WO2021134407A1 - Method for extracting crude product of glycyrrhizic acid and glycyrrhizic flavone from licorice and purifying to obtain glycyrrhizinate - Google Patents
Method for extracting crude product of glycyrrhizic acid and glycyrrhizic flavone from licorice and purifying to obtain glycyrrhizinate Download PDFInfo
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- WO2021134407A1 WO2021134407A1 PCT/CN2019/130346 CN2019130346W WO2021134407A1 WO 2021134407 A1 WO2021134407 A1 WO 2021134407A1 CN 2019130346 W CN2019130346 W CN 2019130346W WO 2021134407 A1 WO2021134407 A1 WO 2021134407A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
- A61K36/484—Glycyrrhiza (licorice)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
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- C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
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Definitions
- the present invention relates to the technical field of extraction and purification of medicinal materials, in particular to a method for extracting crude glycyrrhizic acid and licorice flavonoids from licorice, and purifying and obtaining glycyrrhizinate.
- Licorice is the dried roots and rhizomes of Glycyrrhiza (Glycyrrhizauralensis Fisch.), G. inflata Bat. and G. glabra L. in the leguminous family Glycyrrhiza (Glycyrrhizauralensis Fisch.) Fields such as over-the-counter medicines and licorice extracts.
- Licorice contains saponins, triterpenes, flavonoids, coumarins, sterols, alkaloids, volatile oils, organic acids, amino acids and other ingredients. Glycyrrhizic acid has anti-allergic, anti-tumor, anti-ulcer, anti-inflammatory and other effects.
- Licorice flavonoids have significant anti-ulcer, anti-tumor, anti-oxidation, antibacterial, anti-inflammatory and other pharmacological activities.
- the extraction methods of glycyrrhizic acid mainly include water extraction, dilute ammonia extraction, ammonia alcohol extraction, and microwave-assisted extraction. Due to the low solvent cost, safe application, and high extraction rate of effective ingredients, the dilute ammonia extraction method is the main extraction method of glycyrrhizic acid in the current industrial production.
- the use of ammonia in the extraction process of this process leads to a large amount of ammonia in the extracted drug residue, unpleasant smell during storage of the drug residue, and destruction of nutrients such as starch in the medicinal materials. The subsequent application of the drug residue is affected.
- the process adopts alkali-soluble acid precipitation process to obtain glycyrrhizic acid powder.
- the strong acid solution has about 20% damage to glycyrrhizic acid, and the large amount of acid water produced by the process brings great pressure to environmental protection. .
- Licorice is rich in flavonoids.
- licorice active ingredients only glycyrrhizic acid in licorice has been extracted as the main effective ingredient in industrial production, and licorice residues rich in flavonoids are discarded as waste. To go, resulting in a great waste of resources.
- people Based on the comprehensive utilization of licorice resources, people have also developed the extraction process of flavonoids from licorice dregs, but with little success.
- the current status of the licorice extract industry is as follows: 1) The use of ammonia in the extraction process of glycyrrhizic acid results in a large amount of ammonia in the extracted drug residue, unpleasant smell during storage of the drug residue, and the destruction of nutritional components such as starch in the medicine. The follow-up application of glycyrrhizic acid is greatly restricted; 2) The preparation of glycyrrhizic acid adopts the process of alkali-soluble acid precipitation to obtain glycyrrhizic acid powder.
- the strong acid solution has about 20% damage to glycyrrhizic acid, and the large amount of acid water produced by the process is environmentally friendly Cost brings great pressure; 3)
- the content of water-soluble glycyrrhizin is very small, and on the other hand, the further extraction of flavonoids is affected by the ammonia components of the residues of glycyrrhizic acid, and the production cost is high and limited.
- the application of licorice flavonoids This situation directly led to the active or passive shutdown of domestic glycyrrhizic acid powder manufacturers, the recycling of glycyrrhizinate residues was blocked, and the cost of licorice flavonoids remained high.
- the purpose of the present invention is to provide a method for extracting crude glycyrrhizic acid from licorice, which realizes the efficient extraction of glycyrrhizic acid components and avoids the destruction of glycyrrhizic acid; the organic solvents used can be efficiently recovered by modern means, avoiding the traditional Wastewater treatment and environmental protection issues in the extraction process of glycyrrhizic acid, and the starch, protein and other nutrients in the glycyrrhizinate residue are well preserved, so as to realize the efficient use of licorice resources.
- the present invention provides a method for extracting crude glycyrrhizic acid from licorice, which includes the following steps: pulverizing licorice: pulverizing the licorice material into glycyrrhiza powder, and controlling the particle size of the licorice powder to be 30-200 mesh; and Primary extraction:
- the first solvent is composed of organic solvent and water, and then mixed with the above-mentioned licorice powder at a ratio of (2-20):1 (volume/weight, V/W), stirred and added the first pH regulator, Adjust the pH value, stir and extract for 0.5-3 hours, filter, and collect the first-level extract and the dregs; the first-level extract is the crude glycyrrhizic acid.
- the particle size of the licorice powder is preferably 30-100 mesh.
- the present invention also provides a method for extracting crude glycyrrhizic acid from licorice, which includes the following steps: licorice pulverization: pulverize the licorice material into glycyrrhiza powder, and control the particle size of the glycyrrhiza powder to 30-200 mesh; first-level extraction: The organic solvent and water constitute the first solvent, which is then mixed with the licorice powder in a ratio of (2-20):1 (V/W), stirred and added the first pH adjuster, adjusted the pH value, stirred and extracted 0.5- Filter for 3 hours to collect the first-level extract and the first-level drug residue; and the K-level extraction: the first solvent and the drug residue obtained from the previous stage are in a ratio of (2-20):1(V/W) Mix, stir and add the first pH adjuster, adjust the pH value, stir and extract for 0.5-3 hours, filter, collect the K-level extract and K-level dregs; and perform the K-
- the first solvent in each round of extraction, also includes all levels of extraction liquid in the previous round/batch extraction except for the first-level extraction liquid. This action can make the solution
- the repeated recycling improves the utilization rate of the solvent, and at the same time avoids the waste water treatment and environmental protection problems in the traditional extraction process of glycyrrhizic acid and licorice flavonoids.
- the organic solvent is selected from at least one of alcohols, alcohol derivatives, and ketones.
- the organic solvent is selected from at least one of methanol, ethanol, ethyl acetate and acetone.
- the volume percentage of the water in the first solvent is 0-30%.
- the first solvent is selected from one of methanol-ethanol and ethanol-ethyl acetate.
- the ratio of the first solvent to the glycerin powder is preferably (4-9):1 (volume/weight, V/W).
- the first pH adjusting agent is selected from at least one of the following substances: H 2 SO 4 , HCl, H 3 PO 4 , polyphosphoric acid, CH 3 COOH, citric acid, oxalic acid, cation exchange resin, a buffer system with pH ⁇ 2, and solutions of different concentrations prepared from at least one of the above substances; preferably, the The first pH adjusting agent is H 2 SO 4 -HCl.
- the buffer system with pH ⁇ 2 is preferably a glycine-hydrochloric acid buffer system, and more preferably a 0.1-0.3M glycine-hydrochloric acid buffer system.
- the pH value is adjusted to be ⁇ 4.
- the pH value is adjusted to between 0.5-4.
- the time for stirring and extraction after adding the first pH regulator is preferably 0.3-1 hour.
- the present invention also provides a method for extracting licorice flavonoids from licorice, which includes all the steps of extracting crude glycyrrhizic acid from licorice and subsequent concentration steps, the concentration steps are as follows:
- Concentration Adjust the pH of the first-level extract to between 5-10, stir, generate precipitation, filter, collect the filtrate, concentrate the filtrate, and dry to obtain licorice flavonoids.
- the extraction rate of licorice flavonoids is not less than 95%.
- a second pH adjusting agent is added to the primary extract to adjust the pH to between 5-10.
- the second pH adjusting agent is selected from at least one of the following substances: metals, metal oxides, metal hydroxides, salts formed by metals, anion exchange resins, ammonia, liquids Ammonia, ammonia, ammonium salt, triethylamine, a buffer system with a pH buffer range of 9-13, and solutions of various concentrations prepared from at least one of the above substances;
- the metals are sodium, potassium, and calcium metals .
- the metal oxides are oxides of sodium, potassium, calcium, magnesium, and zinc
- the salts formed by the metals are various acid salts formed by sodium, potassium, calcium, magnesium, and zinc.
- the hydroxide is a hydroxide of sodium, potassium, calcium, magnesium, and zinc.
- the buffer system with a pH buffer range of 9-13 is preferably one of ammonia-ammonium chloride and boric acid-potassium chloride, and is more preferably an ammonia-ammonium chloride solution.
- the present invention also provides a method for obtaining glycyrrhizinate by purification from licorice, which includes all the steps of extracting crude glycyrrhizic acid from licorice and the subsequent purification steps, the purification steps are as follows:
- Purification Add a second pH regulator to the first-level extract, control the pH of the system, filter, collect the precipitate, then wash the precipitate with a lotion, filter, and collect the filtered lotion and precipitate respectively.
- the washed precipitate is dried at 50-80°C to obtain glycyrrhizinate, and the total yield of glycyrrhizinate obtained by the purification method is not less than 90%.
- the first solvent is selected from one of methanol-ethanol-water and ethanol-ethyl acetate-water.
- the first solvent in each round of extraction, also includes the extraction liquids of all levels in the previous round/batch extraction except for the first-level extraction liquid and after filtering in the purification step This action can make the solution reused, thus avoiding the waste water treatment and environmental protection problems in the traditional glycyrrhizic acid extraction process.
- the second pH adjusting agent is selected from at least one of the following substances: metals, metal oxides, metal hydroxides, salts formed by metals, anion exchange Resin, ammonia gas, liquid ammonia, ammonia water, ammonium salt, triethylamine, a buffer system with a pH buffer range of 9-13, and at least one of the above substances are formulated into solutions of various concentrations;
- the metal is a metal Sodium, potassium, calcium, magnesium, zinc;
- the metal oxides are oxides of sodium, potassium, calcium, magnesium, and zinc;
- the metal salts are various acid salts formed by sodium, potassium, calcium, magnesium, and zinc;
- the metal hydroxide is a hydroxide of sodium, potassium, calcium, magnesium, and zinc.
- the corresponding alkali solution or buffer solution prepared by the alkali solution and the corresponding salt is selected according to the type of glycyrrhizinate that is ultimately required.
- the pH value is controlled between 5-10.
- the washing liquid in the purification step, is the same as the first solvent.
- the washing liquid is selected from methanol-ethanol-water and ethanol-ethyl acetate-waterkind of.
- the lotion in the purification step, is washed at room temperature or under heating; preferably, the heating temperature is 40-80°C, and the number of washings is 40-80°C. For 2-3 times.
- the washed precipitate in the purification step, is preferably dried at 50-70°C for later use.
- the present invention has the following beneficial effects:
- the method of extracting crude glycyrrhizic acid and glycyrrhizin flavonoids from licorice and obtaining glycyrrhizinate by purification involved in the present invention uses an organic solvent plus a pH regulator to extract glycyrrhizic acid and glycyrrhizin flavonoids, and at the same time precipitate the glycyrrhizic acid by salt precipitation, The glycyrrhizic acid is further purified, and the filtrate produced during the purification process and the subsequent grade glycyrrhizic acid extract can continue to be used in the next round of extraction of glycyrrhizic acid.
- this method improves the extraction rate of glycyrrhizic acid and avoids the loss and damage of the traditional alkaline solution and acid precipitation step.
- the extraction solvent used in the extraction process is recycled to extract medicinal materials, which improves the utilization rate of organic solvents.
- the traditional process of extracting licorice and the secondary extraction of flavonoids from licorice residues is avoided, and the extraction solvent used in the extraction process is used to extract the medicinal materials, which improves the solvent utilization rate and controls the extraction cost of the medicinal materials.
- the organic solvent used in this method can be efficiently recovered by modern means, avoiding the waste water treatment and environmental protection problems in the traditional extraction process of glycyrrhizic acid and licorice flavonoids, and well preserves the starch and protein in the licorice residue Nutrient ingredients, realize the efficient utilization of licorice resources.
- Fig. 1 is a process flow diagram of a method for extracting crude glycyrrhizic acid and glycyrrhizin flavonoids from licorice and purifying to obtain glycyrrhizinate according to an embodiment of the present invention.
- the first-level extract is used for the separation and purification of glycyrrhizic acid
- the filtrate is solvent recovery after the first-level extract glycyrrhizic acid is purified
- the rest of the extracts are used for the extraction of a new batch of glycyrrhizin materials.
- the first-level extract is used for the separation and purification of glycyrrhizic acid
- the filtrate is solvent recovery after the first-level extract glycyrrhizic acid is purified
- the rest of the extracts are used for the extraction of a new batch of glycyrrhizin materials.
- the first-level extract is used for the separation and purification of glycyrrhizic acid
- the filtrate is solvent recovery after the first-level extract glycyrrhizic acid is purified
- the rest of the extracts are used for the extraction of a new batch of glycyrrhizin materials.
- the first-level extract is used for the separation and purification of glycyrrhizic acid
- the filtrate is solvent recovery after the first-level extract glycyrrhizic acid is purified
- the rest of the extracts are used for the extraction of a new batch of glycyrrhizin materials.
- the present invention further performs statistics on the extraction rate of glycyrrhizic acid in the foregoing Examples 1-4.
- Primary extraction solution accurately pipet 2ml to a 100mL volumetric flask, accurately add 5mL of internal standard solution, and dilute to the mark with dilute ethanol;
- Second to sixth grade extracts accurately pipet 5mL to 100mL volumetric flasks, precisely add 5mL of internal standard solution, and dilute to the mark with dilute ethanol;
- the extraction of glycyrrhizic acid is basically complete, the content of glycyrrhizic acid in the medicine residue is less than 1%, and the nutritional components such as starch and fat in the medicine residue are well protected.
- the follow-up application of glycerin residues provides a better foundation.
- Primary extraction solution accurately pipet 2ml to a 100mL volumetric flask, accurately add 5mL of internal standard solution, and dilute to the mark with dilute ethanol;
- Second to fifth grade extraction solution accurately pipet 5mL to 100mL volumetric flask respectively, accurately add 5mL internal standard solution, and dilute to the mark with dilute ethanol;
- the extraction of glycyrrhizic acid is basically complete, the content of glycyrrhizic acid in the dregs is less than 1%, and the nutritional components such as starch and fat in the dregs are well protected.
- the follow-up application of glycerin residues provides a better foundation.
- the present invention further describes the method for extracting licorice flavonoids from licorice through a number of embodiments.
- the extraction rate of flavonoids in the licorice material is higher than 90%, and the starch, fat and other components in the dregs are protected, which provides a good foundation for the subsequent application of licorice dregs.
- the present invention further performs statistics on the extraction rate of licorice flavonoids in the foregoing Examples 5-8.
- Reagents standard rutin; ethanol: 30%; ethanol: 60%.
- Aluminum nitrate 10%; sodium hydroxide: 1mol/L; sodium nitrite: 5%.
- Preparation of standard solution Weigh 20 mg of rutin standard (accurate to 0.1 mg), dissolve it in a 100 mL volumetric flask with 60% ethanol, and dilute to the mark. Take 20mL of this sample solution in a 50mL volumetric flask, add 5mL of 60% ethanol solution, and dilute to the mark with water to obtain a standard solution (1mL standard solution contains about 0.08mg of rutin standard).
- sample solution Precisely pipette 1 mL of the extract into a 50 mL volumetric flask and dilute to the mark with 60% ethanol to obtain the sample solution.
- the concentration C of total flavonoids is calculated by the following formula
- m1 the weight of flavonoids in the standard product (mg);
- K The dilution factor of the sample.
- the extraction rate of flavonoids in the herbal dregs is higher than 95%, and the starch, fat and other components in the dregs are protected, which provides a good foundation for the follow-up application of the dregs of licorice.
- Reagents standard rutin; ethanol: 30%; ethanol: 60%.
- Aluminum nitrate 10%; sodium hydroxide: 1mol/L; sodium nitrite: 5%.
- Preparation of standard solution Weigh 20 mg of rutin standard (accurate to 0.1 mg), dissolve it in a 100 mL volumetric flask with 60% ethanol, and dilute to the mark. Take 20mL of this sample solution in a 50mL volumetric flask, add 5mL of 60% ethanol solution, and dilute to the mark with water to obtain a standard solution (1mL standard solution contains about 0.08mg of rutin standard).
- sample solution Precisely pipette several milliliters of the extract into a 50 mL volumetric flask, and dilute to the mark with 60% ethanol to obtain the sample solution.
- the concentration C of total flavonoids is calculated by the following formula
- m1 the weight of flavonoids in the standard product (mg);
- K The dilution factor of the sample.
- Example 7 The concentration and extraction rate of glycyrrhiza flavonoids in the extracts at various levels in Example 7 and Example 8 were calculated, and the data are shown in Table 4.
- the extraction rate of the flavonoids in the licorice material is higher than 95%, and the starch, fat and other components in the dregs are protected, which provides a good foundation for the follow-up application of the licorice dregs.
- the present invention further performs statistics on the extraction rate of glycyrrhizic acid in the foregoing Examples 9-12.
- Primary extraction solution accurately pipet 2ml to a 100mL volumetric flask, accurately add 5mL of internal standard solution, and dilute to the mark with dilute ethanol;
- Second to sixth grade extracts accurately pipet 5mL to 100mL volumetric flasks, precisely add 5mL of internal standard solution, and dilute to the mark with dilute ethanol;
- Ammonium glycyrrhizinate Take 25mg of glycyrrhizic acid intermediate, accurately weigh it, and transfer it to a 100ml volumetric flask, accurately add 5mL of internal standard solution, and dilute to the mark with dilute ethanol.
- Example 9 The concentration and extraction rate of glycyrrhizic acid in the extracts at various levels in Example 9 and Example 10 were calculated, and the data are shown in Table 5.
- the glycyrrhizic acid in the glycyrrhizic acid is almost completely extracted after the above-mentioned method is used to extract 6 levels.
- the primary extraction liquid is used to precipitate the ammonium glycyrrhizinate, and the yield of the glycyrrhizinate relative to the transfer rate of the primary extraction liquid is more than 94%, which is higher than the traditional extraction process of glycyrrhizic acid.
- the concentration of the first-level extract is gradually increased, so that the yield of glycyrrhizinate relative to medicinal materials obtained in each round of extraction of medicinal materials is increased, and finally each is achieved.
- the total yield of ammonium glycyrrhizinate in round extraction is over 90%.
- Primary extraction solution accurately pipet 2ml to a 100mL volumetric flask, accurately add 5mL of internal standard solution, and dilute to the mark with dilute ethanol;
- Second to fifth grade extraction solution accurately pipet 5mL to 100mL volumetric flask respectively, accurately add 5mL internal standard solution, and dilute to the mark with dilute ethanol;
- Glycyrrhizinate potassium salt Take 25mg of glycyrrhizic acid intermediate, accurately weigh it, transfer to a 100ml volumetric flask, accurately add 5mL internal standard solution, and dilute to the mark with dilute ethanol.
- the glycyrrhizic acid extraction is almost complete after the glycyrrhizic acid is extracted by the above-mentioned method for 6 levels.
- the primary extraction liquid is used to precipitate the ammonium glycyrrhizinate, and the yield of potassium glycyrrhizinate relative to the transfer rate of the primary extraction liquid is more than 94%, which is higher than the traditional extraction process of glycyrrhizic acid.
- the concentration of the first-level extracting solution increases step by step, so that the yield of potassium glycyrrhizinate relative to the medicinal materials obtained in each round of medicinal materials extraction is improved, and finally realized
- the total yield of potassium glycyrrhizinate in each round of extraction is more than 90%.
- the present invention further carried out specific verification tests on the test conditions, which are detailed as follows.
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Abstract
A method for extracting a crude product of glycyrrhizic acid and glycyrrhizic flavone from licorice and purifying to obtain glycyrrhizinate. In the method, a medicinal material of licorice and an organic solvent are mixed at a certain ratio, a pH regulator is added to the mixing system to control the pH value of the system at ≤4, and glycyrrhizic acid in the medicinal material is extracted in multiple stages; a first stage extraction liquid is used for the further purification of glycyrrhizic acid and for the concentration and enrichment of a glycyrrhizic flavone component, and a subsequent second stage glycyrrhizic acid extraction liquid can be used continuously in the next round/batch of extracting the medicinal material of licorice. The method for extracting a crude product of glycyrrhizic acid achieves the high-efficiency extraction of glycyrrhizic acid and glycyrrhizic flavone components.
Description
本发明关于药材提取及纯化技术领域，特别是关于一种从甘草中提取甘草酸粗品、甘草黄酮、及提纯获得甘草酸盐的方法。The present invention relates to the technical field of extraction and purification of medicinal materials, in particular to a method for extracting crude glycyrrhizic acid and licorice flavonoids from licorice, and purifying and obtaining glycyrrhizinate.
甘草为豆科甘草属甘草(GlycyrrhizauralensisFisch.)、胀果甘草(G.inflata Bat.)、光果甘草(G.glabraL.)的干燥根和根茎，为常用大宗药材，广泛应用于中药饮片、中成药和甘草提取物等领域。甘草中含有皂苷类、三萜类、黄酮类、香豆素、甾醇、生物碱、挥发油、有机酸、氨基酸等多种成分。甘草酸具有抗过敏、抗肿瘤、抗溃疡、消炎等功效，是目前甘草成分中研究得最多、最早的五环三萜皂苷类化合物。甘草黄酮类成分具有显著的抗溃疡、抗肿瘤、抗氧化、抑菌、抗炎等药理活性。Licorice is the dried roots and rhizomes of Glycyrrhiza (Glycyrrhizauralensis Fisch.), G. inflata Bat. and G. glabra L. in the leguminous family Glycyrrhiza (Glycyrrhizauralensis Fisch.) Fields such as over-the-counter medicines and licorice extracts. Licorice contains saponins, triterpenes, flavonoids, coumarins, sterols, alkaloids, volatile oils, organic acids, amino acids and other ingredients. Glycyrrhizic acid has anti-allergic, anti-tumor, anti-ulcer, anti-inflammatory and other effects. It is currently the most studied and earliest pentacyclic triterpene saponin compound among the ingredients of licorice. Licorice flavonoids have significant anti-ulcer, anti-tumor, anti-oxidation, antibacterial, anti-inflammatory and other pharmacological activities.
目前甘草酸的提取方法主要有水提法、稀氨水提取法及氨性醇提取法、微波辅助提取法等。由于溶剂成本低、应用安全、有效成分提取率高，稀氨水提取法是目前工业生产中甘草酸主要的提取方法。然而，此种工艺提取过程使用氨水，一方面导致提取完的药渣中含有大量的氨、药渣存放过程中气味难闻、药材中的淀粉等营养成分被破坏，药渣的后续应用受到了极大的限制；另一方面，工艺采用碱溶酸沉的工艺得到甘草酸粉，强酸溶液对甘草酸有约为20％的破坏作用，且工艺产生的大量酸水给环保带来极大压力。目前由于环保要求、成本提升的限制，国内甘草酸粉生产厂家大多关停。At present, the extraction methods of glycyrrhizic acid mainly include water extraction, dilute ammonia extraction, ammonia alcohol extraction, and microwave-assisted extraction. Due to the low solvent cost, safe application, and high extraction rate of effective ingredients, the dilute ammonia extraction method is the main extraction method of glycyrrhizic acid in the current industrial production. However, the use of ammonia in the extraction process of this process leads to a large amount of ammonia in the extracted drug residue, unpleasant smell during storage of the drug residue, and destruction of nutrients such as starch in the medicinal materials. The subsequent application of the drug residue is affected. Great limitation; on the other hand, the process adopts alkali-soluble acid precipitation process to obtain glycyrrhizic acid powder. The strong acid solution has about 20% damage to glycyrrhizic acid, and the large amount of acid water produced by the process brings great pressure to environmental protection. . At present, due to environmental protection requirements and cost increase restrictions, most domestic glycyrrhizic acid powder manufacturers have shut down.
甘草富含有大量的黄酮成分，然而长期以来，在甘草有效成分提取领域，工业化生产只将甘草中的甘草酸作为主要有效成分进行提取，而富含大量黄酮类化合的甘草渣则作为废料弃去，从而造成资源的极大浪费。人们基于对甘草资源的综合利用，也开展了甘草药渣黄酮成分的提取工艺开发，然而收效甚微。甘草提取物工业现状呈现为：1)甘草酸提取过程使用氨水，导致提取完的药渣中含有大量的氨、药渣存放过程中气味难闻、药材中的淀粉等营养成分被破坏，药渣的后续应用受到了极大的限制；2)甘草酸制备采用碱溶酸沉的工艺得到甘草酸粉，强酸溶液对甘草酸有约为20％的破坏作用，且工艺产生的大量酸水给环保成本带来极大压力；3)提取完甘草酸的甘草药渣，一方面水溶性的甘草苷含量很少，另一方面进一步提取黄酮受甘草药渣氨成分的 影响，生产成本较高，限制了甘草黄酮的应用。这一现状直接导致国内甘草酸粉生产厂家主动或被动关停，甘草药渣循环利用受阻，甘草黄酮成本居高不下。Licorice is rich in flavonoids. However, for a long time, in the field of extraction of licorice active ingredients, only glycyrrhizic acid in licorice has been extracted as the main effective ingredient in industrial production, and licorice residues rich in flavonoids are discarded as waste. To go, resulting in a great waste of resources. Based on the comprehensive utilization of licorice resources, people have also developed the extraction process of flavonoids from licorice dregs, but with little success. The current status of the licorice extract industry is as follows: 1) The use of ammonia in the extraction process of glycyrrhizic acid results in a large amount of ammonia in the extracted drug residue, unpleasant smell during storage of the drug residue, and the destruction of nutritional components such as starch in the medicine. The follow-up application of glycyrrhizic acid is greatly restricted; 2) The preparation of glycyrrhizic acid adopts the process of alkali-soluble acid precipitation to obtain glycyrrhizic acid powder. The strong acid solution has about 20% damage to glycyrrhizic acid, and the large amount of acid water produced by the process is environmentally friendly Cost brings great pressure; 3) After extracting the glycyrrhizic acid residues of glycyrrhizin, on the one hand, the content of water-soluble glycyrrhizin is very small, and on the other hand, the further extraction of flavonoids is affected by the ammonia components of the residues of glycyrrhizic acid, and the production cost is high and limited. The application of licorice flavonoids. This situation directly led to the active or passive shutdown of domestic glycyrrhizic acid powder manufacturers, the recycling of glycyrrhizinate residues was blocked, and the cost of licorice flavonoids remained high.
随着近些年来我国甘草资源的逐年短缺，以及国家对于绿色制造、环境保护的大力提倡，甘草提取行业亟待清洁、高效的甘草有效成分提取工艺开发。With the year-by-year shortage of licorice resources in my country in recent years, and the country’s vigorous promotion of green manufacturing and environmental protection, the licorice extraction industry urgently needs to develop a clean and efficient extraction process for the effective ingredients of licorice.
公开于该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解，而不应当被视为承认或以任何形式暗示该信息构成已为本领域一般技术人员所公知的现有技术。The information disclosed in the background section is only intended to increase the understanding of the overall background of the present invention, and should not be regarded as an acknowledgement or any form of suggestion that the information constitutes the prior art already known to those of ordinary skill in the art.
发明内容Summary of the invention
本发明的目的在于提供一种从甘草中提取甘草酸粗品方法，该方法实现对甘草酸成分的高效提取，避免了甘草酸的破坏；使用的有机溶剂可通过现代手段进行高效回收，避免了传统甘草酸提取工艺中的废水处理和环保问题，且很好的保存了甘草药渣中的淀粉、蛋白等营养成分，实现甘草资源的高效利用。The purpose of the present invention is to provide a method for extracting crude glycyrrhizic acid from licorice, which realizes the efficient extraction of glycyrrhizic acid components and avoids the destruction of glycyrrhizic acid; the organic solvents used can be efficiently recovered by modern means, avoiding the traditional Wastewater treatment and environmental protection issues in the extraction process of glycyrrhizic acid, and the starch, protein and other nutrients in the glycyrrhizinate residue are well preserved, so as to realize the efficient use of licorice resources.
为实现上述目的，本发明提供了一种从甘草中提取甘草酸粗品的方法，包括以下步骤：甘草粉碎：将甘草药材经粉碎处理成甘草药粉，控制甘草药粉的粒度在30～200目；以及一级提取：将有机溶剂和水组成第一溶剂，然后与上述甘草药粉以(2-20)：1(体积/重量，V/W)的配比混合，搅拌并加入第一pH调节剂，调节pH值，搅拌提取0.5-3小时，过滤，收集一级提取液和药渣；所述一级提取液即为甘草酸粗品。In order to achieve the above objective, the present invention provides a method for extracting crude glycyrrhizic acid from licorice, which includes the following steps: pulverizing licorice: pulverizing the licorice material into glycyrrhiza powder, and controlling the particle size of the licorice powder to be 30-200 mesh; and Primary extraction: The first solvent is composed of organic solvent and water, and then mixed with the above-mentioned licorice powder at a ratio of (2-20):1 (volume/weight, V/W), stirred and added the first pH regulator, Adjust the pH value, stir and extract for 0.5-3 hours, filter, and collect the first-level extract and the dregs; the first-level extract is the crude glycyrrhizic acid.
在本发明的一实施方式中，甘草粉碎步骤中，所述甘草药粉的粒度优选为30～100目。In one embodiment of the present invention, in the licorice pulverization step, the particle size of the licorice powder is preferably 30-100 mesh.
本发明还提供了一种从甘草中提取甘草酸粗品的方法，包括以下步骤：甘草粉碎：将甘草药材经粉碎处理成甘草药粉，控制甘草药粉的粒度在30～200目；一级提取：将有机溶剂和水组成第一溶剂，然后与所述甘草药粉以(2-20)：1(V/W)的配比混合，搅拌并加入第一pH调节剂，调节pH值，搅拌提取0.5-3小时过滤，收集一级提取液和一级药渣；以及K级提取：将所述第一溶剂与上一级获取的药渣以(2-20)：1(V/W)的配比混合，搅拌并加入第一pH调节剂，调节pH值，搅拌提取0.5-3小时，过滤，收K级提取液和K级药渣；以及从K＝2开始执行所述K级提取直至N级提取，从而完成完整的一轮提取，N大于等于2小于等于8，其中所述一级提取液即为甘草酸粗品。The present invention also provides a method for extracting crude glycyrrhizic acid from licorice, which includes the following steps: licorice pulverization: pulverize the licorice material into glycyrrhiza powder, and control the particle size of the glycyrrhiza powder to 30-200 mesh; first-level extraction: The organic solvent and water constitute the first solvent, which is then mixed with the licorice powder in a ratio of (2-20):1 (V/W), stirred and added the first pH adjuster, adjusted the pH value, stirred and extracted 0.5- Filter for 3 hours to collect the first-level extract and the first-level drug residue; and the K-level extraction: the first solvent and the drug residue obtained from the previous stage are in a ratio of (2-20):1(V/W) Mix, stir and add the first pH adjuster, adjust the pH value, stir and extract for 0.5-3 hours, filter, collect the K-level extract and K-level dregs; and perform the K-level extraction from K=2 to N-level Extraction to complete a complete round of extraction, N is greater than or equal to 2 and less than or equal to 8, wherein the first-level extract is the crude glycyrrhizic acid.
在本发明的一实施方式中，在每轮提取过程中，所述第一溶剂还包括上一轮/批次提取中除所述一级提取液以外的各级提取液，该举措可以使溶液重复循环利用，提高了溶剂的利用率，同时避免了传统甘草酸和甘草黄酮提取工艺中的废水处理和环保问题。In an embodiment of the present invention, in each round of extraction, the first solvent also includes all levels of extraction liquid in the previous round/batch extraction except for the first-level extraction liquid. This action can make the solution The repeated recycling improves the utilization rate of the solvent, and at the same time avoids the waste water treatment and environmental protection problems in the traditional extraction process of glycyrrhizic acid and licorice flavonoids.
在本发明的一实施方式中，所述一级提取步骤中，所述有机溶剂选自醇类、醇类衍生物、和酮类中的至少一种。In one embodiment of the present invention, in the primary extraction step, the organic solvent is selected from at least one of alcohols, alcohol derivatives, and ketones.
在本发明的一实施方式中，所述一级提取步骤中，所述有机溶剂选自甲醇、乙醇、乙酸乙酯和丙酮中的至少一种。In one embodiment of the present invention, in the primary extraction step, the organic solvent is selected from at least one of methanol, ethanol, ethyl acetate and acetone.
在本发明的一实施方式中，所述一级提取步骤中，所述水在第一溶剂中所占的体积百分比为0-30％。In an embodiment of the present invention, in the primary extraction step, the volume percentage of the water in the first solvent is 0-30%.
在本发明的一实施方式中，所述一级提取步骤中，所述第一溶剂选自甲醇-乙醇和乙醇-乙酸乙酯中的一种。In an embodiment of the present invention, in the primary extraction step, the first solvent is selected from one of methanol-ethanol and ethanol-ethyl acetate.
在本发明的一实施方式中，所述一级提取步骤中，所述第一溶剂与甘草药粉与的配比优选为(4-9)：1(体积/重量，V/W)。In one embodiment of the present invention, in the primary extraction step, the ratio of the first solvent to the glycerin powder is preferably (4-9):1 (volume/weight, V/W).
在本发明的一实施方式中，所述一级提取步骤和所述K级提取步骤中，所述第一pH调节剂选自如下物质中的至少一种：H
2SO
4、HCl、H
3PO
4、多聚磷酸、CH
3COOH、枸橼酸、草酸、阳离子交换树脂、pH≤2的缓冲体系、以及由上述物质中的至少一种配制成的不同浓度的溶液；优选的，所述第一pH调节剂为H
2SO
4-HCl。所述pH≤2的缓冲体系优选为甘氨酸-盐酸缓冲体系，进一步优选为0.1-0.3M的甘氨酸-盐酸缓冲体系。
In an embodiment of the present invention, in the first-level extraction step and the K-level extraction step, the first pH adjusting agent is selected from at least one of the following substances: H 2 SO 4 , HCl, H 3 PO 4 , polyphosphoric acid, CH 3 COOH, citric acid, oxalic acid, cation exchange resin, a buffer system with pH≤2, and solutions of different concentrations prepared from at least one of the above substances; preferably, the The first pH adjusting agent is H 2 SO 4 -HCl. The buffer system with pH≤2 is preferably a glycine-hydrochloric acid buffer system, and more preferably a 0.1-0.3M glycine-hydrochloric acid buffer system.
在本发明的一实施方式中，所述一级提取步骤和所述K级提取步骤中，调节pH值≤4。In an embodiment of the present invention, in the first-level extraction step and the K-level extraction step, the pH value is adjusted to be ≤4.
在本发明的一实施方式中，所述一级提取步骤和所述K级提取步骤中，调节pH值至0.5-4之间。In an embodiment of the present invention, in the first-level extraction step and the K-level extraction step, the pH value is adjusted to between 0.5-4.
在本发明的一实施方式中，所述一级提取步骤和所述K级提取步骤中，加入第一pH调节剂后搅拌提取的时间优选为0.3-1小时。In one embodiment of the present invention, in the first-level extraction step and the K-level extraction step, the time for stirring and extraction after adding the first pH regulator is preferably 0.3-1 hour.
本发明还提供了一种从甘草中提取甘草黄酮的方法，包括上述从甘草中提取甘草酸粗品的所有步骤以及后续的浓缩步骤，所述浓缩步骤如下所示：The present invention also provides a method for extracting licorice flavonoids from licorice, which includes all the steps of extracting crude glycyrrhizic acid from licorice and subsequent concentration steps, the concentration steps are as follows:
浓缩：将所述一级提取液调节pH至5-10之间，搅拌，生成沉淀，过滤，收集滤液，滤液浓缩，烘干，得甘草黄酮，该甘草黄酮的提取率不低于95％。Concentration: Adjust the pH of the first-level extract to between 5-10, stir, generate precipitation, filter, collect the filtrate, concentrate the filtrate, and dry to obtain licorice flavonoids. The extraction rate of licorice flavonoids is not less than 95%.
在本发明的一实施方式中，所述浓缩步骤中，将所述一级提取液中加入第二pH调节剂以调节pH至5-10之间。In one embodiment of the present invention, in the concentration step, a second pH adjusting agent is added to the primary extract to adjust the pH to between 5-10.
在本发明的一实施方式中，所述第二pH调节剂选自如下物质中的至少一种：金属、金属氧化物、金属氢氧化物、金属形成的盐、阴离子交换树脂、氨气、液氨、氨水、铵盐、三乙胺、pH缓冲范围为9～13的缓冲体系、以及由上述物质中的至少一种配制成的各种浓 度的溶液；所述金属为金属钠、钾、钙、镁、锌，所述金属氧化物为钠、钾、钙、镁、锌的氧化物，所述金属形成的盐为钠、钾、钙、镁、锌形成的各种酸盐，所述金属氢氧化物为钠、钾、钙、镁、锌的氢氧化物。所述pH缓冲范围为9～13的缓冲体系优选自氨-氯化铵和硼酸-氯化钾中的一种，进一步优选为氨-氯化铵溶液。In an embodiment of the present invention, the second pH adjusting agent is selected from at least one of the following substances: metals, metal oxides, metal hydroxides, salts formed by metals, anion exchange resins, ammonia, liquids Ammonia, ammonia, ammonium salt, triethylamine, a buffer system with a pH buffer range of 9-13, and solutions of various concentrations prepared from at least one of the above substances; the metals are sodium, potassium, and calcium metals , Magnesium, zinc, the metal oxides are oxides of sodium, potassium, calcium, magnesium, and zinc, and the salts formed by the metals are various acid salts formed by sodium, potassium, calcium, magnesium, and zinc. The hydroxide is a hydroxide of sodium, potassium, calcium, magnesium, and zinc. The buffer system with a pH buffer range of 9-13 is preferably one of ammonia-ammonium chloride and boric acid-potassium chloride, and is more preferably an ammonia-ammonium chloride solution.
本发明还提供了一种从甘草中提纯获得甘草酸盐的方法，包括上述从甘草中提取甘草酸粗品的所有步骤以及后续的纯化步骤，所述纯化步骤如下所示：The present invention also provides a method for obtaining glycyrrhizinate by purification from licorice, which includes all the steps of extracting crude glycyrrhizic acid from licorice and the subsequent purification steps, the purification steps are as follows:
纯化：向所述一级提取液中添加第二pH调节剂，控制体系pH值，过滤，收集沉淀，然后将所述沉淀用洗液洗涤，过滤，分别收集过滤后的洗液和沉淀，所述洗涤后的沉淀于50～80℃烘干，即得甘草酸盐，通过该提纯方法所获得的甘草酸盐的总收率不低于90％。Purification: Add a second pH regulator to the first-level extract, control the pH of the system, filter, collect the precipitate, then wash the precipitate with a lotion, filter, and collect the filtered lotion and precipitate respectively. The washed precipitate is dried at 50-80°C to obtain glycyrrhizinate, and the total yield of glycyrrhizinate obtained by the purification method is not less than 90%.
在本发明的一实施方式中，所述一级提取步骤中，所述第一溶剂选自甲醇-乙醇-水和乙醇-乙酸乙酯-水中的一种。In an embodiment of the present invention, in the primary extraction step, the first solvent is selected from one of methanol-ethanol-water and ethanol-ethyl acetate-water.
在本发明的一实施方式中，在每轮提取过程中，所述第一溶剂还包括上一轮/批次提取中除所述一级提取液以外的各级提取液与纯化步骤中过滤后的洗液所组成的混合液，该举措可以使溶液重复循环利用，从而避免了传统甘草酸提取工艺中的废水处理和环保问题。In an embodiment of the present invention, in each round of extraction, the first solvent also includes the extraction liquids of all levels in the previous round/batch extraction except for the first-level extraction liquid and after filtering in the purification step This action can make the solution reused, thus avoiding the waste water treatment and environmental protection problems in the traditional glycyrrhizic acid extraction process.
在本发明的一实施方式中，所述纯化步骤中，所述第二pH调节剂选自如下物质中的至少一种：金属、金属氧化物、金属氢氧化物、金属形成的盐、阴离子交换树脂、氨气、液氨、氨水、铵盐、三乙胺、pH缓冲范围为9～13的缓冲体系、以及由上述物质中的至少一种配制成各种浓度的溶液；所述金属为金属钠、钾、钙、镁、锌；所述金属氧化物为钠、钾、钙、镁、锌的氧化物；所述金属盐为钠、钾、钙、镁、锌形成的各种酸盐；所述金属氢氧化物为钠、钾、钙、镁、锌的氢氧化物。。实际操作过程中根据最终需要的甘草酸盐的种类选择相应的碱溶液或者碱溶液和对应盐配制而成的缓冲溶液。In one embodiment of the present invention, in the purification step, the second pH adjusting agent is selected from at least one of the following substances: metals, metal oxides, metal hydroxides, salts formed by metals, anion exchange Resin, ammonia gas, liquid ammonia, ammonia water, ammonium salt, triethylamine, a buffer system with a pH buffer range of 9-13, and at least one of the above substances are formulated into solutions of various concentrations; the metal is a metal Sodium, potassium, calcium, magnesium, zinc; the metal oxides are oxides of sodium, potassium, calcium, magnesium, and zinc; the metal salts are various acid salts formed by sodium, potassium, calcium, magnesium, and zinc; The metal hydroxide is a hydroxide of sodium, potassium, calcium, magnesium, and zinc. . In the actual operation process, the corresponding alkali solution or buffer solution prepared by the alkali solution and the corresponding salt is selected according to the type of glycyrrhizinate that is ultimately required.
在本发明的一实施方式中，所述纯化步骤中，将pH值控制在5-10之间。In one embodiment of the present invention, in the purification step, the pH value is controlled between 5-10.
在本发明的一实施方式中，所述纯化步骤中，所述洗液与所述第一溶剂相同，优选的，所述洗液选自甲醇-乙醇-水、和乙醇-乙酸乙酯-水中的一种。In one embodiment of the present invention, in the purification step, the washing liquid is the same as the first solvent. Preferably, the washing liquid is selected from methanol-ethanol-water and ethanol-ethyl acetate-water Kind of.
在本发明的一实施方式中，所述纯化步骤中，所述洗液是在常温或加热的条件下进行洗涤的；优选的，所述加热的温度为40-80℃，所述洗涤的次数为2-3次。In one embodiment of the present invention, in the purification step, the lotion is washed at room temperature or under heating; preferably, the heating temperature is 40-80°C, and the number of washings is 40-80°C. For 2-3 times.
在本发明的一实施方式中，所述纯化步骤中，所述洗涤后的沉淀优选于50～70℃烘干备用。In an embodiment of the present invention, in the purification step, the washed precipitate is preferably dried at 50-70°C for later use.
与现有技术相比，本发明具有如下有益效果：Compared with the prior art, the present invention has the following beneficial effects:
(1)本发明所涉及的从甘草中提取甘草酸粗品、甘草黄酮、及提纯获得甘草酸盐的 方法使用有机溶剂加pH调节剂提取甘草酸和甘草黄酮，同时用盐沉方式沉淀甘草酸，并对甘草酸进行进一步纯化，纯化过程产生的滤液和后续级次甘草酸提取液可以继续应用于下一轮甘草药材的提取。(1) The method of extracting crude glycyrrhizic acid and glycyrrhizin flavonoids from licorice and obtaining glycyrrhizinate by purification involved in the present invention uses an organic solvent plus a pH regulator to extract glycyrrhizic acid and glycyrrhizin flavonoids, and at the same time precipitate the glycyrrhizic acid by salt precipitation, The glycyrrhizic acid is further purified, and the filtrate produced during the purification process and the subsequent grade glycyrrhizic acid extract can continue to be used in the next round of extraction of glycyrrhizic acid.
(2)该方法一方面提高了甘草酸的提取率，避免了传统工艺碱溶酸沉步骤的损失和破坏，另一方面提取过程中使用的提取溶剂循环应用提取药材，提高了有机溶剂利用率，同时避免了传统工艺提取甘草，甘草药渣二次提取黄酮的环节，并且提取过程中使用的提取溶剂循环应用提取药材，提高了溶剂利用率，控制药材提取成本。(2) On the one hand, this method improves the extraction rate of glycyrrhizic acid and avoids the loss and damage of the traditional alkaline solution and acid precipitation step. On the other hand, the extraction solvent used in the extraction process is recycled to extract medicinal materials, which improves the utilization rate of organic solvents. At the same time, the traditional process of extracting licorice and the secondary extraction of flavonoids from licorice residues is avoided, and the extraction solvent used in the extraction process is used to extract the medicinal materials, which improves the solvent utilization rate and controls the extraction cost of the medicinal materials.
(3)该方法使用的有机溶剂可通过现代手段进行高效回收，避免了传统甘草酸、甘草黄酮提取工艺中的废水处理和环保问题，且很好的保存了甘草药渣中的淀粉、蛋白等营养成分，实现甘草资源的全草高效利用。(3) The organic solvent used in this method can be efficiently recovered by modern means, avoiding the waste water treatment and environmental protection problems in the traditional extraction process of glycyrrhizic acid and licorice flavonoids, and well preserves the starch and protein in the licorice residue Nutrient ingredients, realize the efficient utilization of licorice resources.
图1是根据本发明一实施方式的从甘草中提取甘草酸粗品、甘草黄酮以及提纯获得甘草酸盐的方法的工艺流程图。Fig. 1 is a process flow diagram of a method for extracting crude glycyrrhizic acid and glycyrrhizin flavonoids from licorice and purifying to obtain glycyrrhizinate according to an embodiment of the present invention.
下面对本发明的具体实施方式进行详细描述，但应当理解本发明的保护范围并不受具体实施方式的限制。The specific embodiments of the present invention will be described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.
除非另有其它明确表示，否则在整个说明书和权利要求书中，术语“包括”或其变换如“包含”或“包括有”等等将被理解为包括所陈述的元件或组成部分，而并未排除其它元件或其它组成部分。Unless otherwise expressly stated otherwise, throughout the specification and claims, the term "comprising" or its transformations such as "including" or "including" will be understood to include the stated elements or components, and not Other elements or other components are not excluded.
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。The experimental methods used in the following examples are conventional methods unless otherwise specified.
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径可购得。The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.
以下通过多个实施例对从甘草中提取甘草酸粗品的方法进行描述。The method for extracting crude glycyrrhizic acid from licorice is described below through a number of examples.
实施例1Example 1
(1)取甘草药材，破碎粉碎，使药粉粒度为全部通过40目标准筛；(1) Take the licorice material, crush and smash, so that the particle size of the medicinal powder is all passed through a 40-mesh standard sieve;
(2)一级提取：取此甘草药粉1kg，加甲醇-乙醇(V/V，15/85)5L，搅拌均匀，加0.2M甘氨酸-盐酸(V/V，1/4)适量，控制混合体系pH值至2-4之间，搅拌提取30分钟，过滤，分别收集1级提取液和药渣；(2) Primary extraction: Take 1kg of this glycine herbal powder, add 5L of methanol-ethanol (V/V, 15/85), stir evenly, add 0.2M glycine-hydrochloric acid (V/V, 1/4) appropriate amount, control the mixing The pH value of the system is between 2-4, stirring and extracting for 30 minutes, filtering, and collecting the first-level extract and medicine residue;
(3)二～六级提取：采用相同方法，再对药材提取五级，即共提取6级，分别收集各 级提取液和药渣；(3) Two to six levels of extraction: using the same method, and then extracting the medicinal materials for five levels, that is, a total of 6 levels, and collecting the extracts and dregs of each level;
(4)甘草酸分离纯化：1级提取液用于甘草酸的分离纯化，1级提取液甘草酸纯化后滤液进行溶剂回收，其余级次的提取液用于新一批次甘草药材的提取。(4) Glycyrrhizic acid separation and purification: the first-level extract is used for the separation and purification of glycyrrhizic acid, the filtrate is solvent recovery after the first-level extract glycyrrhizic acid is purified, and the rest of the extracts are used for the extraction of a new batch of glycyrrhizin materials.
实施例2Example 2
(1)取甘草药材，破碎粉碎，使药粉粒度为全部通过40目标准筛；(1) Take the licorice material, crush and smash, so that the particle size of the medicine powder is all passed through a 40-mesh standard sieve;
(2)一级提取：取此甘草药粉1kg，加上述实施例1中2～6级提取液混合物共5L，搅拌均匀，加0.2M甘氨酸-盐酸(V/V，1/4)适量，控制混合体系pH值至2-4之间，搅拌提取30分钟，过滤，分别收集1级提取液和药渣；(2) First-level extraction: Take 1kg of this glycine herbal powder, add 5L of the 2-6 level extract mixture in the above example 1, and stir it evenly, add 0.2M glycine-hydrochloric acid (V/V, 1/4) in an appropriate amount, control The pH value of the mixed system is between 2-4, stirred and extracted for 30 minutes, filtered, and collected the first-level extract and the medicine residue respectively;
(3)二～六级提取：采用相同方法，再对药材提取五级，即共提取6级，分别收集各级提取液和药渣；(3) Two to six levels of extraction: the same method is used to extract five levels of medicinal materials, that is, a total of 6 levels, and the extracts and slag of each level are collected respectively;
(4)甘草酸分离纯化：1级提取液用于甘草酸的分离纯化，1级提取液甘草酸纯化后滤液进行溶剂回收，其余级次的提取液用于新一批次甘草药材的提取。(4) Glycyrrhizic acid separation and purification: the first-level extract is used for the separation and purification of glycyrrhizic acid, the filtrate is solvent recovery after the first-level extract glycyrrhizic acid is purified, and the rest of the extracts are used for the extraction of a new batch of glycyrrhizin materials.
实施例3Example 3
(1)取甘草药材，破碎粉碎，使药粉粒度为全部通过60目标准筛；(1) Take the licorice material, crush and smash, make the particle size of the medicine powder pass the 60-mesh standard sieve;
(2)一级提取：取此甘草药粉1kg，加乙醇8L，搅拌均匀，加5M硫酸-盐酸(V/V，1:1)适量，控制混合体系pH值至2-4之间，搅拌提取30分钟，过滤，分别收集1级提取液和药渣；(2) First-level extraction: Take 1kg of this licorice powder, add 8L of ethanol, mix well, add 5M sulfuric acid-hydrochloric acid (V/V, 1:1) in an appropriate amount, control the pH of the mixed system to between 2-4, and stir to extract After 30 minutes, filter and collect the first-level extract and medicine residue respectively;
(3)二～五级提取：采用相同方法，再对药材提取四级，即共提取5级，分别收集各级提取液和药渣；(3) Two to five levels of extraction: using the same method, and then extracting the medicinal materials for four levels, that is, a total of five levels of extraction, and each level of extraction liquid and drug residue are collected respectively;
(4)甘草酸分离纯化：1级提取液用于甘草酸的分离纯化，1级提取液甘草酸纯化后滤液进行溶剂回收，其余级次的提取液用于新一批次甘草药材的提取。(4) Glycyrrhizic acid separation and purification: the first-level extract is used for the separation and purification of glycyrrhizic acid, the filtrate is solvent recovery after the first-level extract glycyrrhizic acid is purified, and the rest of the extracts are used for the extraction of a new batch of glycyrrhizin materials.
实施例4Example 4
(1)取甘草药材，破碎粉碎，使药粉粒度为全部通过60目标准筛；(1) Take the licorice material, crush and smash, make the particle size of the medicine powder pass the 60-mesh standard sieve;
(2)一级提取：取此甘草药粉1kg，加上述实施例3中2～5级提取液混合物共8L，搅拌均匀，加5M硫酸-盐酸(V/V，1:1)适量，控制混合体系pH值至2-4之间，搅拌提取30分钟，过滤，分别收集1级提取液和药渣；(2) First-level extraction: Take 1kg of this licorice powder, add 8L of the 2-5 level extract mixture in the above Example 3, stir it evenly, add 5M sulfuric acid-hydrochloric acid (V/V, 1:1) in an appropriate amount, and control the mixing The pH value of the system is between 2-4, stirring and extracting for 30 minutes, filtering, and collecting the first-level extract and medicine residue;
(3)二～五级提取：采用相同方法，再对药材提取四级，即共提取5级，分别收集各级提取液和药渣；(3) Two to five levels of extraction: using the same method, and then extracting the medicinal materials for four levels, that is, a total of five levels of extraction, and each level of extraction liquid and drug residue are collected respectively;
(4)甘草酸分离纯化：1级提取液用于甘草酸的分离纯化，1级提取液甘草酸纯化后滤液进行溶剂回收，其余级次的提取液用于新一批次甘草药材的提取。(4) Glycyrrhizic acid separation and purification: the first-level extract is used for the separation and purification of glycyrrhizic acid, the filtrate is solvent recovery after the first-level extract glycyrrhizic acid is purified, and the rest of the extracts are used for the extraction of a new batch of glycyrrhizin materials.
本发明进一步对上述实施例1-4的甘草酸提取率进行统计。The present invention further performs statistics on the extraction rate of glycyrrhizic acid in the foregoing Examples 1-4.
(一)实施例1和实施例2甘草酸提取率统计(1) Example 1 and Example 2 Glycyrrhizic acid extraction rate statistics
检测方法：高效液相色谱法Detection method: high performance liquid chromatography
(1)色谱条件与***适用性试验：用十八烷基硅烷键合硅胶为填充剂；乙腈-0.01mol/L磷酸溶液(38：62)为流动相；检测波长为252nm；流速:1ml/min；进样量：10μL；理论板数按甘草酸单铵峰计算应不低于2000，甘草酸单铵盐峰和内标物质峰的分离度应符合要求。(1) Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel is used as filler; acetonitrile-0.01mol/L phosphoric acid solution (38:62) is used as mobile phase; detection wavelength is 252nm; flow rate: 1ml/ min; Injection volume: 10μL; The number of theoretical plates should not be less than 2000 based on the monoammonium glycyrrhizinate peak, and the resolution of the monoammonium glycyrrhizinate peak and the peak of the internal standard substance should meet the requirements.
(2)内标溶液的制备：取对羟基苯甲酸正丁酯约70mg，精密称定，置100ml量瓶中，以稀乙醇溶解并稀释至刻度，摇匀。(2) Preparation of internal standard solution: Take about 70 mg of n-butyl p-hydroxybenzoate, accurately weigh it, and place it in a 100 ml measuring flask, dissolve it with dilute ethanol and dilute to the mark, and shake it well.
(3)对照品溶液的制备：取甘草酸单铵盐对照品约20mg，精密称定，置100ml量瓶中，加稀乙醇溶解，并精密加入内标溶液5ml，用稀乙醇稀释至刻度，摇匀。(3) Preparation of reference solution: take about 20mg of glycyrrhizinate monoammonium salt reference substance, accurately weigh it, put it in a 100ml measuring flask, add dilute ethanol to dissolve, and accurately add 5ml of internal standard solution, dilute to the mark with dilute ethanol, Shake well.
(4)样品溶液制备(4) Sample solution preparation
一级提取液：精密移取2ml至100mL容量瓶中，精密加入内标溶液5mL，用稀乙醇稀释至刻度；Primary extraction solution: accurately pipet 2ml to a 100mL volumetric flask, accurately add 5mL of internal standard solution, and dilute to the mark with dilute ethanol;
二～六级提取液：分别精密移取5mL至100mL容量瓶中，精密加入内标溶液5mL，用稀乙醇稀释至刻度；Second to sixth grade extracts: accurately pipet 5mL to 100mL volumetric flasks, precisely add 5mL of internal standard solution, and dilute to the mark with dilute ethanol;
统计实施例1和实施例2中各级提取液的甘草酸的浓度和提取率，数据见表1。The concentration and extraction rate of glycyrrhizic acid in the extracts of each level in Example 1 and Example 2 were calculated, and the data are shown in Table 1.
表1Table 1
如上表所示，甘草药材经上述方法提取6级后，甘草酸提取基本完全，药渣中甘草酸含量低于1％，并且药渣中的淀粉、脂肪等营养成分得到很好的保护，为甘草药渣的后续应用的提供了较好的基础。As shown in the above table, after glycyrrhizic acid is extracted by the above-mentioned method for 6 levels, the extraction of glycyrrhizic acid is basically complete, the content of glycyrrhizic acid in the medicine residue is less than 1%, and the nutritional components such as starch and fat in the medicine residue are well protected. The follow-up application of glycerin residues provides a better foundation.
(二)实施例3和实施例4甘草酸提取率统计(2) Example 3 and Example 4 Glycyrrhizic acid extraction rate statistics
检测方法：高效液相色谱法Detection method: high performance liquid chromatography
(1)色谱条件与***适用性试验：用十八烷基硅烷键合硅胶为填充剂；乙腈-0.01mol/L磷酸溶液(38︰62)为流动相；检测波长为252nm。理论板数按甘草酸单铵峰计算应不低于2000，甘草酸单铵盐峰和内标物质峰的分离度应符合要求。(1) Chromatographic conditions and system suitability test: use octadecylsilane bonded silica gel as filler; acetonitrile-0.01mol/L phosphoric acid solution (38:62) as mobile phase; detection wavelength is 252nm. The number of theoretical plates calculated based on the monoammonium glycyrrhizinate peak should not be less than 2000, and the resolution of the monoammonium glycyrrhizinate peak and the peak of the internal standard substance should meet the requirements.
(2)内标溶液的制备：取对羟基苯甲酸正丁酯约70mg，精密称定，置100ml量瓶中，以稀乙醇溶解并稀释至刻度，摇匀。(2) Preparation of internal standard solution: Take about 70 mg of n-butyl p-hydroxybenzoate, accurately weigh it, and place it in a 100 ml measuring flask, dissolve it with dilute ethanol and dilute to the mark, and shake it well.
(3)对照品溶液的制备：取甘草酸单铵盐对照品约20mg，精密称定，置100ml量瓶中，加稀乙醇溶解，并精密加入内标溶液5ml，用稀乙醇稀释至刻度，摇匀。(3) Preparation of reference solution: take about 20mg of glycyrrhizinate monoammonium salt reference substance, accurately weigh it, put it in a 100ml measuring flask, add dilute ethanol to dissolve, and accurately add 5ml of internal standard solution, dilute to the mark with dilute ethanol, Shake well.
(4)样品溶液制备(4) Sample solution preparation
一级提取液：精密移取2ml至100mL容量瓶中，精密加入内标溶液5mL，用稀乙醇稀释至刻度；Primary extraction solution: accurately pipet 2ml to a 100mL volumetric flask, accurately add 5mL of internal standard solution, and dilute to the mark with dilute ethanol;
二～五级提取液：分别精密移取5mL至100mL容量瓶中，精密加入内标溶液5mL，用稀乙醇稀释至刻度；Second to fifth grade extraction solution: accurately pipet 5mL to 100mL volumetric flask respectively, accurately add 5mL internal standard solution, and dilute to the mark with dilute ethanol;
统计实施例3和实施例4中各级提取液的甘草酸的浓度和提取率，数据见表2。The concentration and extraction rate of glycyrrhizic acid in the extracts of each level in Example 3 and Example 4 are calculated, and the data are shown in Table 2.
表2Table 2
如上表所示，甘草药材经上述方法提取5级后，甘草酸提取基本完全，药渣中甘草酸含量低于1％，并且药渣中的淀粉、脂肪等营养成分得到很好的保护，为甘草药渣的后续应用的提供了较好的基础。As shown in the above table, after 5 levels of licorice materials are extracted by the above method, the extraction of glycyrrhizic acid is basically complete, the content of glycyrrhizic acid in the dregs is less than 1%, and the nutritional components such as starch and fat in the dregs are well protected. The follow-up application of glycerin residues provides a better foundation.
本发明进一步通过多个实施例对从甘草中提取甘草黄酮的方法进行描述。The present invention further describes the method for extracting licorice flavonoids from licorice through a number of embodiments.
实施例5Example 5
(1)取甘草药材(总黄酮含量2.69％)，破碎粉碎，使药粉粒度为全部通过60目标准 筛；(1) Take the licorice herb material (total flavonoid content 2.69%), crush and pulverize, so that the particle size of the powder is all passed through a 60-mesh standard sieve;
(2)一级提取：取此甘草药粉1kg，加甲醇-乙醇(V/V，1/4)6L，搅拌均匀，加0.2M甘氨酸-盐酸(V/V，1/4)溶液适量，控制混合体系pH值至0.5-4之间，搅拌提取30分钟，过滤，分别收集1级提取液和药渣；(2) Primary extraction: Take 1kg of this glycine herb powder, add 6L of methanol-ethanol (V/V, 1/4), stir evenly, add 0.2M glycine-hydrochloric acid (V/V, 1/4) solution in an appropriate amount, control The pH value of the mixed system is between 0.5-4, stirred and extracted for 30 minutes, filtered, and collected the first-level extract and the medicine residue;
(3)二～五级提取：采用相同方法，再对药材提取四级，即共提取5级，分别收集各级提取液和药渣；(3) Two to five levels of extraction: using the same method, and then extracting the medicinal materials for four levels, that is, a total of five levels of extraction, and each level of extraction liquid and drug residue are collected respectively;
(4)甘草黄酮成分浓缩和富集：1级提取液添加2M氨-氯化铵(V/V，97/3)溶液，以调节pH至5-10之间，搅拌，生成沉淀，过滤，收集滤液，滤液浓缩，烘干，得甘草黄酮。(4) Concentration and enrichment of licorice flavonoids: Add 2M ammonia-ammonium chloride (V/V, 97/3) solution to the first-level extract to adjust the pH to between 5-10, stir to form a precipitate, filter, The filtrate is collected, the filtrate is concentrated, and dried to obtain licorice flavonoids.
实施例6Example 6
(1)取甘草药材，破碎粉碎，使药粉粒度为全部通过60目标准筛；(1) Take the licorice material, crush and smash, make the particle size of the medicine powder pass the 60-mesh standard sieve;
(2)一级提取：取此甘草药粉1kg，加上述实施例5中2～5级提取液混合物共6L，搅拌均匀，加0.2M甘氨酸-盐酸(V/V，1/4)溶液适量，控制混合体系pH值至0.5-4之间，搅拌提取30分钟，过滤，分别收集1级提取液和药渣；(2) First-level extraction: Take 1kg of this glycine herbal powder, add 6L of the 2-5 level extract mixture in the above example 5, stir evenly, add an appropriate amount of 0.2M glycine-hydrochloric acid (V/V, 1/4) solution, Control the pH value of the mixed system to between 0.5-4, stir and extract for 30 minutes, filter, and collect the first-level extract and the dregs;
(3)二～五级提取：采用相同方法，再对药材提取四级，即共提取5级，分别收集各级提取液和药渣；(3) Two to five levels of extraction: using the same method, and then extracting the medicinal materials for four levels, that is, a total of five levels of extraction, and each level of extraction liquid and drug residue are collected respectively;
(4)甘草黄酮成分浓缩和富集：1级提取液添加2M氨-氯化铵(V/V，97/3)溶液，以调节pH至5-10之间，搅拌，生成沉淀，过滤，收集滤液，滤液浓缩，烘干，得甘草黄酮。(4) Concentration and enrichment of licorice flavonoids: Add 2M ammonia-ammonium chloride (V/V, 97/3) solution to the first-level extract to adjust the pH to between 5-10, stir to form a precipitate, filter, The filtrate is collected, the filtrate is concentrated, and dried to obtain licorice flavonoids.
实施例7Example 7
(1)取甘草药材(总黄酮含量2.98％)，破碎粉碎，使药粉粒度为全部通过100目标准筛；(1) Take licorice herbal material (total flavonoid content 2.98%), crush and pulverize, so that the particle size of the medicine powder passes through a standard 100 mesh sieve;
(2)一级提取：取此甘草药粉1kg，加乙酸乙酯-乙醇(V/V，1/2)8L，搅拌均匀，加5M硫酸-盐酸(V/V，1/1)适量，控制混合体系pH值至0.5-4之间，搅拌提取30分钟，过滤，分别收集1级提取液和药渣；(2) First level extraction: Take 1kg of this licorice powder, add 8L of ethyl acetate-ethanol (V/V, 1/2), stir evenly, add 5M sulfuric acid-hydrochloric acid (V/V, 1/1) in an appropriate amount, control The pH value of the mixed system is between 0.5-4, stirred and extracted for 30 minutes, filtered, and collected the first-level extract and the medicine residue;
(3)二～六级提取：采用相同方法，再对药材提取五级，即共提取6级，分别收集各级提取液和药渣。(3) Two to six levels of extraction: using the same method, and then extracting the medicinal materials for five levels, that is, a total of 6 levels, and collecting the extracts and slag of each level.
(4)甘草黄酮成分浓缩和富集：1级提取液添加2M氢氧化钾溶液，以调节pH至5-10之间，搅拌，生成沉淀，过滤，收集滤液，滤液浓缩，烘干，得甘草黄酮；(4) Concentration and enrichment of licorice flavonoids: add 2M potassium hydroxide solution to the first-level extract to adjust the pH to between 5-10, stir to form a precipitate, filter, collect the filtrate, concentrate the filtrate, and dry to obtain licorice Flavonoids
经过6级提取，甘草药材中的黄酮成分的提取率高于90％，且药渣中的淀粉、脂肪等成分得到了保护，为甘草药渣的后续应用提供了较好的基础。After 6 levels of extraction, the extraction rate of flavonoids in the licorice material is higher than 90%, and the starch, fat and other components in the dregs are protected, which provides a good foundation for the subsequent application of licorice dregs.
实施例8Example 8
(1)取甘草药材(总黄酮含量2.98％)，破碎粉碎，使药粉粒度为全部通过100目标准筛；(1) Take licorice herbal material (total flavonoid content 2.98%), crush and pulverize, so that the particle size of the medicine powder passes through a standard 100 mesh sieve;
(2)一级提取：取此甘草药粉1kg，加上述实施例7中2～5级提取液混合物共8L，搅拌均匀，加5M硫酸-盐酸(V/V，1/1)适量，控制混合体系pH值至0.5-4之间，搅拌提取30分钟，过滤，分别收集1级提取液和药渣；(2) First-level extraction: Take 1kg of this licorice powder, add 8L of the 2-5 level extract mixture in Example 7 above, stir evenly, add 5M sulfuric acid-hydrochloric acid (V/V, 1/1) in an appropriate amount, and control the mixing The pH value of the system is between 0.5-4, stirring and extracting for 30 minutes, filtering, and collecting the first-level extract and the medicine residue;
(3)二～六级提取：采用相同方法，再对药材提取五级，即共提取6级，分别收集各级提取液和药渣；(3) Two to six levels of extraction: the same method is used to extract five levels of medicinal materials, that is, a total of 6 levels, and the extracts and slag of each level are collected respectively;
(4)甘草黄酮成分浓缩和富集：1级提取液添加2M氢氧化钾溶液，以调节pH至5-10之间，搅拌，生成沉淀，过滤，收集滤液，滤液浓缩，烘干，得甘草黄酮。(4) Concentration and enrichment of licorice flavonoids: add 2M potassium hydroxide solution to the first-level extract to adjust the pH to between 5-10, stir to form a precipitate, filter, collect the filtrate, concentrate the filtrate, and dry to obtain licorice Flavonoids.
本发明进一步对上述实施例5-8的甘草黄酮提取率进行统计。The present invention further performs statistics on the extraction rate of licorice flavonoids in the foregoing Examples 5-8.
(一)实施例5和实施例6甘草黄酮提取率统计(1) Example 5 and Example 6 Glycyrrhiza flavonoid extraction rate statistics
试剂：芦丁标准品；乙醇：30％；乙醇：60％。硝酸铝：10％；氢氧化钠：1mol/L；亚硝酸钠：5％。Reagents: standard rutin; ethanol: 30%; ethanol: 60%. Aluminum nitrate: 10%; sodium hydroxide: 1mol/L; sodium nitrite: 5%.
分析步骤：Analysis steps:
标准液的制备：称取芦丁标准品20mg(精确到0.1mg)，以60％乙醇溶解于100mL容量瓶中，并稀释至刻度。取此样液20mL于50mL容量瓶中，加60％乙醇溶液5mL，以水稀释至刻度，即得标准液(1mL标准液约含0.08mg芦丁标准品)。Preparation of standard solution: Weigh 20 mg of rutin standard (accurate to 0.1 mg), dissolve it in a 100 mL volumetric flask with 60% ethanol, and dilute to the mark. Take 20mL of this sample solution in a 50mL volumetric flask, add 5mL of 60% ethanol solution, and dilute to the mark with water to obtain a standard solution (1mL standard solution contains about 0.08mg of rutin standard).
试样液的制备：精密移取提取液1mL至50mL容量瓶中，以60％乙醇稀释至刻度，即得试样液。Preparation of sample solution: Precisely pipette 1 mL of the extract into a 50 mL volumetric flask and dilute to the mark with 60% ethanol to obtain the sample solution.
分别取上述两液各1mL于10mL容量瓶中，加5％亚硝酸钠溶液0.3mL，摇匀，静置6min，加10％硝酸铝溶液0.3mL，摇匀，静置6min，加入1mo/L氢氧化钠溶液4mL，摇匀，用30％乙醇定容，静置15min，用1cm比色皿，在波长510nm处测吸光度。Take 1mL each of the above two liquids into a 10mL volumetric flask, add 0.3mL of 5% sodium nitrite solution, shake well, and let stand for 6min, add 0.3mL of 10% aluminum nitrate solution, shake well, let stand for 6min, add 1mo/L 4mL of sodium hydroxide solution, shake well, constant volume with 30% ethanol, stand for 15min, use a 1cm cuvette, measure the absorbance at a wavelength of 510nm.
总黄酮的浓度C，按下式计算The concentration C of total flavonoids is calculated by the following formula
C＝K*m1*A/A0/2500C=K*m1*A/A0/2500
式中：Where:
m1—标准品中黄酮重量(mg)；m1—the weight of flavonoids in the standard product (mg);
A—试样的吸光度；A—The absorbance of the sample;
A0—标准品的吸光度；A0—The absorbance of the standard product;
K—试样的稀释倍数。K—The dilution factor of the sample.
统计实施例5和实施例6中各级提取液的甘草黄酮的浓度和提取率，数据见表3。The concentration and extraction rate of glycyrrhiza flavonoids in the extracts at various levels in Example 5 and Example 6 were calculated, and the data are shown in Table 3.
表3table 3
经过5级提取，甘草药材中的黄酮成分的提取率高于95％，且药渣中的淀粉、脂肪等成分得到了保护，为甘草药渣的后续应用提供了较好的基础。After 5 levels of extraction, the extraction rate of flavonoids in the herbal dregs is higher than 95%, and the starch, fat and other components in the dregs are protected, which provides a good foundation for the follow-up application of the dregs of licorice.
(二)实施例7和实施例8甘草黄酮提取率统计(2) Example 7 and Example 8 Glycyrrhiza flavonoid extraction rate statistics
试剂：芦丁标准品；乙醇：30％；乙醇：60％。硝酸铝：10％；氢氧化钠：1mol/L；亚硝酸钠：5％。Reagents: standard rutin; ethanol: 30%; ethanol: 60%. Aluminum nitrate: 10%; sodium hydroxide: 1mol/L; sodium nitrite: 5%.
分析步骤：Analysis steps:
标准液的制备：称取芦丁标准品20mg(精确到0.1mg)，以60％乙醇溶解于100mL容量瓶中，并稀释至刻度。取此样液20mL于50mL容量瓶中，加60％乙醇溶液5mL，以水稀释至刻度，即得标准液(1mL标准液约含0.08mg芦丁标准品)。Preparation of standard solution: Weigh 20 mg of rutin standard (accurate to 0.1 mg), dissolve it in a 100 mL volumetric flask with 60% ethanol, and dilute to the mark. Take 20mL of this sample solution in a 50mL volumetric flask, add 5mL of 60% ethanol solution, and dilute to the mark with water to obtain a standard solution (1mL standard solution contains about 0.08mg of rutin standard).
试样液的制备：精密移取提取液若干毫升至50mL容量瓶中，以60％乙醇稀释至刻度，即得试样液。Preparation of sample solution: Precisely pipette several milliliters of the extract into a 50 mL volumetric flask, and dilute to the mark with 60% ethanol to obtain the sample solution.
分别取上述两液各1mL于10mL容量瓶中，加5％亚硝酸钠溶液0.3mL，摇匀，静置6min，加10％硝酸铝溶液0.3mL，摇匀，静置6min，加入1mo/L氢氧化钠溶液4mL，摇匀，用30％乙醇定容，静置15min，用1cm比色皿，在波长510nm处测吸光度。Take 1mL each of the above two liquids into a 10mL volumetric flask, add 0.3mL of 5% sodium nitrite solution, shake well, and let stand for 6min, add 0.3mL of 10% aluminum nitrate solution, shake well, let stand for 6min, add 1mo/L 4mL of sodium hydroxide solution, shake well, constant volume with 30% ethanol, stand for 15min, use a 1cm cuvette, measure the absorbance at a wavelength of 510nm.
结果计算Result calculation
总黄酮的浓度C，按下式计算The concentration C of total flavonoids is calculated by the following formula
C＝K*m1*A/A0/2500C=K*m1*A/A0/2500
式中：Where:
m1—标准品中黄酮重量(mg)；m1—the weight of flavonoids in the standard product (mg);
A—试样的吸光度；A—The absorbance of the sample;
A0—标准品的吸光度；A0—The absorbance of the standard product;
K—试样的稀释倍数。K—The dilution factor of the sample.
统计实施例7和实施例8中各级提取液的甘草黄酮的浓度和提取率，数据见表4。The concentration and extraction rate of glycyrrhiza flavonoids in the extracts at various levels in Example 7 and Example 8 were calculated, and the data are shown in Table 4.
表4Table 4
经过6级提取，甘草药材中的黄酮成分的提取率高于95％，且药渣中的淀粉、脂肪等成分得到了保护，为甘草药渣的后续应用提供了较好的基础。After 6 levels of extraction, the extraction rate of the flavonoids in the licorice material is higher than 95%, and the starch, fat and other components in the dregs are protected, which provides a good foundation for the follow-up application of the licorice dregs.
以下进一步通过多个实施例对从甘草中提纯获得甘草酸盐的方法进行描述。The following further describes the method of obtaining glycyrrhizinate from licorice by a number of examples.
实施例9Example 9
(1)取甘草药材，破碎粉碎，使药粉粒度为全部通过40目标准筛；(1) Take the licorice material, crush and smash, so that the particle size of the medicinal powder is all passed through a 40-mesh standard sieve;
(2)一级提取：取此甘草药粉1kg，加甲醇-乙醇-水(V/V/V，10/85/5)5L，搅拌均匀，加5M硫酸-盐酸(V/V，1/1)适量，控制混合体系pH值至2-4之间，搅拌提取30分钟，过滤，分别收集1级提取液和药渣；(2) Primary extraction: Take 1kg of this licorice powder, add 5L of methanol-ethanol-water (V/V/V, 10/85/5), stir evenly, and add 5M sulfuric acid-hydrochloric acid (V/V, 1/1) ) An appropriate amount, control the pH value of the mixed system to between 2-4, stir and extract for 30 minutes, filter, and collect the first-level extract and the medicine residue respectively;
(3)二～六级提取：采用上述相同方法，再对药材提取五级，即共提取6级，分别收集各级提取液和药渣；(3) Two to six levels of extraction: using the same method as above, and then extracting medicinal materials for five levels, that is, a total of 6 levels of extraction, and collecting each level of extraction liquid and medicine residue;
(4)甘草酸分离纯化：向一级提取液中加入0.1M氨-氯化铵混合溶液(V/V，4/1)，控制pH至5-10之间，生成甘草酸盐沉淀，过滤，收集沉淀，沉淀用甲醇-乙醇-水(V/V/V，10/85/5)常温洗涤3次，过滤，分别收集沉淀和滤液；沉淀60℃烘干，得甘草酸铵盐；(4) Separation and purification of glycyrrhizic acid: add 0.1M ammonia-ammonium chloride mixed solution (V/V, 4/1) to the primary extract, control the pH to between 5-10, generate glycyrrhizinate precipitation, filter , Collect the precipitate, wash the precipitate 3 times with methanol-ethanol-water (V/V/V, 10/85/5) at room temperature, filter, and collect the precipitate and the filtrate separately; dry the precipitate at 60°C to obtain ammonium glycyrrhizinate;
(5)溶液的重复利用：上述2～6级提取液和甘草酸盐洗涤滤液用于新一轮药材的提取。(5) Reuse of the solution: The above-mentioned 2-6 level extracts and glycyrrhizinate washing filtrate are used for a new round of extraction of medicinal materials.
实施例10Example 10
(1)取甘草药材，破碎粉碎，使药粉粒度为全部通过40目标准筛；(1) Take the licorice material, crush and smash, so that the particle size of the medicinal powder is all passed through a 40-mesh standard sieve;
(2)一级提取：取此甘草药粉1kg，加实施例9中2～6级提取液和洗涤甘草酸盐滤液共5L，搅拌均匀，加5M硫酸-盐酸(V/V，1/1)适量，控制混合体系pH值至2-4之间，搅拌提取30分钟，过滤，分别收集1级提取液和药渣；(2) First-level extraction: Take 1kg of this licorice powder, add 5L of the 2-6 level extracts and washing glycyrrhizinate filtrate in Example 9 in total, stir evenly, and add 5M sulfuric acid-hydrochloric acid (V/V, 1/1) Appropriate amount, control the pH value of the mixed system to between 2-4, stir and extract for 30 minutes, filter, and collect the first-level extract and medicine residue respectively;
(3)二～六级提取：采用上述相同方法，再对药材提取五级，即共提取6级，分别收集各级提取液和药渣；(3) Two to six levels of extraction: using the same method as above, and then extracting medicinal materials for five levels, that is, a total of 6 levels of extraction, and collecting each level of extraction liquid and medicine residue;
(4)甘草酸分离纯化：向一级提取液中加入0.1M氨-氯化铵混合溶液(V/V，4/1)，控制pH至5-10之间，生成甘草酸盐沉淀，过滤，收集沉淀，沉淀用甲醇-乙醇-水(V/V/V，10/85/5)常温洗涤3次，过滤，分别收集沉淀和滤液；沉淀60℃烘干，得甘草酸铵盐；(4) Separation and purification of glycyrrhizic acid: add 0.1M ammonia-ammonium chloride mixed solution (V/V, 4/1) to the primary extract, control the pH to between 5-10, generate glycyrrhizinate precipitation, filter , Collect the precipitate, wash the precipitate 3 times with methanol-ethanol-water (V/V/V, 10/85/5) at room temperature, filter, and collect the precipitate and the filtrate separately; dry the precipitate at 60°C to obtain ammonium glycyrrhizinate;
(5)溶液的重复利用：上述2～6级提取液和甘草酸盐洗涤滤液用于新一轮药材的提取。(5) Reuse of the solution: The above-mentioned 2-6 level extracts and glycyrrhizinate washing filtrate are used for a new round of extraction of medicinal materials.
实施例11Example 11
(1)取甘草药材，破碎粉碎，使药粉粒度为全部通过80目标准筛；(1) Take the licorice herb material, crush and smash, make the particle size of the medicine powder all pass the 80-mesh standard sieve;
(2)一级提取：取此甘草药粉1kg，加乙醇-水(V/V，90/10)8L，搅拌均匀，加0.2M甘氨酸-盐酸(V/V，1/4)溶液适量，控制混合体系pH值至2-4之间，搅拌提取30分钟，过滤，分别收集1级提取液和药渣；(2) Primary extraction: Take 1kg of this glycine herbal powder, add 8L of ethanol-water (V/V, 90/10), stir evenly, add 0.2M glycine-hydrochloric acid (V/V, 1/4) solution in an appropriate amount, control The pH value of the mixed system is between 2-4, stirred and extracted for 30 minutes, filtered, and collected the first-level extract and the medicine residue respectively;
(3)二～五级提取：采用上述相同方法，再对药材提取四级，即共提取5级，分别收集各级提取液和药渣；(3) Two to five levels of extraction: using the same method as described above, and then extracting medicinal materials for four levels, that is, a total of 5 levels of extraction, and collecting each level of extraction liquid and medicine residue;
(4)甘草酸分离纯化：向一级提取液中加入2M氢氧化钾溶液，控制pH至5-10之间，生成甘草酸钾盐沉淀，过滤，收集沉淀，沉淀用乙醇-水(V/V，90/10)常温洗涤2次，过滤，分别收集沉淀和滤液；沉淀60℃烘干，得甘草酸钾盐；(4) Separation and purification of glycyrrhizic acid: Add 2M potassium hydroxide solution to the primary extract, control the pH to between 5-10, and generate potassium glycyrrhizinate precipitate, filter, collect the precipitate, and use ethanol-water (V/ V, 90/10) Wash twice at room temperature, filter, and collect the precipitate and filtrate separately; dry the precipitate at 60°C to obtain potassium glycyrrhizinate;
(5)溶液的重复利用：上述2～5级提取液和甘草酸盐洗涤滤液用于新一轮药材的提取。(5) Reuse of solution: The above-mentioned 2 to 5 grades of extraction liquid and glycyrrhizinate washing filtrate are used for a new round of extraction of medicinal materials.
实施例12Example 12
(1)取甘草药材，破碎粉碎，使药粉粒度为全部通过80目标准筛；(1) Take the licorice herb material, crush and smash, make the particle size of the medicine powder all pass the 80-mesh standard sieve;
(2)一级提取：取此甘草药粉1kg，加实施例9中2～6级提取液和洗涤甘草酸盐滤液共8L，搅拌均匀，加0.2M甘氨酸-盐酸(V/V，1/4)适量，控制混合体系pH值至2-4之 间，搅拌提取30分钟，过滤，分别收集1级提取液和药渣；(2) First-level extraction: Take 1kg of this glycine herb powder, add 8L of the 2-6 level extract in Example 9 and the washing glycyrrhizinate filtrate, mix well, add 0.2M glycine-hydrochloric acid (V/V, 1/4 ) An appropriate amount, control the pH value of the mixed system to between 2-4, stir and extract for 30 minutes, filter, and collect the first-level extract and the medicine residue respectively;
(3)二～五级提取：采用上述相同方法，再对药材提取四级，即共提取5级，分别收集各级提取液和药渣；(3) Two to five levels of extraction: using the same method as described above, and then extracting medicinal materials for four levels, that is, a total of 5 levels of extraction, and collecting each level of extraction liquid and medicine residue;
(4)甘草酸分离纯化：向一级提取液中加入2M氢氧化钾溶液，控制pH至5-10之间，生成甘草酸钾盐沉淀，过滤，收集沉淀，沉淀用乙醇-水(V/V，90/10)常温洗涤2次，过滤，分别收集沉淀和滤液；沉淀60℃烘干，得甘草酸钾盐；(4) Separation and purification of glycyrrhizic acid: Add 2M potassium hydroxide solution to the primary extract, control the pH to between 5-10, and generate potassium glycyrrhizinate precipitate, filter, collect the precipitate, and use ethanol-water (V/ V, 90/10) Wash twice at room temperature, filter, and collect the precipitate and filtrate separately; dry the precipitate at 60°C to obtain potassium glycyrrhizinate;
(5)溶液的重复利用：上述2～5级提取液和甘草酸盐洗涤滤液用于新一轮药材的提取。(5) Reuse of solution: The above-mentioned 2 to 5 grades of extraction liquid and glycyrrhizinate washing filtrate are used for a new round of extraction of medicinal materials.
本发明进一步对上述实施例9-12的甘草酸提取率进行统计。The present invention further performs statistics on the extraction rate of glycyrrhizic acid in the foregoing Examples 9-12.
(一)实施例9和实施例10甘草酸提取率统计(1) Example 9 and Example 10 Glycyrrhizic acid extraction rate statistics
检测方法：高效液相色谱法Detection method: high performance liquid chromatography
(1)色谱条件与***适用性试验：用十八烷基硅烷键合硅胶为填充剂；乙腈-0.01mol/L磷酸溶液(38：62)为流动相；检测波长为252nm；流速:1ml/min；进样量：10μL；理论板数按甘草酸单铵峰计算应不低于2000，甘草酸单铵盐峰和内标物质峰的分离度应符合要求。(1) Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel is used as filler; acetonitrile-0.01mol/L phosphoric acid solution (38:62) is used as mobile phase; detection wavelength is 252nm; flow rate: 1ml/ min; Injection volume: 10μL; The number of theoretical plates should not be less than 2000 based on the monoammonium glycyrrhizinate peak, and the resolution of the monoammonium glycyrrhizinate peak and the peak of the internal standard substance should meet the requirements.
(2)内标溶液的制备：取对羟基苯甲酸正丁酯约70mg，精密称定，置100ml量瓶中，以稀乙醇溶解并稀释至刻度，摇匀。(2) Preparation of internal standard solution: Take about 70 mg of n-butyl p-hydroxybenzoate, accurately weigh it, and place it in a 100 ml measuring flask, dissolve it with dilute ethanol and dilute to the mark, and shake it well.
(3)对照品溶液的制备：取甘草酸单铵盐对照品约20mg，精密称定，置100ml量瓶中，加稀乙醇溶解，并精密加入内标溶液5ml，用稀乙醇稀释至刻度，摇匀。(3) Preparation of reference solution: take about 20mg of glycyrrhizinate monoammonium salt reference substance, accurately weigh it, put it in a 100ml measuring flask, add dilute ethanol to dissolve, and accurately add 5ml of internal standard solution, dilute to the mark with dilute ethanol, Shake well.
(4)样品溶液制备(4) Sample solution preparation
一级提取液：精密移取2ml至100mL容量瓶中，精密加入内标溶液5mL，用稀乙醇稀释至刻度；Primary extraction solution: accurately pipet 2ml to a 100mL volumetric flask, accurately add 5mL of internal standard solution, and dilute to the mark with dilute ethanol;
二～六级提取液：分别精密移取5mL至100mL容量瓶中，精密加入内标溶液5mL，用稀乙醇稀释至刻度；Second to sixth grade extracts: accurately pipet 5mL to 100mL volumetric flasks, precisely add 5mL of internal standard solution, and dilute to the mark with dilute ethanol;
甘草酸铵盐：取甘草酸中间体25mg，精密称定，移至100ml容量瓶中，精密加入内标溶液5mL，用稀乙醇稀释至刻度。Ammonium glycyrrhizinate: Take 25mg of glycyrrhizic acid intermediate, accurately weigh it, and transfer it to a 100ml volumetric flask, accurately add 5mL of internal standard solution, and dilute to the mark with dilute ethanol.
统计实施例9和实施例10中各级提取液的甘草酸的浓度和提取率，数据见表5。The concentration and extraction rate of glycyrrhizic acid in the extracts at various levels in Example 9 and Example 10 were calculated, and the data are shown in Table 5.
表5table 5
如表5所示，甘草药材经上述方法提取6级后，甘草药材中甘草酸提取基本完全。工艺中用一级提取液进行甘草酸铵盐的沉淀，甘草酸盐的收率相对于一级提取液的转移率94％以上，高于甘草酸传统提取工艺。由于本发明中循环利用溶剂对药材进行提取，随着套用次数的增加，一级提取液的浓度在逐级上升，从而每轮提取药材得到的甘草酸盐相对药材的收率提高，最后实现各轮提取时甘草酸铵盐的总收率在90％以上。As shown in Table 5, the glycyrrhizic acid in the glycyrrhizic acid is almost completely extracted after the above-mentioned method is used to extract 6 levels. In the process, the primary extraction liquid is used to precipitate the ammonium glycyrrhizinate, and the yield of the glycyrrhizinate relative to the transfer rate of the primary extraction liquid is more than 94%, which is higher than the traditional extraction process of glycyrrhizic acid. Since the solvent is recycled to extract medicinal materials in the present invention, as the number of applications increases, the concentration of the first-level extract is gradually increased, so that the yield of glycyrrhizinate relative to medicinal materials obtained in each round of extraction of medicinal materials is increased, and finally each is achieved. The total yield of ammonium glycyrrhizinate in round extraction is over 90%.
(二)实施例11和实施例12甘草酸提取率统计(B) Example 11 and Example 12 Glycyrrhizic acid extraction rate statistics
检测方法：高效液相色谱法Detection method: high performance liquid chromatography
(1)色谱条件与***适用性试验：用十八烷基硅烷键合硅胶为填充剂；乙腈-0.01mol/L磷酸溶液(38：62)为流动相；检测波长为252nm；流速:1ml/min；进样量：10μL；理论板数按甘草酸单铵峰计算应不低于2000，甘草酸单铵盐峰和内标物质峰的分离度应符合要求。(1) Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel is used as filler; acetonitrile-0.01mol/L phosphoric acid solution (38:62) is used as mobile phase; detection wavelength is 252nm; flow rate: 1ml/ min; Injection volume: 10μL; The number of theoretical plates should not be less than 2000 based on the monoammonium glycyrrhizinate peak, and the resolution of the monoammonium glycyrrhizinate peak and the peak of the internal standard substance should meet the requirements.
(2)内标溶液的制备：取对羟基苯甲酸正丁酯约70mg，精密称定，置100ml量瓶中，以稀乙醇溶解并稀释至刻度，摇匀。(2) Preparation of internal standard solution: Take about 70 mg of n-butyl p-hydroxybenzoate, accurately weigh it, and place it in a 100 ml measuring flask, dissolve it with dilute ethanol and dilute to the mark, and shake it well.
(3)对照品溶液的制备：取甘草酸单铵盐对照品约20mg，精密称定，置100ml量瓶 中，加稀乙醇溶解，并精密加入内标溶液5ml，用稀乙醇稀释至刻度，摇匀。(3) Preparation of reference solution: take about 20mg of glycyrrhizinate monoammonium salt reference substance, accurately weigh it, put it in a 100ml measuring flask, add dilute ethanol to dissolve, and accurately add 5ml of internal standard solution, dilute to the mark with dilute ethanol, Shake well.
(4)样品溶液制备(4) Sample solution preparation
一级提取液：精密移取2ml至100mL容量瓶中，精密加入内标溶液5mL，用稀乙醇稀释至刻度；Primary extraction solution: accurately pipet 2ml to a 100mL volumetric flask, accurately add 5mL of internal standard solution, and dilute to the mark with dilute ethanol;
二～五级提取液：分别精密移取5mL至100mL容量瓶中，精密加入内标溶液5mL，用稀乙醇稀释至刻度；Second to fifth grade extraction solution: accurately pipet 5mL to 100mL volumetric flask respectively, accurately add 5mL internal standard solution, and dilute to the mark with dilute ethanol;
甘草酸钾盐：取甘草酸中间体25mg，精密称定，移至100ml容量瓶中，精密加入内标溶液5mL，用稀乙醇稀释至刻度。Glycyrrhizinate potassium salt: Take 25mg of glycyrrhizic acid intermediate, accurately weigh it, transfer to a 100ml volumetric flask, accurately add 5mL internal standard solution, and dilute to the mark with dilute ethanol.
统计实施例11和实施例12中各级提取液的甘草酸的浓度和提取率，数据见表6。The concentration and extraction rate of glycyrrhizic acid in the extracts at various levels in Example 11 and Example 12 were counted, and the data are shown in Table 6.
表6Table 6
如上表所示，甘草药材经上述方法提取6级后，甘草酸提取基本完全。工艺中用一级提取液进行甘草酸铵盐的沉淀，甘草酸钾盐的收率相对于一级提取液的转移率94％以上，高于甘草酸传统提取工艺。由于本发明中循环利用溶剂对药材进行提取，随着套用次数的增加，一级提取液的浓度在逐级上升，从而每轮提取药材得到的甘草酸钾盐相对药材的收率提高，最后实现各轮提取时甘草酸钾盐的总收率在90％以上。As shown in the above table, the glycyrrhizic acid extraction is almost complete after the glycyrrhizic acid is extracted by the above-mentioned method for 6 levels. In the process, the primary extraction liquid is used to precipitate the ammonium glycyrrhizinate, and the yield of potassium glycyrrhizinate relative to the transfer rate of the primary extraction liquid is more than 94%, which is higher than the traditional extraction process of glycyrrhizic acid. Since the solvent is recycled to extract medicinal materials in the present invention, with the increase of the number of applications, the concentration of the first-level extracting solution increases step by step, so that the yield of potassium glycyrrhizinate relative to the medicinal materials obtained in each round of medicinal materials extraction is improved, and finally realized The total yield of potassium glycyrrhizinate in each round of extraction is more than 90%.
本发明进一步对试验条件进行了具体的验证试验，详见如下。The present invention further carried out specific verification tests on the test conditions, which are detailed as follows.
(1)药材粒度：(1) Granularity of medicinal materials:
取甘草药材(甘草酸含量3.14％)，分别粉碎成10目、30目、60目、80目、100目、200目的药粉各50g；Take licorice herbal material (glycyrrhizic acid content 3.14%) and crush them into 10 mesh, 30 mesh, 60 mesh, 80 mesh, 100 mesh, and 200 mesh powder respectively;
每份药材分别加甲醇-乙醇-水(V/V/V，10/85/5)250mL，加5M硫酸-盐酸(V/V，1/1)适量，搅拌各提取30min，提取完毕，过滤，收集滤液，记录提取液体积，依法检测一级提取液浓度，统计粒度对甘草药材甘草酸的提取情况。Add 250mL of methanol-ethanol-water (V/V/V, 10/85/5) to each medicinal material, add 5M sulfuric acid-hydrochloric acid (V/V, 1/1), stir and extract for 30 minutes. After extraction, filter , Collect the filtrate, record the volume of the extract, detect the concentration of the first-level extract in accordance with the law, and calculate the particle size for the extraction of glycyrrhizic acid from the licorice material.
表7Table 7
| 样品sample | 1号-10目1-10 mesh | 2号-30目2-30 mesh | 3号-60目3-60 mesh | 4号-80目No. 4-80 mesh | 5号-100目5-100 mesh | 6号-200目No. 6-200 mesh |
| 提取率Extraction rate | 42.94％42.94% | 60.28％60.28% | 65.77％65.77% | 72.98％72.98% | 74.96％74.96% | 75.19％75.19% |
从表7可知，控制甘草药粉目数在合理的范围内，以获得更佳的甘草酸提取率。It can be seen from Table 7 that the mesh number of the glycyrrhizinate powder is controlled within a reasonable range to obtain a better extraction rate of glycyrrhizic acid.
(2)提取时间(2) Extraction time
取甘草药材，粉碎至80目，取5份，100g/份，各加入甲醇-乙醇-水(V/V/V，10/85/5)500mL，加5M硫酸-盐酸(V/V，1/1)适量，搅拌，提取，分别一级提取15min、30min、60min、90min、120min、180min过滤，收集滤液，记录提取液体积，依法检测一级提取液浓度，统计不同提取时间对甘草药材甘草酸的提取情况。Take the licorice material, crush it to 80 mesh, take 5 portions, 100g/portion, add methanol-ethanol-water (V/V/V, 10/85/5) 500mL, add 5M sulfuric acid-hydrochloric acid (V/V, 1 /1) Appropriate amount, stir, extract, extract 15min, 30min, 60min, 90min, 120min, 180min at the first level, filter, collect the filtrate, record the volume of the extract, detect the concentration of the first level extract according to law, and count the effects of different extraction time The extraction of acid.
表8Table 8
从表8可知，控制甘草药粉的一级提取时间在合理的范围内，以获得更佳的甘草酸提取率。It can be seen from Table 8 that the primary extraction time of the licorice powder is controlled within a reasonable range to obtain a better extraction rate of glycyrrhizic acid.
(3)提取级次(3) Extraction level
取甘草药材，粉碎至100目，100kg，一级提取加入乙醇-水(V/V；90/10)500L，加5M硫酸-盐酸(V/V，1/1)适量，搅拌，提取30min，过滤，收集一级提取液体积，2～5级提取：采用上述相同方法，再对药材提取四级，即共提取5级，分别收集各级提取液，记录提取液体积，依法检测各级提取液浓度，统计提取级次对甘草药材甘草酸的提取情况。Take the licorice material, crush it to 100 mesh, 100kg, add 500L of ethanol-water (V/V; 90/10), add 5M sulfuric acid-hydrochloric acid (V/V, 1/1) appropriate amount, stir and extract for 30min, Filter, collect the volume of the first-level extract, and extract the level 2-5: use the same method as above, and then extract the medicinal materials at four levels, that is, a total of 5 levels. Collect each level of the extract, record the volume of the extract, and test each level of extraction in accordance with the law The concentration of the liquid, the extraction of glycyrrhizic acid from the glycyrrhizic acid by the extraction grades.
表9Table 9
| 级次level | 浓度(％)concentration(%) | 提取率(％)Extraction rate (%) | 综合提取率(％)Comprehensive extraction rate (%) |
| 11 | 7.627.62 | 84.25％84.25% | 84.25％84.25% |
| 22 | 1.151.15 | 12.14％12.14% | 96.39％96.39% |
| 33 | 0.220.22 | 2.39％2.39% | 98.78％98.78% |
| 44 | 0.060.06 | 0.64％0.64% | 99.42％99.42% |
| 55 | 0.020.02 | 0.18％0.18% | 99.60％99.60% |
从表9可知，控制甘草药粉的提取级次在合理范围内，以获得更佳的甘草酸提取率。It can be seen from Table 9 that the extraction grade of the glycyrrhizinate powder is controlled within a reasonable range to obtain a better extraction rate of glycyrrhizic acid.
(4)提取液酸度(4) Acidity of extract
取甘草药材，粉碎至80目，取5份，100g/份，各加入甲醇-乙醇-水(V/V/V，10/85/5)500mL，加5M硫酸-盐酸(V/V，1/1)适量，分别控制体系pH值在1.0、1.5、2.0、2.5、3、4、5、6左右，搅拌，提取30min，过滤，收集滤液，记录提取液体积，依法检测提取液浓度，统计不同酸度对对甘草药材甘草酸的提取影响情况。Take the licorice material, crush it to 80 mesh, take 5 portions, 100g/portion, add 500mL methanol-ethanol-water (V/V/V, 10/85/5), add 5M sulfuric acid-hydrochloric acid (V/V, 1 /1) Appropriate amount, respectively control the pH value of the system at about 1.0, 1.5, 2.0, 2.5, 3, 4, 5, 6, stir, extract for 30 minutes, filter, collect the filtrate, record the volume of the extract, detect the concentration of the extract according to law, and make statistics The effect of different acidity on the extraction of glycyrrhizic acid from licorice herbal material.
表10Table 10
| 编号Numbering | 体系pHSystem pH | 浓度(mg/mL)Concentration (mg/mL) | 提取率(％)Extraction rate (%) |
| 11 | 1.101.10 | 5.83％5.83% | 76.7％76.7% |
| 22 | 1.531.53 | 5.92％5.92% | 77.9％77.9% |
| 33 | 2.082.08 | 5.76％5.76% | 76.2％76.2% |
| 44 | 2.612.61 | 5.87％5.87% | 77.2％77.2% |
| 55 | 3.043.04 | 5.62％5.62% | 73.9％73.9% |
| 66 | 4.214.21 | 2.34％2.34% | 30.8％30.8% |
| 77 | 5.025.02 | 1.01％1.01% | 13.3％13.3% |
| 88 | 6.116.11 | 0.11％0.11% | 1.5％1.5% |
从表10可知，控制体系pH在合理范围内，保证药材适宜的提取率。It can be seen from Table 10 that the pH of the control system is within a reasonable range to ensure the appropriate extraction rate of the medicinal materials.
(5)甘草酸盐制备pH值的确定(5) Determination of pH value of glycyrrhizinate preparation
取甘草药材一级提取液5份，200ml/份，分别加入0.1M氨-氯化铵混合溶液(V/V，4/1)适量，分别调节体系pH至5.0、6.0、7.0、8.0、9.0、10.0左右，过滤，收集滤液，检测滤液甘草酸盐残留占比。Take 5 parts of the first-level extract of Glycyrrhiza medicinal materials, 200ml/part, add appropriate amount of 0.1M ammonia-ammonium chloride mixed solution (V/V, 4/1) respectively, adjust the pH of the system to 5.0, 6.0, 7.0, 8.0, 9.0 respectively , 10.0, filter, collect the filtrate, and detect the proportion of glycyrrhizinate residue in the filtrate.
表11Table 11
从表11可知，控制体系pH在合理范围内，使甘草酸盐更多的沉淀，使滤液残留浓度尽可能低。It can be seen from Table 11 that by controlling the pH of the system within a reasonable range, more glycyrrhizinate can be precipitated, and the residual concentration of the filtrate can be as low as possible.
前述对本发明的具体示例性实施方案的描述是为了说明和例证的目的。这些描述并非想将本发明限定为所公开的精确形式，并且很显然，根据上述教导，可以进行很多改变和变化。对示例性实施例进行选择和描述的目的在于解释本发明的特定原理及其实际应用，从而使得本领域的技术人员能够实现并利用本发明的各种不同的示例性实施方案以及各种不同的选择和改变。本发明的范围意在由权利要求书及其等同形式所限定。The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and illustration. These descriptions are not intended to limit the invention to the precise form disclosed, and it is obvious that many changes and changes can be made based on the above teachings. The purpose of selecting and describing exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can implement and use various exemplary embodiments and various different embodiments of the present invention. Choice and change. The scope of the present invention is intended to be defined by the claims and their equivalents.
Claims (17)
- 一种从甘草中提取甘草酸粗品的方法，其特征在于，包括以下步骤：A method for extracting crude glycyrrhizic acid from licorice is characterized in that it comprises the following steps:甘草粉碎：将甘草药材经粉碎处理成甘草药粉，控制甘草药粉的粒度在30～200目；以及Licorice pulverization: the licorice material is crushed into glycyrrhiza powder, and the particle size of the licorice powder is controlled to be 30-200 mesh; and一级提取：将有机溶剂和水组成第一溶剂，然后与所述甘草药粉以(2-20)：1(V/W)的配比混合，搅拌并加入第一pH调节剂，调节pH值，搅拌提取0.5-3小时，过滤，收集一级提取液和药渣；所述一级提取液即为甘草酸粗品。Primary extraction: The first solvent is composed of organic solvent and water, and then mixed with the glycerin powder in a ratio of (2-20):1 (V/W), stirring and adding the first pH regulator to adjust the pH value , Stir and extract for 0.5-3 hours, filter, and collect the first-level extract and medicine residue; the first-level extract is the crude glycyrrhizic acid.
- 一种从甘草中提取甘草酸粗品的方法，其特征在于，包括以下步骤：A method for extracting crude glycyrrhizic acid from licorice is characterized in that it comprises the following steps:甘草粉碎：将甘草药材经粉碎处理成甘草药粉，控制甘草药粉的粒度在30～200目；Licorice crushing: the licorice material is crushed into licorice powder, and the particle size of the licorice powder is controlled to be 30-200 mesh;一级提取：将有机溶剂和水组成第一溶剂，然后与所述甘草药粉以(2-20)：1(V/W)的配比混合，搅拌并加入第一pH调节剂，调节pH值，搅拌提取0.5-3小时，过滤，收集一级提取液和一级药渣；以及Primary extraction: The first solvent is composed of organic solvent and water, and then mixed with the glycerin powder in a ratio of (2-20):1 (V/W), stirring and adding the first pH regulator to adjust the pH value , Stir and extract for 0.5-3 hours, filter, collect the first-level extract and the first-level medicine residue; andK级提取：将所述第一溶剂与上一级获取的药渣以(2-20)：1(V/W)的配比混合，搅拌并加入第一pH调节剂，调节pH值，搅拌提取0.5-3小时，过滤，收集K级提取液和K级药渣；以及K-level extraction: mix the first solvent and the dregs obtained from the previous level at a ratio of (2-20):1 (V/W), stir and add the first pH regulator, adjust the pH value, and stir Extract for 0.5-3 hours, filter, and collect K-level extract and K-level medicine residue; and从K＝2开始执行所述K级提取直至N级提取，从而完成完整的一轮提取，N大于等于2小于等于8，From K=2, perform the K-level extraction until the N-level extraction, so as to complete a complete round of extraction, N is greater than or equal to 2 and less than or equal to 8,其中所述一级提取液即为甘草酸粗品。The first-level extract is the crude glycyrrhizic acid.
- 如权利要求2所述的从甘草中提取甘草酸粗品的方法，其特征在于，在每轮提取过程中，所述第一溶剂还包括上一轮提取中除所述一级提取液以外的各级提取液。The method for extracting crude glycyrrhizic acid from licorice according to claim 2, characterized in that, in each round of extraction, the first solvent also includes all the components in the previous round of extraction except for the first-level extract. Grade extraction liquid.
- 如权利要求2所述的从甘草中提取甘草酸粗品的方法，其特征在于，所述一级提取步骤中，所述有机溶剂选自醇类、醇类衍生物、和酮类中的至少一种。The method for extracting crude glycyrrhizic acid from licorice according to claim 2, wherein in the primary extraction step, the organic solvent is selected from at least one of alcohols, alcohol derivatives, and ketones. Kind.
- 如权利要求2所述的从甘草中提取甘草酸粗品的方法，其特征在于，所述一级提取步骤中，所述有机溶剂选自甲醇、乙醇、乙酸乙酯和丙酮中的至少一种。The method for extracting crude glycyrrhizic acid from licorice according to claim 2, characterized in that, in the primary extraction step, the organic solvent is selected from at least one of methanol, ethanol, ethyl acetate and acetone.
- 如权利要求2所述的从甘草中提取甘草酸粗品的方法，其特征在于，所述一级提取步骤中，所述水在第一溶剂中所占的体积百分比为0-30％。The method for extracting crude glycyrrhizic acid from licorice according to claim 2, characterized in that, in the primary extraction step, the volume percentage of the water in the first solvent is 0-30%.
- 如权利要求2所述的从甘草中提取甘草酸粗品的方法，其特征在于，所述一级提取步骤和所述K级提取步骤中，所述第一pH调节剂选自如下物质中的至少一种：H 2SO 4、HCl、H 3PO 4、多聚磷酸、CH 3COOH、枸橼酸、草酸、阳离子交换树脂、pH≤2的缓冲体系、以及由上述物质中的至少一种配制成的不同浓度的溶液。 The method for extracting crude glycyrrhizic acid from licorice according to claim 2, wherein in the first-level extraction step and the K-level extraction step, the first pH adjusting agent is selected from at least the following substances One kind: H 2 SO 4 , HCl, H 3 PO 4 , polyphosphoric acid, CH 3 COOH, citric acid, oxalic acid, cation exchange resin, buffer system with pH≤2, and formulated with at least one of the above substances Into different concentrations of solutions.
- 如权利要求2所述的从甘草中提取甘草酸粗品的方法，其特征在于，所述一级提取步骤和所述K级提取步骤中，调节pH值≤4。The method for extracting crude glycyrrhizic acid from licorice according to claim 2, characterized in that, in the first-level extraction step and the K-level extraction step, the pH value is adjusted to be ≤4.
- 如权利要求2所述的从甘草中提取甘草酸粗品的方法，其特征在于，所述一级提取步骤和所述K级提取步骤中，调节pH值至0.5-4之间。The method for extracting crude glycyrrhizic acid from licorice according to claim 2, characterized in that, in the first-level extraction step and the K-level extraction step, the pH value is adjusted to between 0.5-4.
- 一种从甘草中提取甘草黄酮的方法，其特征在于，包括权利要求2中的所有步骤以及后续的浓缩步骤，所述浓缩步骤如下所示：A method for extracting licorice flavonoids from licorice, characterized in that it comprises all the steps in claim 2 and subsequent concentration steps, and the concentration steps are as follows:浓缩：将所述一级提取液调节pH至5-10之间，搅拌，生成沉淀，过滤，收集滤液，滤液浓缩，烘干，得甘草黄酮。Concentration: Adjust the pH of the first-level extract to between 5-10, stir, generate precipitation, filter, collect the filtrate, concentrate the filtrate, and dry to obtain licorice flavonoids.
- 如权利要求10所述的从甘草中提取甘草黄酮的方法，其特征在于，所述浓缩步骤中，将所述一级提取液中加入第二pH调节剂以调节pH至5-10之间。The method for extracting licorice flavonoids from licorice according to claim 10, wherein in the concentration step, a second pH regulator is added to the primary extract to adjust the pH to between 5-10.
- 如权利要求11所述的从甘草中提取甘草黄酮的方法，其特征在于，所述第二pH调节剂选自如下物质中的至少一种：金属、金属氧化物、金属氢氧化物、金属形成的盐、阴离子交换树脂、氨气、液氨、氨水、铵盐、三乙胺、pH缓冲范围为9～13的缓冲体系、以及由上述物质中的至少一种配制成的各种浓度的溶液；其中，所述金属为金属钠、钾、钙、镁、锌，所述金属氧化物为钠、钾、钙、镁、锌的氧化物，所述金属形成的盐为钠、钾、钙、镁、锌形成的各种酸盐，所述金属氢氧化物为钠、钾、钙、镁、锌的氢氧化物。The method for extracting licorice flavonoids from licorice according to claim 11, wherein the second pH adjusting agent is selected from at least one of the following substances: metals, metal oxides, metal hydroxides, metal formation Salt, anion exchange resin, ammonia gas, liquid ammonia, ammonia water, ammonium salt, triethylamine, a buffer system with a pH buffer range of 9-13, and solutions of various concentrations prepared from at least one of the above substances Wherein, the metal is the metal sodium, potassium, calcium, magnesium, zinc, the metal oxide is the oxide of sodium, potassium, calcium, magnesium, zinc, the salt formed by the metal is sodium, potassium, calcium, Various acid salts formed by magnesium and zinc, and the metal hydroxides are hydroxides of sodium, potassium, calcium, magnesium, and zinc.
- 一种从甘草中提纯获得甘草酸盐的方法，其特征在于，包括权利要求2中的所有步骤以及后续的纯化步骤，所述纯化步骤如下所示：A method for purifying and obtaining glycyrrhizinate from licorice, characterized in that it comprises all the steps in claim 2 and subsequent purification steps, and the purification steps are as follows:纯化：向所述一级提取液中添加第二pH调节剂，控制体系pH值，过滤，收集沉淀，然后将所述沉淀用洗液洗涤，过滤，分别收集过滤后的洗液和沉淀，所述洗涤后的沉淀于50～80℃烘干，即得甘草酸盐。Purification: Add a second pH regulator to the first-level extract, control the pH of the system, filter, collect the precipitate, then wash the precipitate with a lotion, filter, and collect the filtered lotion and precipitate respectively. The precipitate after washing is dried at 50-80°C to obtain glycyrrhizinate.
- 如权利要求13所述的从甘草中提纯获得甘草酸盐的方法，其特征在于，在每轮提取过程中，所述第一溶剂还包括上一轮提取中除所述一级提取液以外的各级提 取液与纯化步骤中过滤后的洗液所组成的混合液。The method for obtaining glycyrrhizinate by purification from licorice according to claim 13, characterized in that, in each round of extraction, the first solvent also includes the first-level extract in the previous round of extraction. The mixed liquid composed of various levels of extraction liquid and the filtered washing liquid in the purification step.
- 如权利要求13所述的从甘草中提纯获得甘草酸盐的方法，其特征在于，所述纯化步骤中，所述第二pH调节剂选自如下物质中的至少一种：金属、金属氧化物、金属氢氧化物、金属形成的盐、阴离子交换树脂、氨气、液氨、氨水、铵盐、三乙胺、pH缓冲范围为9～13的缓冲体系、以及由上述物质中的至少一种配制成的各种浓度的溶液；其中，所述金属为金属钠、钾、钙、镁、锌；所述金属氧化物为钠、钾、钙、镁、锌的氧化物；所述金属形成的盐为钠、钾、钙、镁、锌形成的各种酸盐；所述金属氢氧化物为钠、钾、钙、镁、锌的氢氧化物。The method for purifying and obtaining glycyrrhizinate from licorice according to claim 13, characterized in that, in the purification step, the second pH adjusting agent is selected from at least one of the following substances: metals, metal oxides , Metal hydroxide, metal salt, anion exchange resin, ammonia gas, liquid ammonia, ammonia water, ammonium salt, triethylamine, a buffer system with a pH buffer range of 9-13, and at least one of the above substances Prepared solutions of various concentrations; wherein the metal is sodium, potassium, calcium, magnesium, and zinc; the metal oxide is an oxide of sodium, potassium, calcium, magnesium, and zinc; the metal is formed Salts are various acid salts formed by sodium, potassium, calcium, magnesium, and zinc; the metal hydroxides are hydroxides of sodium, potassium, calcium, magnesium, and zinc.
- 如权利要求13所述的从甘草中提纯获得甘草酸盐的方法，其特征在于，所述纯化步骤中，将pH值控制在5-10之间。The method for obtaining glycyrrhizinate by purification from licorice according to claim 13, characterized in that, in the purification step, the pH value is controlled between 5-10.
- 如权利要求13所述的从甘草中提纯获得甘草酸盐的方法，其特征在于，所述纯化步骤中，所述洗液与所述第一溶剂相同，所述洗液是在常温或加热的条件下进行洗涤的；优选的，所述加热的温度为40-80℃，所述洗涤的次数为2-3次。The method for purifying glycyrrhizinate from licorice according to claim 13, characterized in that, in the purification step, the washing liquid is the same as the first solvent, and the washing liquid is heated at room temperature or heated. The washing is performed under the conditions; preferably, the heating temperature is 40-80° C., and the washing times are 2-3 times.
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