WO2012061984A1 - 制备芍药内酯苷和芍药苷的方法 - Google Patents

制备芍药内酯苷和芍药苷的方法 Download PDF

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WO2012061984A1
WO2012061984A1 PCT/CN2010/078597 CN2010078597W WO2012061984A1 WO 2012061984 A1 WO2012061984 A1 WO 2012061984A1 CN 2010078597 W CN2010078597 W CN 2010078597W WO 2012061984 A1 WO2012061984 A1 WO 2012061984A1
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paeoniflorin
alumina
column
chromatography
silica gel
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PCT/CN2010/078597
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English (en)
French (fr)
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张作光
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Zhang Zuoguang
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Priority to US13/884,422 priority Critical patent/US9453041B2/en
Priority to ES10859598.4T priority patent/ES2670820T3/es
Priority to EP10859598.4A priority patent/EP2650301B1/en
Priority to NO10859598A priority patent/NO2650301T3/no
Priority to DK10859598.4T priority patent/DK2650301T3/en
Priority to CN201080069710.XA priority patent/CN103180334B/zh
Priority to PCT/CN2010/078597 priority patent/WO2012061984A1/zh
Publication of WO2012061984A1 publication Critical patent/WO2012061984A1/zh

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04Heterocyclic radicals containing only oxygen as ring hetero atoms
    • 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

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 the plant natural physiological active substances paeoniflorin and paeoniflorin. Background technique
  • Peony is a perennial herb of the genus Paeonia, which is commonly used as a dry root.
  • Two Chinese medicines were included in the 2010 edition of the Chinese Pharmacopoeia.
  • One is Radix Paeoniae Alba, which is the dried root of Paeow'a lactiflora Pall.; the other is Radix Paeoniae Rubra, the dry root of Paeonia veitchii Lynch, Ranunculaceae .
  • traditional Chinese medicine preparations containing peony such as cerebral thrombosis tablets, Yinaofujian capsules, hemiplegia recovery tablets, Huisheng reconstituted pills, and Dahuoluo pills.
  • Peony contains mainly paeonif lorin, albif lorin, 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 analgesic, sedative, liver protection, anti-inflammatory, immune regulation, dilatation 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.
  • Albiflorin is a monoterpenoid compound with a molecular formula of C 23 H 28 O U and a molecular weight of 480.46.
  • the molecular structure is as shown in formula (I).
  • Paeoniflorin is a monoterpenoid compound.
  • CH OU For CH OU,
  • paeoniflorin has analgesic, sedative, anticonvulsant effects, effects on the immune system, effects on smooth muscle, anti-inflammatory effects, anti-pathogenic microorganisms, liver protection, clinically It should be used for anti-epilepsy, analgesia, detoxification, vertigo, treatment of rheumatoid arthritis, treatment of bacterial dysentery and enteritis, treatment of viral hepatitis, senile diseases, anti-sulphate flocculation and dissolution of mucus.
  • the invention patent application with the application number 200510045840.0 discloses a kind of paeonia from the white peony (Panonia peony peony) Lactifora Pal l) extracting and separating 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 composition The ratio is 1:10 to 10:1.
  • 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, which has simple operation process and short production cycle.
  • the method of the invention 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, the method comprising the steps of: a) using a percolation extraction method for percolating and extracting a paeoniflorin; Peony osmosis solution; b) The sputum osmosis solution is sequentially subjected to macroporous resin column separation, alumina column chromatography and silica gel column chromatography.
  • the percolation extraction is carried out by using water, ethanol or methanol as the permeation extraction solution, preferably water.
  • the ratio of the volume of the percolating extract solution to the weight of the paeoniflorin is 5-20:1, that is, when the weight of the paeoniflorin (dry weight) is 1 kg, the volume of the percolating extract solution is 5-20 L, when When the weight of the peony drug (dry weight) is lg, the volume of the osmotic extraction solution is 5-20 ml.
  • the ratio of the flow rate of the percolating solution per hour to the weight of the peony medicinal material during the percolation extraction process is 0.5-8:1, preferably 1-5:1, that is, when the weight (dry weight) of the peony drug is lkg.
  • the flow rate of percolating solution per hour during the percolation extraction process is 0.5-8L, and when the weight of the drug (dry weight) is lg, the flow rate of the percolating solution is 0.5-8ml.
  • the method further comprises: after soaking the peony medicinal material with water, performing the osmotic extraction, wherein the ratio of the volume of water immersed in the peony medicinal material to the weight of the peony medicinal material is 2-3:1, that is, when the weight of the medicinal material is When the dry weight is lkg, the volume of the osmotic extraction solution is 2-3L, and when the weight of the medicinal herb (dry weight) is lg, the volume of the osmotic extraction solution is 2-3ml.
  • the separation of the macroporous resin column described in the step b) comprises the following steps:
  • the macroporous resin described in the step 1) is one selected from the group consisting of D101, D-20K AB-8 or HP-20 type nonpolar macroporous adsorption resins.
  • the ratio of the volume of the macroporous adsorption resin to the weight of the peony drug in the macroporous resin column is 0.5 to 5: 1, that is, when the weight of the medicinal herb (dry weight) is lkg, the volume of the macroporous adsorption resin When it is 0.5-5 L, when the weight (dry weight) of the medicinal herb is lg, the volume of the macroporous adsorption resin is 0.5 to 5 ml, preferably 1-2:1.
  • the flow rate of the eluent during the separation of the macroporous resin column is 0.5-5 times the bed volume/h, that is, the flow rate of the eluate per hour is 0.5-5 times the volume of the macroporous adsorption resin in the macroporous resin column.
  • the ratio of the volume of the eluent water to the macroporous resin in the step 2) is 3-5:1; the mass percentage concentration of the ethanol solution described in the step 3) is 30-70%, preferably 40-60. %;
  • the ratio of the volume of the ethanol solution to the macroporous resin in the step 3) is 3-5:1.
  • the alumina column chromatography described in step b) comprises the following steps:
  • Alumina chromatography sample is placed on top of the alumina column, and ethyl acetate is used as an eluent to perform alumina chromatography, and the eluate is collected.
  • the combined eluate contains paeoniflorin and/or paeoniflorin. The fraction of the obtained alumina chromatography eluate;
  • step 1) the ratio of the macroporous resin column separation mixture to the alumina is 1:0.5-5; in step 2), the alumina in the alumina column is separated from the macroporous resin column by the weight of the mixture.
  • the ratio is 5-30: 1, preferably 8-25: 1, further preferably 10-25:1.
  • the ratio of the column diameter of the alumina column to the height of the alumina in the column is 1:6-10.
  • silica gel column chromatography described in the step b) comprises the following steps:
  • the ratio of the alumina chromatography extract to the silica gel in step 1) is 1: 0.5-5; the ratio of the weight of the silica gel to the alumina chromatography mixture in the silica gel column in step 2) is 5-30: 1, preferably 12-30: 1.
  • the ratio of the column diameter of the silica gel column to the height of the silica gel in the column is 1:6-10, preferably 1:8-10.
  • Another aspect of the invention provides a method of 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 step a) is 5-12: 1 (ie if the weight (dry weight) of the medicinal material is 1 kg, the volume of the extraction solvent is 5-12 liters; if the weight (dry weight) of the medicinal material is 1 gram, the volume of the extraction solvent is 5-12 ml); the number of extractions is 2-3 times, and the extraction time is 2-4 small Hours / times.
  • the ratio of the volume of the dilute alcohol extract diluent to the weight of the paeoniflorin (dry weight) in step b) is from 1 to 8: 1, preferably from 2 to 8: 1, that is, when the weight (dry weight) of the medicine is At 1 kg, the volume of the dilute solution of paeoniflorin is 1-8 L.
  • the weight (dry weight) of the medicine is 1 g
  • the volume of the dilute solution of paeoniflorin is 1-8 ml.
  • step c) comprises the following sequence of steps:
  • step c) eluting the macroporous resin column with an ethanol solution as an elution solvent, collecting the eluate, and combining the fractions containing paeoniflorin and/or paeoniflorin in the eluate, and drying to obtain a macroporous resin column.
  • the mixture was separated.
  • the alumina column chromatography described in step c) comprises the following sequence of steps:
  • Alumina chromatography sample is placed on top of the alumina column, and ethyl acetate is used as an eluent to perform alumina chromatography, and the eluate is collected.
  • the combined eluate contains paeoniflorin and/or paeoniflorin. The fraction of the obtained alumina chromatography eluate;
  • silica gel column chromatography described in step c) comprises the following sequence of steps:
  • paeoniflorin and paeoniflorin can be prepared in the same process, and the baical-level paeoniflorin and kilogram-class paeoniflorin can be respectively obtained, 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 adopts water as an extract of paeoniflorin and paeoniflorin, and uses non-toxic and harmless ethanol and ethyl acetate as purification and elution solvent, which not only saves cost, but also is environmentally friendly and conforms. Green economy, environmental protection Development requirements for protection.
  • 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.
  • FIG. 1 is a flow chart of a preparation process of paeoniflorin and paeoniflorin according to the present invention
  • Figure 5 is a HPLC detection spectrum of the obtained paeoniflorin prepared in Example 4.
  • FIG. 7 is a HPLC detection spectrum of the obtained paeoniflorin prepared in Example 5.
  • the volume of pure water is 10L; when the weight of white peony (dry weight) is 1 gram, pure water The volume is 10ml.
  • 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 4L ( The diameter of the column is 8cm and the height is 1.5m.
  • the ratio of the volume of the resin to the weight of the medicinal material (dry weight) is 4:3, that is, if the medicinal material (dry weight) is 3 kg, the volume of the macroporous resin is 4 liters; Weight) 30 g, the volume of the macroporous resin is 40 ml.
  • TLC method thin layer chromatography
  • the volume ratio of chloroform to methanol was 4:1, and after the ascending expansion, Sprayed with 5% solution of sulfuric acid in ethanol, and heated at 150 ° C for 5 minutes to develop color; 3)
  • the resin column eluate containing paeoniflorin and paeoniflorin components was placed in a vacuum decompression concentration tank for concentration under reduced pressure. After concentrating, the water bath was evaporated to dryness and pulverized to obtain 210 g of a macroporous resin column separation mixture, wherein the temperature under reduced pressure was 70 ° C, and the relative pressure was -0.09 to -0.075 MPa.
  • Alumina column chromatography sample is placed on top of the alumina column, and ethyl acetate is used as an eluent to perform alumina column chromatography.
  • the particle size of the adsorbent alumina in the alumina column is 100-200 mesh.
  • the ratio of the column diameter of the alumina column to the alumina height in the column is 1:10; the weight ratio of the adsorbent alumina to the macroporous resin column separation mixture is 20:1, the eluent is ethyl acetate, and the amount is 100L. Collect a first-class share per 1L of eluent;
  • TLC method thin layer chromatography
  • the silica gel chromatography sample 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 of the silica gel column is 2
  • the ratio of the height to the silica gel in the column is 1:8; the weight ratio of the adsorbent silica gel to the alumina column chromatography mixture is 24:1, and the flow rate is 10 L/h.
  • the amount of ethyl acetate used in the eluent was 96 L, and each 1 L of the eluate was collected as a first-class fraction;
  • TLC method thin layer chromatography (TLC method) to detect the content of paeoniflorin and paeoniflorin in the elution fraction of silica gel column, and combine the elution fraction containing only the single component of paeoniflorin into the first part (ie, paeoniflorin) Partially; combining the elution fraction containing only the single component of paeoniflorin into the second part (ie, the paeoniflorin moiety), 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, a volume ratio of chloroform to methanol of 4: 1, after the ascending expansion, sprayed with a 5% solution of sulfuric acid in ethanol, heated at 150 ° C for 5 minutes and then developed;
  • TLC method thin layer chromatography
  • the osmotic extraction step of the medicinal material is the same as that of the first embodiment except that the ratio of the volume of the purified water of the osmotic extract to the weight of the medicinal material is 5:1, and the flow rate of the osmotic solution is 4 L/h;
  • the macroporous resin column coarse separation step except for the HP-20 type macroporous adsorption resin, the ratio of the resin volume to the weight of the drug (dry weight) is 1:1, the mass percentage concentration of the eluent ethanol is 30%, and the eluent The amount of ethanol used was 20 L; the macroporous resin column differentiation mixture obtained by crude separation was 189 g, and the rest was the same as in Example 1;
  • the alumina column chromatography step except that the weight ratio of the adsorbent alumina to the macroporous resin column separation mixture is 10:1; the ratio of the column diameter of the alumina column to the alumina in the column is 1:6; ethyl acetate
  • the dosage is 53 L; the elution flow rate is 8 L/h; the obtained alumina column chromatography mixture is 105 g, and the rest is the same as in Example 1;
  • the silica gel column chromatography step except that the weight ratio of the adsorbent silica gel to the alumina column chromatography mixture is 15:1, the eluent ethyl acetate amount is 62 L, the elution flow rate is 6 L/h; the column diameter of the silica gel column The ratio of the height to the silica gel in the column was 1:10; the weight was 74.3 g of paeoniflorin; the weight was 23.6 g of paeoniflorin, and the rest was the same as in Example 1.
  • the osmotic extraction step of the medicinal material except that the ratio of the volume of the purified water of the osmotic extract to the weight of the medicinal material is 15:1, the flow rate of the osmotic solution is 3 L/h, and the rest is the same as in the first embodiment;
  • the macroporous resin column coarse separation step except for the D-201 type macroporous adsorption resin, the ratio of the resin volume to the weight of the medicine (dry weight) is 5:3, and the mass percentage concentration of the eluent ethanol is 70%, the eluent The amount of ethanol used was 12 L, and the elution flow rate was 4 L/h; the macroporous resin column differentiation mixture obtained by crude separation was 243 g, and the rest was the same as in Example 1;
  • the alumina column chromatography step except that the weight ratio of the adsorbent alumina to the macroporous resin column separation mixture is 25:1; the amount of ethyl acetate is 168 L, and the elution flow rate is 8 L/h;
  • the analysis mixture was 124 g, and the rest was the same as in Example 1;
  • the silica gel column chromatography step except that the weight ratio of the adsorbent silica gel to the alumina column chromatography mixture is 30: 1, the eluent ethyl acetate is used in an amount of 120 L, and the elution flow rate is 6 L/h, and the weight is 84.3 g. Paeoniflorin; the same as Example 1 except that the weight was 27.9 g of paeoniflorin.
  • a vacuum decompression concentration tank (produced by Wuxi Huaxin Pharmaceutical Equipment Co., Ltd.) is used to concentrate under reduced pressure, ethanol is removed, and the mixture is concentrated to a non-alcoholic flavor to prepare a concentrated drug of peony, which is concentrated under reduced pressure.
  • the temperature is 70 ° C, the relative vacuum is -0.09 ⁇ -0.075MPa ;
  • the extraction solvent for the heating and reflux extraction treatment of the medicinal material is not limited to an ethanol solution having a mass percentage concentration of 50%, and other ethanol solutions having a mass percentage concentration ranging from 30 to 100% are suitable for use in the present invention.
  • large pore resin column coarse separation 1 The dilute extract of paeoniflorin is separated by a macroporous adsorption resin column, wherein the macroporous adsorption resin selects D-201 macroporous adsorption resin, and the macroporous adsorption resin column D-201 macroporous adsorption resin
  • the column volume is 1.5L (the diameter of the column is 5.5cm and the height is 1200cm).
  • the ratio of the volume of the resin to the weight of the medicinal material (dry weight) is 1.5: 1, that is, the medicinal material (dry weight) is 1 kg, and the volume of the macroporous resin is 1.5.
  • the weight ratio of the adsorbent alumina to the macroporous resin column separation mixture is 8:1; the diameter of the alumina chromatography column is 5.5 cm, and the ratio of the column diameter of the alumina column to the alumina in the column is 1:10; acetic acid
  • the amount of the ethyl ester was 48 L; the elution flow rate was 4 L/h; the obtained alumina column chromatography mixture was 41 g, and the rest was the same as in Example 1.
  • the weight ratio of the adsorbent silica gel to the alumina column chromatography mixture is 12: 1, the diameter of the column is 7.6 cm, and the ratio of the column diameter of the silica column to the height of the silica gel in the column is 1:10; eluent acetate B The ester was used in an amount of 36 L, the elution flow rate was 3 L/h, and the weight was 26.13 g of paeoniflorin; the weight was 9.12 g of paeoniflorin, and the rest was the same as in Example 1.
  • the hot reflux extraction step of the medicinal material except that the extraction solvent is selected from a 70% by mass ethanol solution, and the hot reflux is extracted twice, the amount of the solvent extracted twice is 12L, 10L, and the extraction time is 4h, 3h, decompression
  • the ethanol was recovered to an alcohol-free taste, and diluted with water to obtain a total volume of 8 L of the dilute extract of paeoniflorin, and the rest was the same as in Example 4;
  • the macroporous resin column coarse separation step except for the AB-8 type macroporous adsorption resin, the ratio of the resin volume to the weight of the medicine (dry weight) is 2: 1, and the dilute extract of the paeoniflorin completely flows into the resin column at a flow rate of 2 L/h.
  • the macroporous resin column differentiation mixture obtained by the crude separation is The rest is the same as in Embodiment 1 except for 83g;
  • the alumina column chromatography step except that the weight ratio of the adsorbent alumina to the macroporous resin column separation mixture is 20:1; the diameter of the alumina chromatography column is 7.6 cm, and the column diameter of the alumina column is higher than the alumina in the column.
  • the ratio is 1:10; the amount of ethyl acetate is 66 L; the elution flow rate is 4 L/h; the obtained alumina column chromatography mixture is 37.2 g, and the rest is the same as in Example 1;
  • the silica gel column chromatography step except that the weight ratio of the adsorbent silica gel to the alumina column chromatography mixture is 28:1, the diameter of the silica gel column is 10 cm, and 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 eluent ethyl acetate was used in an amount of 86 L, the elution flow rate was 3 L/h, and the weight was 25.12 g of paeoniflorin; the weight was 8.38 g of paeoniflorin, and the rest was the same as in Example 1.
  • paeoniflorin The content of paeoniflorin was 98.5% by HPLC, see Figure 6; the content of paeoniflorin was 94.1%, see Figure 7.

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Description

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 Paeow'a lactiflora Pall. 的干燥根; 一种是赤芍 (Radix Paeoniae Rubra) , 为毛茛科植物川赤芍 Paeonia veitchii Lynch 的干燥根。 目前市场上己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 (paeonif lorin) , 芍药内酯苷 (albif lorin)、 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。
芍药内酯苷(Albiflorin)为单萜类化合物,其分子式为 C23H28OU,分子量为 480.46, 分子结构如式( I )所示,芍药苷(Paeoniflorin)为单萜类化合物,其分子式为 C H OU ,
Figure imgf000002_0001
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, 止眩暈, 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 lactifora Pal l)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 方法包括如下顺序进行的步骤: a) 采用渗漉提取法对芍药药材进行渗漉提取, 得到芍 药渗漉液; b) 将芍药渗漉液依次进行大孔树脂柱分离、 氧化铝柱层析和硅胶柱层析。
其中, 步骤 a) 中采用水、 乙醇或甲醇为渗漉提取溶液进行所述的渗漉提取, 优选 为水。
特别是, 所述渗漉提取溶液的体积与芍药药材的重量之比为 5-20: 1, 即当芍药药 材重量 (干重) 为 1kg时, 渗漉提取溶液的体积为 5-20L, 当芍药药材重量 (干重) 为 lg时, 渗漉提取溶液的体积为 5-20ml。
尤其是, 渗漉提取过程中每小时渗漉液的流量与芍药药材的重量之比为 0.5-8: 1, 优选为 1-5: 1, 即当芍药药材重量(干重) 为 lkg时, 渗漉提取过程中每小时渗漉液的 流量为 0.5-8L, 当芍药药材重量 (干重) 为 lg时, 渗漉液的流量为 0.5-8ml。
特别是, 还包括将芍药药材用水浸泡处理后, 在进行所述的渗漉提取, 其中浸泡芍 药药材的水的体积与芍药药材的重量之比为 2-3 : 1, 即当芍药药材重量 (干重) 为 lkg 时, 渗漉提取溶液的体积为 2-3L, 当芍药药材重量 (干重) 为 lg时, 渗漉提取溶液的 体积为 2-3ml。
其中, 步骤 b) 中所述的大孔树脂柱分离包括如下顺序进行的步骤:
1 ) 将所述芍药渗漉液加入到大孔树脂柱内;
2) 采用水为洗脱剂对大孔树脂柱进行洗脱, 除杂;
3 )采用乙醇溶液为洗脱溶剂对大孔树脂柱进行洗脱, 收集洗脱液, 合并洗脱液 中含有芍药苷和 /或芍药内酯苷的流份, 干燥后, 得到大孔树脂柱分离混合物。 特别是, 步骤 1 ) 中所述的大孔树脂选择 D101、 D-20K AB-8或 HP-20型非极性大 孔吸附树脂中的一种。
尤其是, 大孔树脂柱内所述的大孔吸附树脂的体积与芍药药材的重量之比为 0.5-5: 1, 即当芍药药材重量 (干重) 为 lkg时, 大孔吸附树脂的体积为 0.5-5L, 当芍药药材 重量 (干重) 为 lg时, 大孔吸附树脂的体积为 0.5-5ml, 优选为 1-2 : 1。
特别是, 大孔树脂柱分离过程中洗脱液流速为 0.5-5 倍柱床体积 /h, 即每小时洗脱 液的流量为大孔树脂柱内大孔吸附树脂的体积的 0.5-5倍。
特别是, 步骤 2) 中洗脱剂水与大孔树脂的体积之比为 3-5: 1; 步骤 3 ) 中所述的 乙醇溶液的质量百分比浓度为 30-70%, 优选为 40-60%; 步骤 3 ) 中所述乙醇溶液与大 孔树脂的体积之比为 3-5: 1。 其中, 步骤 b) 中所述的氧化铝柱层析包括如下顺序进行的步骤:
1 ) 将大孔树脂柱分离混合物溶解后, 拌入氧化铝, 干燥得到氧化铝层析样品;
2)将氧化铝层析样品置于氧化铝柱顶部, 以乙酸乙酯为洗脱剂, 进行氧化铝层 析, 收集洗脱液, 合并洗脱液中含有芍药苷和 /或芍药内酯苷的流份, 得到氧化铝层 析洗脱液;
3 ) 将氧化铝层析洗脱液蒸干后, 得到氧化铝层析混合物。
特别是, 步骤 1 ) 中大孔树脂柱分离混合物与氧化铝拌合的比例为 1 : 0.5-5; 步骤 2) 中所述氧化铝柱内的氧化铝与大孔树脂柱分离混合物的重量之比为 5-30: 1, 优选为 8-25: 1, 进一步优选为 10-25 : 1。
尤其是, 氧化铝柱的柱直径与柱内氧化铝的高度之比为 1 :6-10。
其中, 步骤 b) 中所述的硅胶柱层析包括如下顺序进行的步骤:
1 ) 将氧化铝层析提取物溶解后, 拌入硅胶, 干燥得到硅胶层析样品;
2)将硅胶层析样品置于硅胶柱顶部, 以乙酸乙酯为洗脱剂, 进行硅胶层析, 收 集洗脱液, 分别合并只含有芍药苷或芍药内酯苷的洗脱液流份;
3 )将只含有芍药苷或芍药内酯苷的洗脱液分别蒸干后, 得到芍药苷或芍药内酯 苷。
特别是, 步骤 1 ) 中氧化铝层析提取物与硅胶拌合的比例为 1 : 0.5-5; 步骤 2) 中 硅胶柱内的硅胶与氧化铝层析混合物的重量之比为 5-30: 1, 优选为 12-30: 1。
尤其是, 硅胶柱的柱直径与柱内硅胶的高度之比为 1 :6-10, 优选为 1 :8-10。
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:
a) 以乙醇或甲醇溶液为提取溶剂, 对芍药药材进行加热回流提取, 得到芍药提取 液;
b) 浓缩芍药提取液, 去除乙醇或甲醇后, 加水稀释得到芍药醇提稀释液; c) 将芍药醇提稀释液依次进行大孔树脂柱分离、 氧化铝柱层析和硅胶柱层析。 其中, 步骤 a) 中所述的乙醇溶液的质量百分比浓度为 30-100%; 所述的甲醇醇溶 液的质量百分比浓度为 30-100%。
特别是, 步骤 a)中所述的提取溶剂的体积与药材的重量(干重)之比为 5-12: 1 (即 如果药材的重量(干重)为 1公斤, 则提取溶剂的体积为 5-12升; 如果药材的重量(干 重) 为 1克, 则提取溶剂的体积为 5-12毫升); 提取次数为 2-3次, 提取时间为 2-4小 时 /次。
特别是, 步骤 b) 中所述芍药醇提稀释液体积与芍药药材重量 (干重) 之比为 1-8: 1, 优选为 2-8: 1, 即当药材的重量(干重)为 1公斤时, 芍药醇提稀释液的体积为 1-8L, 当药材的重量 (干重) 为 1克时, 芍药醇提稀释液的体积为 l-8ml。
特别是, 步骤 c) 中所述的大孔树脂柱分离包括如下顺序进行的步骤:
1 ) 将所述芍药醇提稀释液加入到大孔树脂柱内;
2) 采用水为洗脱剂对大孔树脂柱进行洗脱, 除杂;
3 )采用乙醇溶液为洗脱溶剂对大孔树脂柱进行洗脱, 收集洗脱液, 合并洗脱液 中含有芍药苷和 /或芍药内酯苷的流份, 干燥后, 得到大孔树脂柱分离混合物。 特别是, 步骤 c) 中所述的氧化铝柱层析包括如下顺序进行的步骤:
1 ) 将大孔树脂柱分离混合物溶解后, 拌入氧化铝, 干燥得到氧化铝层析样品;
2)将氧化铝层析样品置于氧化铝柱顶部, 以乙酸乙酯为洗脱剂, 进行氧化铝层 析, 收集洗脱液, 合并洗脱液中含有芍药苷和 /或芍药内酯苷的流份, 得到氧化铝层 析洗脱液;
3 ) 将氧化铝层析洗脱液蒸干后, 得到氧化铝层析混合物。
特别是, 步骤 c) 中所述的硅胶柱层析包括如下顺序进行的步骤:
1 ) 将氧化铝层析提取物溶解后, 拌入硅胶, 干燥得到硅胶层析样品;
2)将硅胶层析样品置于硅胶柱顶部, 以乙酸乙酯为洗脱剂, 进行硅胶层析, 收 集洗脱液, 分别合并只含有芍药苷或芍药内酯苷的洗脱液流份;
3 )将只含有芍药苷或芍药内酯苷的洗脱液分别蒸干后, 得到芍药苷或芍药内酯 苷。 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、 本发明制备方法采用水为芍药苷和芍药内酯苷的提取液, 以无毒、 无害的乙醇、 乙酸乙酯为纯化洗脱溶剂, 既节约了成本, 而且还对环境友好, 符合绿色经济、 环境保 护的发展要求。
4、 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。
5、 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为本发明芍药苷、 芍药内酯苷制备工艺流程图;
图 2为实施例 1制备所得芍药苷的 HPLC检测图谱;
图 3为实施例 1制备所得芍药内酯苷的 HPLC检测图谱;
图 4为实施例 4制备所得芍药苷的 HPLC检测图谱;
图 5为实施例 4制备所得芍药内酯苷的 HPLC检测图谱;
图 6为实施例 5制备所得芍药苷的 HPLC检测图谱;
图 7为实施例 5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
1 ) 干燥的白芍药材 3公斤, 粉碎成粗粉后, 装入不锈钢中药渗漉罐 (容积为 12L) 中, 加入 6L纯净水浸泡 2小时;
2) 加入纯净水, 对芍药粗粉进行渗漉提取, 获得芍药渗漉液, 其中, 渗漉液的流 速为 6L/h, 渗漉提取液纯净水的体积与药材白芍的重量之比为 10: 1, 即当白芍的重量
(干重) 为 1公斤时, 纯净水的体积为 10L; 白芍的重量 (干重) 为 1克时, 纯净水的 体积为 10ml。
2、 大孔树脂柱粗分离
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 4L (层析柱的 直径 8cm, 高 1.5m), 树脂体积与药材重量 (干重) 之比为 4: 3, 即如果药材 (干重) 3公斤, 大孔树脂的体积是 4L; 如果药材(干重) 30克, 则大孔树脂的体积为 40毫升, 待渗漉液以 8L/h的流速完全流入树脂柱后, 先用 12L的纯净水洗涤至流出液澄清后, 弃去水洗脱的流出液; 然后再用 12L质量百分比浓度为 50%的乙醇洗脱, 洗脱流速为 8L/h, 收集乙醇洗脱的流出液, 每 3L流出液收集为一流份;
2)采用薄层色谱法(TLC法)检测收集的大孔树脂柱乙醇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; 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, 得到大孔树脂柱分离混合物 210g, 其中, 减压浓缩的温 度为 70°C, 相对压力为 -0.09〜- 0.075MPa。
3、 氧化铝柱层析
1 ) 将大孔树脂柱分离混合物用适量甲醇全部溶解后加入 210g氧化铝粉 (100-200 目), 搅拌均匀, 干燥, 磨碎, 得到氧化铝柱层析样品;
2) 将氧化铝柱层析样品置于氧化铝柱顶部, 以乙酸乙酯为洗脱剂, 进行氧化铝柱 层析, 其中, 氧化铝柱内的吸附剂氧化铝的粒度为 100-200目, 氧化铝柱的柱直径与柱 内氧化铝高之比 1 : 10; 吸附剂氧化铝与大孔树脂柱分离混合物的重量之比为 20: 1, 洗脱剂为乙酸乙酯, 用量 100L, 每 1L洗脱液收集一流份;
3 ) 采用薄层色谱法 (TLC法) 检测氧化铝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、 芍药内酯苷的流份合并, 制得氧化铝层析洗脱液, 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) 将氧化铝层析洗脱液蒸干, 得到氧化铝层析混合物 121g。
4、 硅胶柱层析
1 )将氧化铝层析混合物用质量百分比浓度为 95%的乙醇溶解后加入 120g层析用硅 胶粉 (200-300目), 搅拌均匀, 干燥, 磨碎, 得到硅胶层析样品;
2) 将硅胶层析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为 1: 8; 吸附剂硅胶与氧化铝柱层析混合物的重量之比为 24: 1, 流速为 10L/h。 洗脱剂 乙酸乙酯的用量为 96L, 每 1L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, 将只含有芍药苷单一成分的洗脱流份合并为第一部分 (即芍药苷部分); 将只含 有芍药内酯苷单一成分的洗脱流份合并为第二部分 (即芍药内酯苷部分), 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,重量为 83.9g芍药苷;重量为 25.3g的芍药内酯苷。 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: Hyper ODS2 C18; 流动相: 乙腈: 0.1%磷酸溶液 ( 14: 86); 检测波长: 230nm; 流速: 1.0ml/min。
经 HPLC检测芍药苷含量为 96.7%, 见附图 2; 芍药内酯苷的含量为 98.6%, 见附 图 3。 实施例 2
药材的渗漉提取步骤,除了渗漉提取液纯净水的体积与药材白芍的重量之比为 5: 1, 渗漉液的流速为 4L/h之外, 其余与实施例 1相同;
大孔树脂柱粗分离步骤,除了选用 HP-20型大孔吸附树脂,树脂体积与药材重量(干 重)之比为 1 : 1, 洗脱液乙醇的质量百分比浓度为 30%, 洗脱液乙醇的用量为 20L; 粗 分离得到的大孔树脂柱分化混合物为 189g之外, 其余与实施例 1相同;
氧化铝柱层析步骤, 除了吸附剂氧化铝与大孔树脂柱分离混合物的重量之比为 10: 1 ; 氧化铝柱的柱直径与柱内氧化铝高之比 1 : 6; 乙酸乙酯的用量为 53L; 洗脱流速为 8L/h; 得到的氧化铝柱层析混合物为 105g之外, 其余与实施例 1相同;
硅胶柱层析步骤, 除了吸附剂硅胶与氧化铝柱层析混合物的重量之比为 15: 1, 洗 脱剂乙酸乙酯的用量为 62L,洗脱流速为 6L/h;硅胶柱的柱直径与柱内硅胶高之比为 1 : 10; 得到重量为 74.3g的芍药苷; 重量为 23.6g的芍药内酯苷之外, 其余与实施例 1相 同。 实施例 3
药材的渗漉提取步骤, 除了渗漉提取液纯净水的体积与药材白芍的重量之比为 15: 1, 渗漉液的流速为 3L/h之外, 其余与实施例 1相同;
大孔树脂柱粗分离步骤,除了选用 D-201型大孔吸附树脂,树脂体积与药材重量(干 重)之比为 5: 3, 洗脱液乙醇的质量百分比浓度为 70%, 洗脱液乙醇的用量为 12L, 洗 脱流速为 4L/h; 粗分离得到的大孔树脂柱分化混合物为 243g之外, 其余与实施例 1相 同;
氧化铝柱层析步骤, 除了吸附剂氧化铝与大孔树脂柱分离混合物的重量之比为 25: 1 ; 乙酸乙酯的用量为 168L, 洗脱流速为 8L/h; 得到的氧化铝柱层析混合物为 124g之 外, 其余与实施例 1相同;
硅胶柱层析步骤, 除了吸附剂硅胶与氧化铝柱层析混合物的重量之比为 30: 1, 洗 脱剂乙酸乙酯的用量为 120L, 洗脱流速为 6L/h, 得到重量为 84.3g的芍药苷; 重量为 27.9g的芍药内酯苷之外, 其余与实施例 1相同。
经 HPLC检测芍药苷含量为 97.5%; 芍药内酯苷的含量为 96.7%。 实施例 4
1、 药材的提取
1 ) 干燥的白芍药材 1公斤, 粉碎成粗粉后, 装入 10L圆底烧瓶中, 加热进行乙醇 热回流提取, 共提起三次, 其中提取溶剂乙醇的质量百分比浓度 50%, 三次提取溶剂乙 醇的用量为 5L, 5L, 5L, 三次提取时间为 3h, 3h, 2h;
2) 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,去除乙醇,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;
3 ) 向芍药浓缩液中加入纯净水至总体积为 2L, 制成芍药醇提稀释液, 备用, 其中, 芍药醇提稀释液的体积与芍药药材的重量之比为 2: 1。
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 30-100%的乙醇溶液均适用于本发明。
2、 大孔树脂柱粗分离 1 )将芍药醇提稀释液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 D-201型大孔吸附树脂,大孔吸附树脂柱内的 D-201型大孔吸附树脂的柱体积为 1.5L (层 析柱的直径 5.5cm, 高 1200cm), 树脂体积与药材重量 (干重) 之比为 1.5: 1, 即药材 (干重) 1公斤, 大孔树脂的体积是 1.5L, 待芍药醇提稀释液以 lL/h的流速完全流入树 脂柱后, 先用 5L的纯净水洗涤至流出液清澈透明后, 弃去水流出液, 再用 5L质量百分 比浓度为 50%的乙醇洗脱, 洗脱流速为 1.5L/h, 收集乙醇洗脱液;
2 )将含芍药苷和芍药内酯苷组分的树脂柱乙醇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, 得到大孔树脂柱分离混合物 71g, 其中, 减压浓缩的 温度为 70。C, 相对压力为 -0.09〜 - 0.075MPa。
3、 氧化铝柱层析
除了吸附剂氧化铝与大孔树脂柱分离混合物的重量之比为 8: 1; 氧化铝层析柱的直 径 5.5cm, 氧化铝柱的柱直径与柱内氧化铝高之比 1 : 10; 乙酸乙酯的用量为 48L; 洗 脱流速为 4L/h; 得到的氧化铝柱层析混合物为 41g之外, 其余与实施例 1相同。
4、 硅胶柱层析
除了吸附剂硅胶与氧化铝柱层析混合物的重量之比为 12: 1, 层析柱的直径 7.6cm, 硅胶柱的柱直径与柱内硅胶高之比为 1 : 10; 洗脱剂乙酸乙酯的用量为 36L, 洗脱流速 为 3L/h, 得到重量为 26.13g的芍药苷; 重量为 9.12g的芍药内酯苷之外, 其余与实施例 1相同。
经 HPLC检测芍药苷含量为 94.05%, 见附图 4; 芍药内酯苷的含量为 92.25%, 见 附图 5。 实施例 5
药材的热回流提取步骤, 除了提取溶剂选用质量百分比浓度为 70%的乙醇溶液, 热 回流提取 2次, 2次提取溶剂乙醇的用量为 12L, 10L, 2次提取时间为 4h, 3h, 减压回 收乙醇至无醇味, 加水稀释制得总体积为 8L的芍药醇提稀释液之外, 其余与实施例 4 相同;
大孔树脂柱粗分离步骤, 除了选用 AB-8型大孔吸附树脂,树脂体积与药材重量(干 重) 之比为 2: 1, 芍药醇提稀释液以 2L/h的流速完全流入树脂柱, 纯净水洗脱时, 水 的用量为 8L; 乙醇洗脱时乙醇溶液的质量百分比浓度为 70%, 用量为 6L, 洗脱流速为 2L/h; 粗分离得到的大孔树脂柱分化混合物为 83g之外, 其余与实施例 1相同; 氧化铝柱层析步骤, 除了吸附剂氧化铝与大孔树脂柱分离混合物的重量之比为 20: 1 ; 氧化铝层析柱的直径 7.6cm, 氧化铝柱的柱直径与柱内氧化铝高之比 1 : 10; 乙酸乙 酯的用量为 66L; 洗脱流速为 4L/h; 得到的氧化铝柱层析混合物为 37.2g之外, 其余与 实施例 1相同;
硅胶柱层析步骤, 除了吸附剂硅胶与氧化铝柱层析混合物的重量之比为 28: 1, 硅 胶层析柱的直径 10cm, 硅胶柱的柱直径与柱内硅胶高之比为 1 : 10; 洗脱剂乙酸乙酯的 用量为 86L, 洗脱流速为 3L/h, 得到重量为 25.12g的芍药苷; 重量为 8.38g的芍药内酯 苷之外, 其余与实施例 1相同。
经 HPLC检测芍药苷含量为 98.5%, 见附图 6; 芍药内酯苷的含量为 94.1%, 见附 图 7。

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: a)采用渗 漉提取法对芍药药材进行渗漉提取, 得到芍药提取液; b) 将芍药提取液依次进 行大孔树脂柱分离、 氧化铝柱层析和硅胶柱层析。
2、 如权利要求 1所述的制备方法, 其特征是步骤 a) 中采用水、 乙醇或甲醇为 渗漉提取溶液进行所述的渗漉提取。
3、 如权利要求 2所述的制备方法, 其特征是渗漉提取溶液的体积与芍药药材的 重量之比为 5-15: 1。
4、 如权利要求 1或 2所述的制备方法, 其特征是步骤 b) 中所述的大孔树脂柱 分离包括如下顺序进行的步骤:
1 ) 将所述芍药提取液加入到大孔树脂柱内;
2) 采用水为洗脱剂对大孔树脂柱进行洗脱, 除杂;
3 ) 采用乙醇溶液为洗脱溶剂对大孔树脂柱进行洗脱, 收集洗脱液, 合并洗 脱液中含有芍药苷和 /或芍药内酯苷的流份, 干燥后, 得到大孔树脂柱分离混合 物。
5、如权利要求 4所述的制备方法,其特征是步骤 1 )中所述的大孔树脂选择 D101、 D-20K AB-8或 HP-20型非极性大孔吸附树脂中的一种。
6、 如权利要求 4或 5所述的制备方法, 其特征是大孔树脂柱内所述的大孔吸附 树脂的体积与芍药药材的重量之比为 0.5-5: 1。
7、 如权利要求 4或 5所述的制备方法, 其特征是步骤 3 ) 中所述的乙醇溶液的
8、 如权利要求 1或 2所述的制备方法, 其特征是步骤 b) 中所述的氧化铝柱层 析包括如下顺序进行的步骤:
1 ) 将经所述大孔树脂柱分离的混合物溶解后, 拌入氧化铝, 干燥得到氧化 铝层析样品;
2 ) 将氧化铝层析样品置于氧化铝柱顶部, 以乙酸乙酯为洗脱剂, 进行氧化 铝层析, 收集洗脱液, 合并洗脱液中含有芍药苷和 /或芍药内酯苷的流份, 得到 氧化铝层析洗脱液;
3 ) 将氧化铝层析洗脱液蒸干后, 得到氧化铝层析混合物。
9、 如权利要求 8所述的制备方法, 其特征是步骤 2) 中氧化铝柱内的氧化铝与 大孔树脂柱分离混合物的重量之比为 5-30: 1。
10、 如权利要求 1或 2所述的制备方法, 其特征是步骤 b) 中所述的硅胶柱层析 包括如下顺序进行的步骤:
1 ) 将经所述氧化铝层析的提取物溶解后, 拌入硅胶, 干燥得到硅胶层析样
P
ρπ ;
2)将硅胶层析样品置于硅胶柱顶部, 以乙酸乙酯为洗脱剂, 进行硅胶层析, 收集洗脱液, 分别合并只含有芍药苷或芍药内酯苷的洗脱液流份;
3 ) 将只含有芍药苷或芍药内酯苷的洗脱液分别蒸干后, 得到芍药苷或芍药 内酯苷。
11、 如权利要求 10所述的制备方法, 其特征是步骤 2) 中硅胶柱内的硅胶与氧 化铝层析混合物的重量之比为 5-30: 1。
12、 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: a) 以乙 醇或甲醇为提取溶剂, 对芍药药材进行加热回流提取, 得到芍药提取液; b) 浓 缩芍药提取液去除乙醇或甲醇后, 加水稀释得到芍药醇提稀释液; c) 将芍药醇 提稀释液依次进行大孔树脂柱分离、 氧化铝柱层析和硅胶柱层析。
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