即 500L) 的水洗涤至流出液清澈 透明后, 再用 5倍柱体积(即 1000L)的质量百分比浓度为 60%的乙醇溶液洗脱, 收集洗脱 液, 洗脱流速为 150L/h, 每 50L洗脱液收集为一流份;
3 )采用薄层色谱法(TLC法)检测收集的大孔树脂柱洗脱流份中芍药苷和芍药内酯苷 的含有情况, 将含有芍药苷或 /和芍药内酯苷的洗脱液流份合并, 得到树脂柱洗脱液, 其中, TLC法使用的薄层板为硅胶 G板, 展开剂为氯仿、 甲醇的混合液, 氯仿与甲醇的体积比为 4: 1, 上行展开后, 喷以 5%的硫酸乙醇溶液, 150°C加热 5分钟后显色;
4 )将含芍药苷与芍药内酯苷组分的树脂柱洗脱液置于真空减压浓缩罐(无锡市华新药 化设备有限公司产) 内进行减压浓缩, 浓缩后水浴蒸干、 粉碎, 得到芍药苷和芍药内酯苷的 混合物粗品 7.5公斤, 其中, 减压浓缩的温度为 70°C, 相对真空度为 -0.095- -0.08Mpa;
3、 纯化处理
1 ) 将芍药苷和芍药内酯苷的混合物粗品用 30L 甲醇全部溶解后加入 8.9 公斤硅胶粉 ( 100-200目), 搅拌均匀, 干燥, 磨碎, 得到混合物样品;
2 )将混合物样品置于硅胶柱顶部, 以乙酸乙酯与甲醇的混合物为洗脱剂, 进行硅胶柱 层析, 其中, 硅胶柱内的吸附剂硅胶的粒度为 100-200目, 硅胶柱的柱直径与柱内硅胶高之 比 1 : 8; 吸附剂硅胶与芍药苷和芍药内酯苷的混合物粗品的重量之比为 16: 1, 洗脱剂为乙 酸乙酯与甲醇的混合溶剂(体积比为 20: 1 ),洗脱剂用量 5000L,洗脱流速为 200L/h,每 50L 洗脱液收集一流份;
3 )采用薄层色谱法(TLC法)检测硅胶柱洗脱流份中芍药苷和芍药内酯苷的含有情况, 将洗脱液合并为三个部分:第一部分为只含有芍药苷单一成分、第二部分为含芍药苷和芍药 内酯苷的混合部分、 第三部分为只含芍药内酯苷单一成分, 其中, TLC法使用的薄层板为 硅胶 G板, 展开剂为氯仿、 甲醇的混合液, 氯仿与甲醇的体积比为 4: 1, 上行展开后, 喷以 5%的硫酸乙醇溶液, 150°C加热 5分钟后显色;
4 ) 将 3部分分别蒸干, 第一部分的重量为 1.74公斤 (即获得的芍药苷纯品的重量为 1.74公斤)、第二部分的重量为 0.37公斤(即芍药苷和芍药内酯苷混合物的重量为 0.37公斤)、 第三部分的重量为 0.86公斤 (即获得的芍药内酯苷纯品的重量为 0.86公斤)。
采用 HPLC法检查制得的芍药苷、芍药内酯苷的含量, HPLC检测条件为:仪器: Water 515泵, 2487检测器; 色谱柱: Kromasil RP-C18; 流动相: 乙腈: 0.1%磷酸水溶液( 13:87) ; 检测波长: 230nm; 流速: 1.0ml/min。
经 HPLC检测第一部分中芍药苷含量为 88.26%, 见附图 1 ; 第三部分中芍药内酯苷含 量为 92.23%, 见附图 2。 实施例 2 1、 药材的提取
1 ) 新鲜的杭白芍根 300公斤 (含水率为 50%), 切碎后, 装入不锈钢中药提取罐 (容 积为 3M3 )中, 进行加热回流提取 3次, 其中回流提取过程中的提取溶剂为质量百分比浓度 为 50%的乙醇溶液, 提取溶剂的体积依次为 2200L, 1800L, 1800L, 提取时间依次为 3h, 3h, 2h;
2 )将三次提取液合并后, 采用真空减压浓缩罐进行减压浓缩, 浓缩至无醇味, 制得芍 药浓缩液, 其中减压浓缩的温度为 60°C, 相对真空度为 -0.09- -0.08MPa, 芍药浓缩液的相 对密度为 1.21。
本发明对药材进行加热回流提取处理时的提取溶剂除了选用质量百分比浓度为 50%的 乙醇溶液之外, 其他质量百分比浓度范围为 50-70%的乙醇溶液、 质量百分比浓度范围为 50-70%的甲醇溶液均适用于本发明。
2、 分离处理
1 )向芍药浓缩液加入质量百分比浓度为 95%的乙醇, 使浓缩液中乙醇的质量百分比含 量为 70%后, 静止放置 8-12小时, 然后取上清液, 其中乙醇溶液与芍药浓缩液的重量之比 为 2.8 : 1;
2 ) 将上清液浓缩至无乙醇味 (即浓缩后的上清液中不含乙醇), 然后采用大孔吸附树 脂柱进行分离处理, 其中, 大孔吸附树脂选择 HP-20 型大孔吸附树脂, 大孔吸附树脂柱内 HP-20型大孔吸附树脂的柱体积为 150L (层析柱的直径 0.30米, 高 2.5米, 内装树脂高度 为 2.12米), 树脂柱内树脂的体积与药材重量(干重)之比为 1 : 1 (即如果药材(干重) 150 公斤, 大孔树脂的体积是 150L; 如果药材(干重) 150克, 则大孔树脂的体积为 150毫升), 待浓缩后的上清液完全流入树脂柱后, 先用 450L水洗涤至流出液清澈透明后, 再 750L质 量百分比浓度为 50%的乙醇溶液洗脱, 收集洗脱液, 洗脱流速为 200L/h, 每 50L洗脱液收 集为一流份;
3 ) 采用薄层色谱法 (TLC法) 检测收集的大孔树脂柱洗脱流份中芍药苷和芍药内酯苷 的含有情况, 将含有芍药苷或 /和芍药内酯苷的洗脱液流份合并到一起, 得到树脂柱洗脱液, 其中, TLC法使用的薄层板为硅胶 G板, 展开剂为氯仿、 甲醇的混合液, 氯仿与甲醇的体 积比为 4: 1, 上行展开后, 喷以 5%的硫酸乙醇溶液, 150°C加热 5分钟后显色;
4 )将含芍药苷和芍药内酯苷组分的树脂柱洗脱液置于真空减压浓缩罐(无锡市华新药 化设备有限公司产) 内进行减压浓缩, 浓缩后水浴蒸干、 粉碎, 得到芍药苷和芍药内酯苷的 混合物粗品 8.1公斤, 其中, 减压浓缩的温度为 70°C, 相对真空度为 -0.09~ -0.075MPa;
3、 纯化处理
1 )将芍药苷和芍药内酯苷的混合物粗品用质量百分比浓度为 70%的乙醇溶液 30L全部 溶解后加入 10公斤层析用硅胶粉 (200-300目), 搅拌均匀, 干燥, 磨碎, 得到混合物样品;
2 ) 将混合物样品置于硅胶柱顶部, 以乙酸乙酯与乙醇混合溶剂为洗脱剂, 梯度洗脱, 进行硅胶柱层析, 其中, 硅胶柱内的吸附剂硅胶的粒度为 100-200目, 硅胶柱的柱直径与柱 内硅胶高之比为 1 : 8; 吸附剂硅胶与芍药苷和芍药内酯苷的混合物粗品的重量之比为 20: 1, 洗脱剂中乙酸乙酯与乙醇的体积比为 10: 1, 洗脱剂用量 6000L, 洗脱流速为每小时 200L/h, 每 50L洗脱液收集为一流份;
3 )采用薄层色谱法(TLC法)检测硅胶柱洗脱流份中芍药苷和芍药内酯苷的含有情况, 将洗脱液合并为两个部分:第一部分为含芍药苷部分(即包括只含有芍药苷单一成分部分和 含芍药苷与芍药内酯苷混合部分); 第二部分只含有芍药内酯苷单一成分, 其中, TLC法使 用的薄层板为硅胶 G板, 展开剂为氯仿、 甲醇的混合液, 氯仿与甲醇的体积比为 4: 1, 上行 展开后, 喷以 5%的硫酸乙醇溶液, 150°C加热 5分钟后显色;
4 )将合并后的 2部分分别蒸干, 第一部分的重量为 2.42公斤(即含芍药苷和芍药内酯 苷的混合物重量为 2.42公斤),第二部分的重量为 1.16公斤(即获得的芍药内酯苷纯品的重 量为 1.16公斤)。
采用 HPLC法检查制得的芍药苷、芍药内酯苷的含量, HPLC检测条件为:仪器: Water 515泵, 2487检测器; 色谱柱: Kromasil RP-C18; 流动相: 乙腈: 0.1%磷酸水溶液( 13:87); 检测波长: 230nm; 流速: 1.0ml/min。
经 HPLC检测第一部分中芍药苷含量为 84.78%, 见附图 3 ; 第二部分中芍药内酯苷的 含量为 94.71%, 见附图 4。 实施例 3
1、 药材的提取
干燥的芍药根 100公斤, 粉碎成粗粉后, 用 400L去离子水浸泡 5小时后, 装入不锈钢 渗漉罐 (容积为 1M3) 中, 用去离子水渗漉, 收集渗漉液约 1500L。
提取溶剂除了选用水之外, 还可以选用质量百分比浓度范围为 20-50%的乙醇溶液、 质 量百分比浓度范围为 20-50%的甲醇溶液。
2、 分离处理
1 ) 将渗漉液直接采用大孔吸附树脂柱进行分离处理, 其中, 大孔吸附树脂选择 D101 型大孔吸附树脂, 大孔吸附树脂柱内的 D101 型大孔吸附树脂的柱体积为 150L (层析柱的 直径 0.30米, 高 2.5米, 内装树脂高度为 2.12米), 树脂的体积与药材重量(干重)之比为 3:2, 即如果药材 (干重) 100公斤, 大孔树脂的体积是 150L; 如果药材 (干重) 100克, 则大孔树脂的体积为 150毫升, 待浓缩后的上清液完全流入树脂柱后, 先用 500L水洗涤至 流出液清澈透明后, 再用 800L质量百分比浓度为 50%的乙醇洗脱, 洗脱流速为 100L/h, 收 集洗脱液, 每 50L洗脱液收集为一流份;
2) 采用薄层色谱法 (TLC法) 检测收集的大孔树脂柱洗脱流份中芍药苷和芍药内酯苷 的含有情况, 将含有芍药苷或 /和芍药内酯苷的洗脱液流份合并到一起, 得到树脂柱洗脱液, 其中, TLC法使用的薄层板为硅胶 G板, 展开剂为氯仿、 甲醇的混合液, 氯仿与甲醇的体 积比为 4:1, 上行展开后, 喷以 5%的硫酸乙醇溶液, 150°C加热 5分钟后显色;
3 ) 将含芍药苷和芍药内酯苷组分的树脂柱洗脱液置于真空减压浓缩罐内进行减压浓 缩, 浓缩后水浴蒸干、 粉碎, 得到芍药苷和芍药内酯苷的混合物粗品 4.81 公斤, 其中, 减 压浓缩的温度为 70°C, 相对真空度为 -0.09- -0.075MPa;
3、 纯化处理
1 ) 将芍药苷和芍药内酯苷的混合物粗品用适量甲醇全部溶解后加入 6.4 公斤硅胶粉 ( 100-200目), 搅拌均匀, 干燥, 磨碎, 得到混合物样品;
2)将混合物样品置于硅胶柱顶部, 以乙酸乙酯为洗脱剂, 进行硅胶柱层析, 其中, 硅 胶柱内的吸附剂硅胶的粒度为 100-200目, 硅胶柱的柱直径与柱内硅胶高之比 1 : 10; 吸附 剂硅胶与芍药苷和芍药内酯苷的混合物粗品的重量之比为 20:1, 洗脱剂为乙酸乙酯, 用量 6000L, 洗脱流速为 300L/h, 每 50L洗脱液收集一流份;
3 )采用薄层色谱法(TLC法)检测硅胶柱洗脱流份中芍药苷和芍药内酯苷的含有情况, 将洗脱液合并为 3个部分:第一部分为只含有芍药苷单一成分、第二部分为含芍药苷和芍药 内酯苷的混合部分、 第三部分为只含芍药内酯苷单一成分, 其中, TLC法使用的薄层板为 硅胶 G板, 展开剂为氯仿、 甲醇的混合液, 氯仿与甲醇的体积比为 4:1, 上行展开后, 喷以 5%的硫酸乙醇溶液, 150°C加热 5分钟后显色;
4)将第一部分、 第三部分的洗脱液分别蒸干, 第一部分的重量为 1.74公斤(即获得的 芍药苷纯品的重量为 1.74公斤),第三部分的重量为 1.02公斤(即获得芍药内酯苷纯品的重 量为 1.02公斤)。 经 HPLC 检测第一部分中芍药苷含量为 93.42%; 第三部分中芍药内酯苷的含量为 92.77%。 实施例 4
以芍药提取物 (芍药总苷) 为原料 (经测定芍药苷含量 24.2%、 芍药内酯苷 15.9%)。 1、 纯化处理
1 ) 芍药提取物 10公斤, 以质量百分比浓度为 70%乙醇全部溶解后加入 12.1公斤硅胶 粉 (100-200目), 搅拌均匀, 蒸干, 得混合物样品;
2 )将混合物样品置于硅胶柱顶部, 以乙酸乙酯与水的混合物为洗脱剂, 进行硅胶柱层 析, 其中, 硅胶柱内的吸附剂硅胶的粒度为 100-200目, 硅胶柱的柱直径与柱内硅胶高之比 为 1 : 6; 吸附剂硅胶与芍药提取物的重量之比为 16:1, 洗脱剂中乙酸乙酯与水的体积比为 100: 3.5, 洗脱剂用量为 6000L, 洗脱流速为 200L/h, 每 50L洗脱液收集一流份;
3 )采用薄层色谱法(TLC法)检测硅胶柱洗脱流份中芍药苷和芍药内酯苷的含有情况, 将洗脱液合并为两个部分:第一部分只含有芍药苷单一成分;第二部分为含芍药内酯苷部分
(即包括只含有芍药内酯苷单一成分部分和含芍药苷与芍药内酯苷混合部分), 其中, TLC 法使用的薄层板为硅胶 G板, 展开剂为氯仿、 甲醇的混合液, 氯仿与甲醇的体积比为 4: 1, 上行展开后, 喷以 5%的硫酸乙醇溶液, 150°C加热 5分钟后显色;
4 )将合并后的 2部分分别蒸干, 第一部分的重量为 2.53公斤(即获得的芍药苷纯品的 重量为 2.53公斤),第二部分的重量为 1.08公斤(即包括只含有芍药内酯苷单一成分部分和 含芍药苷与芍药内酯苷混合部分的混合物总重量为 1.08公斤)。
经 HPLC检测第一部分中芍药苷含量为 93.4%; 第二部分中芍药内酯苷含量为 82.9%。 实施例 5
1、 药材的提取
干燥的亳白芍根 100公斤, 粉碎后, 装入中药提取罐 (容积为 2M3 ) 中, 进行加热回 流提取 3次, 其中回流提取的提取溶剂为质量百分比浓度为 95%的含水乙醇溶液, 提取溶 剂的体积依次为 800L, 600L, 600L, 提取时间依次为 3h, 3h, 2h;
将三次提取液合并后, 采用真空减压浓缩罐进行减压浓缩, 浓缩至干, 制得芍药提取 物 18公斤, 其中减压浓缩的温度为 60°C, 相对真空度为 -0.09- -0.08MPa。
加热回流提取溶剂除了选择质量百分比浓度为 95%的含水乙醇溶液之外, 提取溶剂还 可以选择质量百分比浓度范围为 90-100%的乙醇溶液、质量百分比浓度范围为 95-100%的甲 醇溶液。
2、 纯化处理
1 )向芍药提取物中加入质量百分比浓度为 70%的乙醇 25L,将芍药提取物全部溶解后, 加入 25公斤层析用硅胶粉 (100-200目), 搅拌均匀, 干燥, 磨碎, 得到混合物样品;
2 )将混合物样品置于硅胶柱顶部, 以乙酸乙酯与水混合溶剂为洗脱剂, 进行硅胶柱层 析, 其中, 硅胶柱内的吸附剂硅胶的粒度为 100-200目, 硅胶柱的柱直径与柱内硅胶高之比 为 1 : 8; 吸附剂硅胶与芍药提取物的重量之比为 6: 1, 洗脱剂中乙酸乙酯与水的体积比为 100:2.5, 洗脱剂用量 6000L, 洗脱流速为每小时 200L/h, 每 50L洗脱液收集为一流份;
3 )采用薄层色谱法(TLC法)检测硅胶柱洗脱流份中芍药苷和芍药内酯苷的含有情况, 将洗脱液合并为两个部分:第一部分只含有芍药苷单一成分;第二部分为含芍药内酯苷部分
(即包括只含有芍药内酯苷单一成分部分和含芍药苷与芍药内酯苷混合部分), 其中, TLC 法使用的薄层板为硅胶 G板, 展开剂为氯仿、 甲醇的混合液, 氯仿与甲醇的体积比为 4: 1, 上行展开后, 喷以 5%的硫酸乙醇溶液, 150°C加热 5分钟后显色; 4 )将合并后的 2部分分别蒸干, 第一部分的重量为 3.61公斤(即获得的芍药苷纯品的 重量为 3.61公斤),第二部分的重量为 2.16公斤(即包括只含有芍药内酯苷单一成分部分和 含芍药苷与芍药内酯苷混合部分的混合物总重量为 2.16公斤)。
经 HPLC检测第一部分中芍药苷含量为 72.83%;第二部分中芍药内酯苷含量为 64.59%。 3、 精制处理
1 ) 取步骤 2中获得的第二部分中的芍药内酯苷粗品 2公斤,以质量百分比浓度为 70% 的乙醇 3.5L完全溶解后, 加入 2.5公斤层析用硅胶粉 ( 100-200目), 搅拌均匀, 干燥, 磨 碎, 得到芍药内酯苷样品;
2 )将芍药内酯苷样品置于硅胶柱顶部, 以乙酸乙酯与丙酮混合溶剂为洗脱剂, 进行硅 胶柱层析, 其中, 硅胶柱内的吸附剂硅胶的粒度为 100-200目, 硅胶柱的柱直径与柱内硅胶 高之比为 1 : 10; 吸附剂硅胶与芍药内酯苷粗品的重量之比为 15: 1, 洗脱剂中乙酸乙酯与丙 酮的体积比为 10: 1, 洗脱剂用量 1400L, 洗脱流速为每小时 200L/h, 每 50L洗脱液收集为 一流份;
3 ) 采用薄层色谱法 (TLC法) 检测第二次硅胶柱洗脱流份中芍药内酯苷的含有情况, 将只含有芍药内酯苷单一成分的流份合并, 得到芍药内酯苷洗脱液, 将洗脱液蒸干, 得芍药 内酯苷 1.17公斤, 其中, TLC法使用的薄层板为硅胶 G板, 展开剂为氯仿、 甲醇的混合液, 氯仿与甲醇的体积比为 4: 1, 上行展开后, 喷以 5%的硫酸乙醇溶液, 150°C加热 5分钟后显 色。 经 HPLC检测芍药内酯苷含量为 93.24%。 实施例 6
除了药材提取步骤采用含水率为 61%的新鲜的川赤芍枝叶、 根的混合物 500公斤为原 料, 切碎后, 装入渗漉罐 (容积为 3M3 ) 中, 以质量百分比浓度为 30%的乙醇溶液为渗漉 提取液进行渗漉提取, 渗漉提取液的体积为 3800L, 渗漉提取的流速为 50L/h; 渗漉液合并 后进行减压浓缩, 浓缩去除乙醇, 制得芍药浓缩液, 其中减压浓缩的温度为 80°C, 相对真 空度为 -0.095- -0.08MPa, 芍药浓缩液的相对密度为 1.25之外, 其余与实施例 3相同, 精制 得到的芍药苷 1.96公斤、 芍药内酯苷 0.67公斤。 经 HPLC检测, 第一部分中的芍药苷含量 为 79.4%; 第一部分中的芍药内酯苷含量为 73.7%。
提取溶剂除了选用质量百分比浓度为 30%的乙醇溶液之外, 还可以选用水、 质量百分 比浓度范围为 20-50%的乙醇溶液、 质量百分比浓度范围为 20-50%的甲醇溶液。 实施例 7
1 ) 取实施例 3得到的芍药内酯苷 (纯度 92.77%) 1.0公斤作为试样, 用少量甲醇将芍 药内酯苷试样全部溶解后加入 1.2公斤硅胶粉(100-200目), 搅拌均匀, 干燥, 制得芍药内 酯苷样品;
2 ) 将芍药内酯苷样品进行硅胶柱层析, 以乙酸乙酯 -水的混合物为洗脱剂进行洗脱, 其中,硅胶柱内的吸附剂硅胶的粒度为 100-200目,硅胶柱的柱直径与柱内硅胶高之比为 1 : 5; 吸附剂硅胶与芍药内酯苷试样的重量之比为 30: 1, 洗脱剂中乙酸乙酯与水的体积比为 100: 3, 洗脱体积为 2000L, 洗脱流速为 80L/h, 每 50L洗脱液收集一流份;
3 )采用薄层色谱法(TLC法)检测洗脱液中芍药内酯苷的含有情况, 合并含有芍药内 酯苷的洗脱液, 蒸干后得芍药内酯苷 0.73公斤, 其中, TLC法使用的薄层板为硅胶 G板, 展开剂为氯仿、 甲醇的混合液, 氯仿与甲醇的体积比为 4: 1, 上行展开后, 喷以 5%的硫酸 乙醇溶液, 150°C加热 5分钟后显色。
经 HPLC检测, 芍药内酯苷含量为 99.3%, 见附图 5。
HPLC检测条件为: 仪器: Water 515泵, 2487检测器; 色谱柱: Kromasil RP-C18; 流 动相: 乙腈: 0.1%磷酸水溶液 ( 13:87); 检测波长: 230nm; 流速: 1.0ml/min„ 实施例 8 芍药内酯苷片
取实施例 2所得的芍药内酯苷 400克, 加入淀粉 100克, 硬脂酸镁 30克, 混合均匀, 在压片机上压制 10000粒。 每粒含芍药内酯苷约 40mg, 患者每日服用 3粒, 用于治疗冠状 动脉硬化性心脏病及心绞痛。 实施例 9 芍药苷复方制剂的制备
芍药苷 (纯度 93.4%) 180克
丹参酚酸 B (纯度 98.1 ) 70克
人参皂苷 Rgl (纯度 99.3 ) 50克
冰片 45克
淀粉 120克
上述组分混合均匀, 装入硬明胶胶囊中, 制得 4000粒胶囊, 用于治疗冠状动脉硬化性 心脏病及心绞痛。

Claims

权 利 要 求 书
1、 一种制备芍药内酯苷和芍药苷的方法, 包括如下顺序进行的步骤:
1 ) 将芍药药材加入到提取溶剂中, 对药材进行提取处理, 得到芍药提取液;
2 ) 将芍药提取液采用大孔树脂柱进行分离处理, 收集、 合并洗脱液, 得到含有芍药苷 和芍药内酯苷的树脂柱洗脱液;
3 )将树脂柱洗脱液干燥后进行硅胶柱层析处理, 分段收集洗脱液, 洗脱液分别干燥后, 得到芍药苷、 芍药内酯苷。
2、 如权利要求 1所述的方法, 其特征是步骤 1 ) 中所述的提取溶剂选择水、 乙醇溶液或甲 醇溶液中的一种。
3、 如权利要求 1所述的方法, 其特征是步骤 1 ) 中采用加热回流提取处理法或渗漉提取处 理法进行所述的提取处理。
4、 一种制备芍药内酯苷和芍药苷的方法, 包括如下顺序进行的步骤:
1 ) 将芍药药材加入到提取溶剂中, 对药材进行加热回流提取处理, 得到芍药提取液, 其中提取溶剂选择质量百分比浓度为 50-70%的甲醇溶液或质量百分比浓度为 50-70%的乙 醇溶液;
2 ) 将芍药提取液浓缩, 去除乙醇或甲醇后, 进行大孔树脂柱分离处理, 收集、 合并洗 脱液, 得到含有芍药苷和芍药内酯苷的树脂柱洗脱液;
3 ) 将树脂柱洗脱液干燥后的样品进行硅胶柱层析处理, 分段收集洗脱液, 洗脱液分别 干燥后, 分别得到芍药苷、 芍药内酯苷。
5、 一种制备芍药内酯苷和芍药苷的方法, 包括如下顺序进行的步骤:
1 ) 将芍药药材加入到提取溶剂中, 对药材进行渗漉提取处理, 得到芍药提取液, 其中 提取溶剂选择水;
2 ) 将芍药提取液进行大孔树脂柱分离处理, 收集、 合并洗脱液, 得到含有芍药苷和芍 药内酯苷的树脂柱洗脱液;
3 )将树脂柱洗脱液干燥后的样品进行硅胶柱层析处理, 分段收集洗脱液, 洗脱液分别 干燥后, 分别得到芍药苷、 芍药内酯苷。
6、 一种制备芍药内酯苷和芍药苷的方法, 包括如下顺序进行的步骤:
1 ) 将芍药药材加入到提取溶剂中, 对药材进行渗漉提取处理, 得到芍药提取液, 其中 提取溶剂选择质量百分比浓度为 10-50%的甲醇溶液或质量百分比浓度为 10-50%的乙醇溶 液;
2 ) 将芍药提取液浓缩, 去除乙醇或甲醇后, 进行大孔树脂柱分离处理, 收集、 合并洗 脱液, 得到含有芍药苷和芍药内酯苷的树脂柱洗脱液;
3 )将树脂柱洗脱液干燥后的样品进行硅胶柱层析处理, 分段收集洗脱液, 洗脱液分别 干燥后, 分别得到芍药苷、 芍药内酯苷。
7、 如权利要求 1、 4、 5或 6任一所述的方法, 其特征是还包括还包括步骤 4) : 将步骤 3 ) 制得的芍药苷或芍药内酯苷分别进行第二次硅胶柱层析处理,分别分段收集洗脱液,洗脱液 干燥后, 得到纯度更高的芍药苷或纯度更高的芍药内酯苷。
8、 如权利要求 7所述的方法, 其特征是步骤 4 ) 中所述的第二次硅胶柱层析处理过程中的 吸附剂硅胶与步骤 3 ) 制得的芍药苷或芍药内酯苷的样品的重量之比为 15-30: 1。
9、 如权利要求 7所述的方法, 其特征是步骤 4 ) 中所述的硅胶柱层析处理过程中选择乙酸 乙酯与丙酮或乙酸乙酯与水的混合溶剂为洗脱剂。
10、 如权利要求 1、 4、 5或 6任一所述的方法, 其特征是步骤 2) 中所述的大孔树脂选择 D101、 AB-8、 DM130H、 XAD-2、 D201、 HP-20、 X-5、 H103、 DM11或 ADS-17型树脂。
11、 如权利要求 1、 4、 5或 6任一所述的方法, 其特征是步骤 2) 中所述的大孔树脂的体积 与芍药药材重量之比为 0.5-2: 1。
12、 如权利要求 1、 4、 5或 6任一所述的方法, 其特征是步骤 3 ) 中所述的硅胶柱层析处理 过程中的层析吸附剂硅胶与树脂柱洗脱液干燥后的样品的重量之比为 5-50: 1。
13、 如权利要求 1、 4、 5或 6任一所述的方法, 其特征是步骤 3 ) 中所述的硅胶柱层析处理 过程中选择乙酸乙酯、 乙酸乙酯与甲醇、 乙酸乙酯与乙醇、 乙酸乙酯与丙酮或乙酸乙酯与水 的混合溶剂为洗脱剂。
14、 如权利要求 13所述的方法, 其特征是所述的乙酸乙酯与甲醇的体积之比为 10-20: 1 ; 所 述的乙酸乙酯与乙醇的体积之比为 10-20: 1 ; 所述的乙酸乙酯与水的体积之比为 100: 2-4; 所述的乙酸乙酯与丙酮的体积之比为 10-20: 1。
15、 一种制备芍药内酯苷和芍药苷的方法, 包括如下顺序进行的步骤:
( 1 )将芍药药材加入到提取溶剂中, 对药材进行加热回流提取处理, 得到芍药提取液;
(2 ) 芍药提取液干燥后进行第一次硅胶柱层析处理, 分段收集洗脱液, 洗脱液分别干 燥后, 得到芍药苷粗品、 芍药内酯苷粗品;
(3 )将步骤(2 )得到的芍药苷粗品或芍药内酯苷粗品分别进行第二次硅胶柱层析, 分 别分段收集洗脱液, 洗脱液干燥后, 得到单一成分的芍药苷或单一成分的芍药内酯苷。
16、 如权利要求 15所述方法, 其特征是步骤(1 ) 中所述的提取溶剂选择质量百分比浓度为 90%- 100%乙醇溶液或质量百分比浓度为 95%- 100%甲醇溶液。
17、 如权利要求 15所述方法, 其特征是步骤(2 ) 中所述的第一次硅胶柱层析处理过程中的 层析吸附剂硅胶与步骤 (1 ) 中芍药提取液干燥后的样品的重量之比为 5-50: 1。
18、 如权利要求 15所述方法, 其特征是步骤(3 ) 中所述的第二次硅胶柱层析处理过程中层 析吸附剂硅胶与步骤 (2 ) 制得的芍药苷或芍药内酯苷的粗品的重量之比为 5-50: 1。
19、 如权利要求 15所述方法, 其特征是步骤(2)、 步骤(3 ) 中所述的硅胶柱层析处理过程 中选择乙酸乙酯、 乙酸乙酯与甲醇、 乙酸乙酯与乙醇、 乙酸乙酯与水或乙酸乙酯与丙酮的混 合溶剂为洗脱剂。
20、 如权利要求 19所述的方法, 其特征是所述的乙酸乙酯与甲醇的体积之比为 10-20: 1 ; 所 述的乙酸乙酯与乙醇的体积之比为 10-20: 1 ; 所述的乙酸乙酯与水的体积之比为 100: 2-4; 所述的乙酸乙酯与丙酮的体积之比为 10-20: 1。
21、一种制备芍药内酯苷和芍药苷的方法, 其特征是将芍药提取物进行硅胶柱层析处理, 分 段收集洗脱液, 洗脱液干燥后, 分别得到单一成分的芍药苷、 芍药内酯苷。
22、 如权利要求 21所述的方法, 其特征是所述的硅胶柱层析处理过程中的层析吸附硅胶与 芍药提取物的样品的重量之比为 5-50: 1。
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