Plant extraction and purification method of glycyrrhizic coumarin

Abstract

The invention discloses a plant extraction and purification method of glycyrrhizic coumarin, and belongs to the technical field of natural product extraction. The method comprises a purification method suitable for industrial production of glycyrrhizic coumarin and a purification method for laboratory preparation of glycyrrhizic coumarin. According to the laboratory purification method, licorice extract sequentially passes through a silica gel column and a C18 column for coarse separation and then is refined through semi-preparative liquid chromatography, the glycyrrhizic coumarin with the purity higher than 98% is obtained and can be used as a standard substance for licorice quality research, and an important basis is provided for further deep research and development of glycyrrhizic coumarin. According to the present invention, the industrial production method is based on the laboratory extraction step, the steps are simplified, such that the method is suitable for the pilot scale production, the steps are simple, the efficiency is high, the yield is high, the glycyrrhizic coumarin with the purity of more than 98% can be obtained through the separation, and the advantages are provided for the further research and the wide application of the glycyrrhizic coumarin.

Description

Technical Field

[0001] The present invention relates to the technical field of natural product extraction, and particularly to a method for extracting and purifying glycyrrhizin coumarin from plants. Background Art

[0002] Licorice is a perennial herb of the genus Glycyrrhiza in the family Leguminosae and belongs to traditional Chinese medicine in China. The Chinese Pharmacopoeia includes three varieties, Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat., and Glycyrrhiza glabra L., as the sources of medicinal licorice. Due to its characteristics of being both medicine and food, it is listed as a traditional Chinese medicine that is both medicine and food. Licorice has the effects of invigorating the spleen and replenishing qi, clearing heat and detoxifying, resolving phlegm and relieving cough, alleviating spasm and pain, and coordinating various medicines. It is used for spleen and stomach weakness, lassitude and weakness, palpitation and shortness of breath, cough with copious phlegm, abdominal and limb spasmodic pain, carbuncles and sores, and relieving the toxicity and potency of drugs.

[0003] Numerous studies on licorice at home and abroad have shown that licorice contains flavonoids, triterpenoids, coumarins, saponins, organic acids, stilbenoids, etc. Licorice has anti-inflammatory, antioxidant, antibacterial, antiviral, antitumor, immunomodulatory, and liver-protecting effects and is widely used in the pharmaceutical industry, cosmetics industry, and food industry. The many pharmacological activities of licorice are closely related to its various chemical components.

[0004] Coumarins are an important class of chemical components contained in licorice, mainly including glycyrrhizin furanocoumarin, glycyrrhizin pyranocoumarin, scopoletin, esculetin, Liquiritcoumarin, glycyrrhizin aromatic coumarin, glycyrrhizin coumarin, isoglycyrrhizin coumarin, glycyrrhizinol, isoglycyrrhizinol, etc. Modern pharmacological research has shown that coumarins have anti-inflammatory, analgesic, antioxidant, antitumor, liver-protecting, and central nerve-protecting effects.

[0005] Glycycoumarin is one of the most representative coumarin compounds in licorice, mainly existing in Glycyrrhiza uralensis Fisch. Modern research shows that glycycoumarin has antioxidant, anti-inflammatory, antiviral, and antibacterial effects. However, the content of glycycoumarin in licorice is very low, resulting in a high extraction cost of glycycoumarin. At present, there is no article or patent reporting the extraction and purification method of pure glycycoumarin, especially the extraction and purification method of glycycoumarin for industrial production. For example, the prior art (application number 202411056766.1) discloses an extract of Glycyrrhiza uralensis Fisch. and its preparation method and application. This patent extracts Glycyrrhiza uralensis Fisch. by heating with a solvent, concentrates it under reduced pressure to obtain a concentrated solution of Glycyrrhiza uralensis Fisch., then adds an appropriate amount of solvent to the concentrated solution of Glycyrrhiza uralensis Fisch. for ultrasonic extraction, takes the supernatant for centrifugation, and obtains the test licorice extract. The relative percentage content of glycycoumarin in this test licorice extract is only 0.26%.

[0006] It can be seen that there is currently no purification method for the refined extraction of pure glycycoumarin, let alone an industrial production method for glycycoumarin. This application creates a new plant extraction and purification method for glycycoumarin on this basis, enabling it to not only obtain a method for producing pure glycycoumarin in the laboratory, but also achieve the production and preparation of glycycoumarin at a pilot scale or above, which is of great significance for the further development and wide application of glycycoumarin. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a plant extraction and purification method for glycycoumarin, aiming at industrial production, overcoming the technical problems of low content and difficult extraction of glycycoumarin, and obtaining a method for producing high-purity glycycoumarin that meets the requirements of laboratory and industrial preparation, which is of great significance for the further development and wide application of glycycoumarin.

[0008] To solve the above technical problems, the present invention provides a plant extraction and purification method for glycycoumarin. The method is suitable for the industrial production of glycycoumarin and specifically includes the following steps:

[0009] Q1: Select licorice raw materials, dry the moisture, and cut the licorice into thin slices or crush it;

[0010] Q2: Add the sliced or crushed licorice into an extraction tank and extract it by heating under reflux with ethyl acetate;

[0011] Q3: Combine the extraction solutions, concentrate them into an extract, and redissolve the obtained extract with ethyl acetate;

[0012] Q4: Add silica gel to the ethyl acetate re-dissolved solution obtained in Q3. The amount of silica gel used is 1 / 30 to 1 / 5 of the extract paste. After stirring and standing, take the supernatant and concentrate it, and dissolve it with a mixed solvent of ethyl acetate - n - hexane, where the content of ethyl acetate in the ethyl acetate - n - hexane mixed solvent is 30% to 60%;

[0013] Q5: Wet-load the mixed solution of Q4 onto a silica gel column and elute it with a n - hexane - ethyl acetate system. The ratios of n - hexane and ethyl acetate during the elution process are 100:0, 80:20, and 70:30 in sequence. Collect the eluates in fractions, and use a high - performance liquid chromatograph to detect the collected eluates. Combine the eluates with a purity of glycyrrhizin coumarin ≥ 85% and concentrate them under reduced pressure to obtain a paste;

[0014] Q6: Dissolve the concentrated paste obtained in Q5 with a solvent, and then use the method of evaporation crystallization or cooling crystallization to obtain glycyrrhizin coumarin crystals. After pulverization, obtain glycyrrhizin coumarin powder, where the purity of glycyrrhizin coumarin ≥ 98%.

[0015] Further improvement: In step Q1, the thickness of the licorice slices is 0.5 to 4 mm; in step Q2, the amount of ethyl acetate used is 5 to 20 mL / g, the extraction time is 30 to 120 min, the extraction temperature is 60 to 90 °C, and the number of extraction times is 2 to 3 times; in step Q3, the amount of ethyl acetate for re - dissolution is 6 to 15 mL / g.

[0016] Further improvement: In step Q4, the silica gel added is 300 - 400 mesh chromatography silica gel, and the amount of the ethyl acetate - n - hexane mixed solvent used is 5 to 10 mL / g, and the standing time is 30 to 120 min.

[0017] Further improvement: In step Q5, the sample loading amount of the silica gel column is 1 / 15 to 1 / 40 of its silica gel amount. Among them, 100:0 n - hexane and ethyl acetate are used to rinse 3 to 4 column volumes, 80:20 n - hexane and ethyl acetate are used to rinse 5 to 6 column volumes, and 70:30 n - hexane and ethyl acetate are used to rinse 10 to 12 column volumes.

[0018] Further improvement: In step Q6, the solvent is one or more of petroleum ether, diethyl ether, methyl tert - butyl ether, n - pentane, ethyl acetate, n - hexane, cyclohexane, dichloromethane, ethanol, and the amount of the solvent used is 3 to 10 times that of the concentrated paste.

[0019] The present invention also provides a method for plant extraction and purification of glycyrrhizin coumarin, which is applicable to the laboratory preparation of glycyrrhizin coumarin, and includes the following steps:

[0020] S1: Select licorice raw materials, dry the moisture, and cut the licorice into thin slices or pulverize it;

[0021] S2: Add the sliced or crushed licorice into the extraction tank, and perform heat reflux extraction with ethyl acetate.

[0022] S3: Concentrate the extract obtained in S2, dissolve it with dichloromethane. After complete dissolution, let it stand and filter.

[0023] S4: Load the solution filtered in S3 onto a silica gel column by wet method, and elute with a dichloromethane - methanol system. The ratios of dichloromethane to methanol during the elution process are 100:0 and 95:5 in sequence. Collect the eluate in fractions, and detect the collected eluate with a high - performance liquid chromatograph. Combine the eluate with a glycyrrhizin content ≥ 20%, concentrate it under reduced pressure until the solvent is removed, and then dissolve it with n - hexane.

[0024] S5: Load the n - hexane solution of glycyrrhizin collected in S4 onto a silica gel column by wet method, and elute with a n - hexane - ethyl acetate system. The ratios of n - hexane to ethyl acetate during the elution process are 80:20 and 70:30 in sequence. Collect the eluate in fractions, and detect the collected eluate with a high - performance liquid chromatograph. Combine the eluate with a glycyrrhizin content ≥ 20%, concentrate it under reduced pressure until the solvent is removed, and then dissolve it with methanol.

[0025] S6: Load the methanol solution of glycyrrhizin collected in S5 onto a C18 column for separation by wet method, and elute with a methanol - water system. The ratios of methanol to water during the elution process are 50:50 and 70:30 in sequence. Collect the eluate in fractions, and detect the collected eluate with a high - performance liquid chromatograph. Combine the eluate with a glycyrrhizin content ≥ 20%, concentrate it under reduced pressure until the solvent is removed, and then dissolve it with methanol.

[0026] S7: Load the methanol solution of glycyrrhizin collected in S6 onto a semi - preparative liquid chromatograph, and wash it with a methanol - water system with a ratio of methanol to water of 70:30. Individually collect the fractions when glycyrrhizin peaks appear. After combination, concentrate it under reduced pressure until there is no methanol smell, and then perform freeze - drying to obtain glycyrrhizin powder with a purity ≥ 98%.

[0027] For further improvement, the thickness of the sliced licorice in step S1 is 0.5 - 4 mm; the dosage of ethyl acetate in step S2 is 5 - 20 mL / g, the extraction time is 30 - 120 min, the extraction temperature is 60 - 90 °C, and the extraction times are 2 - 3 times; the dosage of dichloromethane in step S3 is 4 - 15 mL / g, and the standing time is 20 - 120 min.

[0028] For further improvement, the silica gel selected in step S4 is 300 - 400 - mesh chromatography silica gel. The sample loading amount on the silica gel column is 1 / 2 - 1 / 10 of the silica gel dosage. Among them, the 100:0 dichloromethane and methanol mixture is used to wash 6 - 7 column volumes, and the 95:5 dichloromethane and methanol mixture is used to wash 5 - 6 column volumes.

[0029] In step S5, the silica gel selected is chromatography silica gel with a mesh size of 300 - 400. The sample loading amount of the silica gel column is 1 / 2 - 1 / 10 of the silica gel usage amount. Among them, a mixture of n - hexane and ethyl acetate with a ratio of 80:20 is used to rinse for 16 - 17 column volumes, and a mixture of n - hexane and ethyl acetate with a ratio of 70:30 is used to rinse for 8 - 9 column volumes.

[0030] Further improvement: In step S6, the sample loading amount of the C18 column is 1 / 10 - 1 / 20 of its packing amount. Among them, a mixture of methanol and water with a ratio of 50:50 is used to rinse for 5 - 6 column volumes, and a mixture of methanol and water with a ratio of 70:30 is used to rinse for 5 - 6 column volumes again.

[0031] Further improvement: In step S7, the injection volume of the semi - preparative liquid chromatography is 10 - 100 μL.

[0032] After adopting such a design, the present invention has at least the following advantages:

[0033] The present invention is a plant extraction and purification method suitable for extracting glycocoumarin in the laboratory. By passing the licorice extract through a silica gel column and a C 18 column for rough fractionation in sequence, and then refining it through semi - preparative liquid chromatography, glycocoumarin with a purity higher than 98% is obtained. The operation is simple, the extraction efficiency is high, and the cost is low. It can be used as a reference substance for the quality research of licorice, providing an important basis for the further in - depth research and development of glycocoumarin, and facilitating the application of glycocoumarin in different industries.

[0034] The present invention is a plant extraction and purification method suitable for industrial production of glycocoumarin. Based on the laboratory extraction steps, through simplifying the steps, it is suitable for production on a scale above pilot scale. The steps are simpler, the efficiency is high, the yield is high, and the purity of the separated glycocoumarin can also reach more than 98%. It truly overcomes the disadvantage of high cost in extracting glycocoumarin from licorice, providing favorable conditions for the further research and wide application of glycocoumarin. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The above is only an overview of the technical solution of the present invention. In order to understand the technical means of the present invention more clearly, the following provides a more detailed description of the present invention in combination with the drawings and specific embodiments.

[0036] Figure 1 It is the high - performance liquid chromatography diagram of glycocoumarin in the glycocoumarin powder obtained in Example 1 of the present invention.

[0037] Figure 2 It is the nuclear magnetic resonance hydrogen spectrum of glycocoumarin in the glycocoumarin powder obtained in Example 1 of the present invention.

[0038] Figure 3 It is the nuclear magnetic resonance carbon spectrum of glycocoumarin in the glycocoumarin powder obtained in Example 1 of the present invention. Detailed Embodiments

[0039] Exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although the exemplary embodiments of the present invention are shown in the drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided so that the present invention can be more thoroughly understood and the scope of the present invention can be completely conveyed to those skilled in the art.

[0040] Embodiment 1

[0041] The extraction and separation process of glycyrrhizin coumarin in this embodiment is used to process less than 2 kg of licorice to achieve the preparation of glycyrrhizin coumarin in small doses. The specific extraction and purification steps include:

[0042] (1) Raw material pretreatment: Select licorice from Korla, Xinjiang, dry the moisture, and cut it into thin slices with a thickness of 1 mm.

[0043] After subjecting the pretreated licorice to alcohol extraction, the content of glycyrrhizin coumarin in it was detected by high performance liquid chromatography. The content of glycyrrhizin coumarin in the treated licorice was 0.14%.

[0044] (2) Extraction: Take 1 kg of the treated licorice, place it in a heating reflux extractor, add 10 L of ethyl acetate, heat and reflux for extraction. The extraction temperature is 75 °C. After extraction three times, the extraction liquids are combined. Among them, the first extraction time is 60 min, and the second and third extraction times are 75 min.

[0045] (3) Concentration: Concentrate the extraction liquid obtained in the above (2) until the solvent is removed, dissolve it with dichloromethane at 6 mL / g. After complete dissolution, let it stand for 40 min, and filter it with gauze to obtain the filtered dichloromethane solution.

[0046] (4) Primary separation by silica gel column: Wet-load the dichloromethane solution obtained in the above (3) onto a silica gel column for separation. The silica gel used is 300 - 400 mesh chromatography silica gel. The diameter-to-height ratio of the silica gel column is 1:4, and the sample loading amount is 1 / 3 of the silica gel amount. Elute with a dichloromethane-methanol system. First, rinse with a dichloromethane-methanol system for 6 - 7 column volumes, where the volume fraction of methanol in the dichloromethane-methanol system is 0%; then rinse with a dichloromethane-methanol system for 5 - 6 column volumes, where the volume fraction of methanol in the dichloromethane-methanol system is 5%, and collect the eluate in fractions.

[0047] At the same time, use a high performance liquid chromatograph to detect glycyrrhizin coumarin in each fraction of the eluate, combine the eluates with a glycyrrhizin coumarin content ≥ 20%, concentrate under reduced pressure until the solvent is removed, and dissolve with a small amount of n-hexane.

[0048] Among them, the chromatographic conditions of the high performance liquid chromatograph are: C18 Chromatographic column, 4.6×250 mm, 5 μm, mobile phase A is methanol, mobile phase B is pure water, gradient elution with 70% A - 90% A, elution flow rate is 1 mL / min, column temperature is 25 °C, injection volume is 10 μL, detection wavelength is 254 nm.

[0049] (5) Secondary separation on silica gel column: The hexane solution obtained in the above (4) is loaded onto the silica gel column for separation by wet method. The silica gel used is also 300 - 400 mesh chromatography silica gel, the diameter - height ratio of the silica gel column is 1:4, the sample loading amount is 1 / 3 of the silica gel amount, eluted with a hexane - ethyl acetate system. First, wash with a hexane - ethyl acetate system for 16 - 17 column volumes, where the volume fraction of ethyl acetate in the hexane - ethyl acetate system is 20%; then wash with a hexane - ethyl acetate system for 8 - 9 column volumes, where the volume fraction of ethyl acetate in the hexane - ethyl acetate system is 30%, and collect the eluate in fractions.

[0050] At the same time, detect glycocoumarin in each fraction of the eluate under the same high - performance liquid chromatography conditions, combine the eluates with glycocoumarin content ≥ 20%, concentrate under reduced pressure until solvent - free, and dissolve with a small amount of methanol.

[0051] (6) C 18 Separation on chromatography column: The methanol solution obtained in the above (5) is loaded onto column C for separation by wet method. 18 Column C 18 Column C is a finished column, model number SW - 8222 - 330 - SP, the sample loading amount is 1 / 15 of the packing amount. First, wash with a methanol - water system for 5 - 6 column volumes, where the volume fraction of methanol in the methanol - water system is 50%; then wash with a methanol - water system for 5 - 6 column volumes, where the volume fraction of methanol in the methanol - water system is 70%, and collect the eluate in fractions.

[0052] At the same time, detect glycocoumarin in each fraction of the eluate under the same high - performance liquid chromatography conditions, combine the eluates with glycocoumarin content ≥ 20%, concentrate under reduced pressure until solvent - free, and then dissolve with a small amount of methanol.

[0053] (7) Purification: The methanol solution obtained in the above (6) is filtered through a needle - type filter, then injected into semi - preparative liquid chromatography, with an injection volume of 20 μL each time, washed with a methanol - water system, and collect the fractions when glycocoumarin elutes separately. After combination, concentrate under reduced pressure until there is no methanol smell, and freeze - dry to obtain glycocoumarin powder, a total of 0.7240 g.

[0054] Then, detect the content of glycocoumarin in the obtained glycocoumarin powder under the same high - performance liquid chromatography conditions, and the content of glycocoumarin is 98.67%.

[0055] That is, the yield of glycyrrhizin coumarin obtained by the extraction and purification method reaches 51.7%, and the purity ≥ 98%.

[0056] Appendix Figure 1 Appendix shows the high performance liquid chromatography (HPLC) chromatogram of glycyrrhizin coumarin in the glycyrrhizin coumarin powder obtained in this example. Figure 2 Appendix shows the 1H NMR spectrum of glycyrrhizin coumarin in the glycyrrhizin coumarin powder obtained in this example. Figure 3 Appendix shows the 13C NMR spectrum of glycyrrhizin coumarin in the glycyrrhizin coumarin powder obtained in this example. The chemical structural formula of glycyrrhizin coumarin is obtained as follows:

[0057]

[0058] Example 2

[0059] The extraction and separation process of glycyrrhizin coumarin in this example specifically includes the following steps:

[0060] (1) Raw material pretreatment: Licorice from Zhangye, Gansu is selected, its moisture is dried, and then it is crushed by a pulverizer.

[0061] After the pretreatment of licorice, after alcohol extraction, the content of glycyrrhizin coumarin in it is detected by high performance liquid chromatography. The content of glycyrrhizin coumarin in the treated licorice is 0.14%.

[0062] (2) Extraction: Take 1 kg of the treated licorice, place it in a heating reflux extractor, add 15 L of ethyl acetate, heat and reflux for extraction. The extraction temperature is 80 °C. After extraction for 3 times, the extraction solutions are combined. Among them, the first extraction time is 45 min, and the second and third extraction times are 60 min.

[0063] (3) Concentration: Concentrate the extraction solution obtained in (2) above until the solvent is removed, dissolve it with dichloromethane at 8 mL / g. After complete dissolution, let it stand for 60 min, and filter it with gauze to obtain the filtered dichloromethane solution.

[0064] (4) Primary separation by silica gel column: Wet-load the dichloromethane solution obtained in (3) above onto a silica gel column for separation. The silica gel used is 300 - 400 mesh chromatography silica gel. The diameter-height ratio of the silica gel column is 1:4, and the sample loading amount is 1 / 4 of the silica gel amount. Elute with a dichloromethane - methanol system. First, rinse with the dichloromethane - methanol system for 6 - 7 column volumes, where the volume fraction of methanol in the dichloromethane - methanol system is 0%; then rinse with the dichloromethane - methanol system for 5 - 6 column volumes, where the volume fraction of methanol in the dichloromethane - methanol system is 5%, and collect the eluate in fractions.

[0065] At the same time, use a high performance liquid chromatograph to detect glycyrrhizin coumarin in each fraction of the eluate, combine the eluate with glycyrrhizin coumarin content ≥ 20%, concentrate it under reduced pressure until the solvent is removed, and dissolve it with a small amount of n-hexane.

[0066] Among them, the chromatographic conditions of the high-performance liquid chromatograph are as follows: C 18 chromatographic column, 4.6×250 mm, 5 μm, mobile phase A is methanol, mobile phase B is pure water, gradient elution with 70% A - 90% A, elution flow rate is 1 mL / min, column temperature is 25°C, injection volume is 10 μL, and detection wavelength is 254 nm.

[0067] (5) Secondary separation on silica gel column: The hexane solution obtained in the above (4) is separated by wet loading on a silica gel column. The silica gel used is also 300 - 400 mesh chromatography silica gel. The diameter-height ratio of the silica gel column is 1:4, the sample loading amount is 1 / 4 of the silica gel amount, and it is eluted with a hexane-ethyl acetate system. First, rinse with a hexane-ethyl acetate system for 16 - 17 column volumes, where the volume fraction of ethyl acetate in the hexane-ethyl acetate system is 20%; then rinse with a hexane-ethyl acetate system for 8 - 9 column volumes, where the volume fraction of ethyl acetate in the hexane-ethyl acetate system is 30%, and collect the eluate in fractions.

[0068] At the same time, detect glycocoumarin in each fraction of the eluate under the same high-performance liquid chromatography conditions, combine the eluates with a glycocoumarin content ≥ 20%, concentrate under reduced pressure until the solvent is removed, and dissolve with a small amount of methanol.

[0069] (6) C 18 Column chromatography separation: The methanol solution obtained in the above (5) is separated by wet loading on a C 18 column. The C 18 column is a finished column, model SW-8222-330-SP. The sample loading amount is 1 / 10 of the packing amount. First, rinse with a methanol-water system for 5 - 6 column volumes, where the volume fraction of methanol in the methanol-water system is 50%; then rinse with a methanol-water system for 5 - 6 column volumes, where the volume fraction of methanol in the methanol-water system is 70%, and collect the eluate in fractions.

[0070] At the same time, detect glycocoumarin in each fraction of the eluate under the same high-performance liquid chromatography conditions, combine the eluates with a glycocoumarin content ≥ 20%, concentrate under reduced pressure until the solvent is removed, and then dissolve with a small amount of methanol.

[0071] (7) Purification: After filtering the methanol solution obtained in the above (6) with a syringe filter, inject it into semi-preparative liquid chromatography. The injection volume each time is 30 μL, rinse with a methanol-water system, and separately collect the fractions when glycocoumarin elutes. After combining, concentrate under reduced pressure until there is no methanol smell, and freeze-dry to obtain glycocoumarin powder, totaling 0.8056 g.

[0072] Then, detect the content of glycocoumarin in the obtained glycocoumarin powder under the same high-performance liquid chromatography conditions, and it is found that the glycocoumarin content is 98.73%.

[0073] That is, the yield of glycyrrhizin coumarin obtained by this extraction and purification method reaches 57.5%, and the purity is ≥98%.

[0074] Example 3

[0075] The extraction and separation process of glycyrrhizin coumarin in this example is used to process more than 2 kg of licorice to realize the pilot-scale production of glycyrrhizin coumarin. The specific extraction and purification steps include:

[0076] (1) Raw material pretreatment: In this example, the pretreated licorice from Korla, Xinjiang in Example 1 is still used.

[0077] (2) Extraction: Take 5 kg of the treated licorice, place it in a heating reflux extractor, add 50 L of ethyl acetate, and carry out heating reflux extraction. The extraction temperature is 75°C. After extracting 3 times, combine the extraction solutions. Among them, the first extraction time is 60 min, and the second and third extraction times are 75 min.

[0078] (3) Concentration: Concentrate the extraction solution obtained in the above (2) into an extract to obtain an extract of licorice extract, and the extract is redissolved with ethyl acetate at 10 mL / g.

[0079] (4) Impurity removal: Add silica gel accounting for 1 / 10 of the weight of the extract to the ethyl acetate solution obtained in the above (3). The silica gel is 300-400 mesh chromatography silica gel. After stirring, let it stand for 60 min, take the supernatant, concentrate the supernatant into an extract, and then dissolve it with a mixed solvent of ethyl acetate - n-hexane. The dosage of the mixed solvent is 6 mL / g, and the content of ethyl acetate in the ethyl acetate - n-hexane mixed solvent is 40% to obtain a sample for column chromatography.

[0080] (5) Silica gel column separation: Wet-load the sample for column chromatography obtained in the above (4) onto a silica gel column for separation. The silica gel is 300-400 mesh chromatography silica gel. The diameter-to-height ratio of the silica gel column is 1:8, and the sample loading amount is 1 / 30 of the silica gel amount. First, rinse with a n-hexane - ethyl acetate system for 3-4 column volumes, where the volume fraction of ethyl acetate in the n-hexane - ethyl acetate system is 0%; then rinse with a n-hexane - ethyl acetate system for 5-6 column volumes, where the volume fraction of ethyl acetate in the n-hexane - ethyl acetate system is 20%; finally, rinse with a n-hexane - ethyl acetate system for 10-12 column volumes, where the volume fraction of ethyl acetate in the n-hexane - ethyl acetate system is 30%, and collect the eluate in fractions.

[0081] At the same time, use a high-performance liquid chromatograph to detect glycyrrhizin coumarin in each fraction of the eluate, combine the eluates with a glycyrrhizin coumarin content ≥85%, and concentrate under reduced pressure until the solvent is removed to obtain an extract of glycyrrhizin coumarin.

[0082] Among them, the chromatographic conditions of the high-performance liquid chromatograph are: C18 Chromatographic column, 4.6×250 mm, 5 μm, mobile phase A is methanol, mobile phase B is pure water, gradient elution with 70% A - 90% A, elution flow rate is 1 mL / min, column temperature is 25 °C, injection volume is 10 μL, detection wavelength is 254 nm.

[0083] (6) Crystallization: Dissolve the glycocoumarin extract obtained in the above (5) with 6 times of ethyl acetate, and use the evaporation crystallization method to obtain glycocoumarin crystals, which are pulverized to obtain glycocoumarin powder, totaling 4.83 g.

[0084] Then, detect the content of glycocoumarin in the glycocoumarin powder obtained under the same high - performance liquid chromatography conditions, and it is found that the content of glycocoumarin is 98.42%.

[0085] That is, the yield of glycocoumarin obtained by this extraction and purification method reaches 69%, and the purity ≥ 98%.

[0086] Example 4

[0087] The extraction and separation process of glycocoumarin in this example is used to process more than 2 kg of licorice to realize the pilot - scale production of glycocoumarin. The specific extraction and purification steps are as follows:

[0088] (1) Raw material pretreatment: In this example, the licorice from Zhangye, Gansu, pretreated in Example 2 is used.

[0089] (2) Extraction: Take 5 kg of the treated licorice, place it in a heating reflux extractor, add 100 L of ethyl acetate, heat and reflux for extraction. The extraction temperature is 80 °C. After extraction 3 times, the extraction solutions are combined. Among them, the first extraction time is 60 min, and the second and third extraction times are 75 min.

[0090] (3) Concentration: Concentrate the extraction solution obtained in the above (2) into an extract to obtain a licorice extract, and the extract is redissolved with 8 mL / g of ethyl acetate.

[0091] (4) Impurity removal: Add silica gel with a weight of 1 / 20 of the extract to the ethyl acetate solution obtained in the above (3). The silica gel is 300 - 400 - mesh chromatography silica gel. After stirring and standing for 90 min, take the supernatant, concentrate the supernatant into an extract, and then dissolve it with a mixed solvent of ethyl acetate - n - hexane. The dosage of the mixed solvent is 8 mL / g, and the content of ethyl acetate in the ethyl acetate - n - hexane mixed solvent is 40% to obtain a sample for column chromatography.

[0092] (5) Silica gel column separation: The column sample obtained in the above (4) is separated by wet loading on a silica gel column. The silica gel used is 300 - 400 mesh chromatography silica gel. The ratio of the diameter to the height of the silica gel column is 1:8, and the sample loading amount is 1 / 25 of the silica gel amount. First, rinse with a n - hexane - ethyl acetate system for 3 - 4 column volumes, where the volume fraction of ethyl acetate in the n - hexane - ethyl acetate system is 0%; then rinse with a n - hexane - ethyl acetate system for 5 - 6 column volumes, where the volume fraction of ethyl acetate in the n - hexane - ethyl acetate system is 20%; finally, rinse with a n - hexane - ethyl acetate system for 10 - 12 column volumes, where the volume fraction of ethyl acetate in the n - hexane - ethyl acetate system is 30%, and collect the eluate in fractions.

[0093] At the same time, use a high - performance liquid chromatograph to detect glycocoumarin in each fraction of the eluate, combine the eluates with a glycocoumarin content ≥ 85%, and concentrate under reduced pressure until the solvent is removed to obtain a glycocoumarin extract.

[0094] Among them, the chromatographic conditions of the high - performance liquid chromatograph are: C 18 chromatographic column, 4.6×250 mm, 5 μm, mobile phase A is methanol, mobile phase B is pure water, gradient elution with 70%A - 90%A, elution flow rate is 1 mL / min, column temperature is 25 °C, injection volume is 10 μL, and detection wavelength is 254 nm.

[0095] (6) Crystallization: Dissolve the glycocoumarin extract obtained in the above (5) with 6 times the amount of ethyl acetate, and use the cooling crystallization method to obtain glycocoumarin crystals, which are pulverized to obtain glycocoumarin powder, totaling 4.52 g.

[0096] Then, detect the content of glycocoumarin in the obtained glycocoumarin powder under the same high - performance liquid chromatographic conditions, and it is found that the glycocoumarin content is 98.85%.

[0097] That is, the yield of glycocoumarin obtained by this extraction and purification method reaches 64.6%, and the purity ≥ 98%.

[0098] It can be seen from the above Examples 1 - 2 that using this laboratory method for extracting and purifying glycocoumarin can obtain a pure glycocoumarin product with a yield greater than 50% and a purity ≥ 98%. This method is feasible, simple, and has stable results in the laboratory, and can be used as a reference substance for licorice quality research. It can also be seen from the above Examples 3 and 4 that using this pilot - scale production method for extracting and purifying glycocoumarin from plants, by simplifying the impurity - removal steps, only one silica gel column separation and solvent crystallization method are required, which can simply and conveniently achieve the preparation of pure glycocoumarin, and the comprehensive cost is lower, and the loss rate of glycocoumarin is lower, which is suitable for pilot - scale production and above - scale industrial production.

[0099] The extraction and separation method of glycyrrhizin coumarin of the present invention comprehensively considers the economic cost and time cost of preparing glycyrrhizin coumarin, and can achieve the extraction and separation of glycyrrhizin coumarin with a purity ≥ 98% from licorice, which is beneficial to the industrial production and wide application of glycyrrhizin coumarin, and is of great significance to the industrial application of glycyrrhizin coumarin.

[0100] As mentioned above, it is only a preferred embodiment of the present invention, and there is no restriction on the present invention in any form. Those skilled in the art make some simple modifications, equivalent changes or decorations using the disclosed technical content above, and all fall within the protection scope of the present invention.

Claims

1. A method for extracting and purifying glycyrrhizic coumarin from plants, characterized in that: The method is suitable for the industrial production of glycyrrhizic coumarin, and specifically comprises the following steps: Q1: Select licorice raw material, dry the water, and cut the licorice into thin slices or crush it; Q2: Add sliced ​​or crushed licorice into an extraction tank and heat and reflux with ethyl acetate for extraction; Q3: Combine the extracts, concentrate into an extract, and reconstitute the obtained extract with ethyl acetate; Q4: Add silica gel to the ethyl acetate reconstituted solution obtained in Q3, with the amount of silica gel being 1 / 30 to 1 / 5 of the extract, stir and let stand, take the supernatant and concentrate it, and dissolve it with a mixed solvent of ethyl acetate and n-hexane, wherein the content of ethyl acetate in the mixed solvent of ethyl acetate and n-hexane is 30 to 60%; Q5: The mixed solution of Q4 was wet-applied to a silica gel column and eluted with a n-hexane-ethyl acetate system. The ratio of n-hexane to ethyl acetate during the elution process was 100:0, 80:20, and 70:30, respectively. The eluate was collected in sections and detected by a high performance liquid chromatography. The eluate with a glycyrrhizin purity of ≥85% was combined and concentrated under reduced pressure to obtain an extract. Q6: The concentrated extract obtained in Q5 is dissolved in a solvent, and then evaporation crystallization or cooling crystallization is used to obtain glycyrrhizin coumarin crystals, which are crushed to obtain glycyrrhizin coumarin powder, wherein the purity of glycyrrhizin coumarin is ≥98%.

2. The method for extracting and purifying glycyrrhizic coumarin from plants according to claim 1, characterized in that: The thickness of the licorice slices in step Q1 is 0.5-4 mm; the amount of ethyl acetate used in step Q2 is 5-20 mL / g, the extraction time is 30-120 min, the extraction temperature is 60-90° C., and the extraction times are 2-3 times; and the amount of ethyl acetate used for re-dissolution in step Q3 is 6-15 mL / g.

3. The method for extracting and purifying glycyrrhizic coumarin from plants according to claim 2, characterized in that: The silica gel added in step Q4 is 300-400 mesh chromatography silica gel, and the amount of ethyl acetate-n-hexane mixed solvent is 5-10 mL / g, and the standing time is 30-120 min.

4. The method for extracting and purifying glycyrrhizic coumarin from plants according to claim 3, characterized in that: The amount of sample loaded on the silica gel column in step Q5 is 1 / 15 to 1 / 40 of the amount of silica gel used, wherein 3 to 4 column volumes are washed with 100:0 n-hexane and ethyl acetate, 5 to 6 column volumes are washed with 80:20 n-hexane and ethyl acetate, and 10 to 12 column volumes are washed with 70:30 n-hexane and ethyl acetate.

5. The method for extracting and purifying glycyrrhizic coumarin from plants according to claim 4, characterized in that: In step Q6, the solvent is one or more of petroleum ether, ethyl ether, methyl tert-butyl ether, n-pentane, ethyl acetate, n-hexane, cyclohexane, dichloromethane, and ethanol, and the amount of the solvent is 3 to 10 times that of the concentrated extract.

6. A method for extracting and purifying glycyrrhizic coumarin from plants, characterized in that: The method comprises the following steps: S1: Select licorice raw material, dry the water, and cut the licorice into thin slices or crush it; S2: adding the sliced ​​or crushed licorice into an extraction tank, and heating and refluxing with ethyl acetate for extraction; S3: Concentrate the extract obtained in S2, dissolve it with dichloromethane, and after it is completely dissolved, let it stand and filter; S4: wet-loading the filtered solution of S3 onto a silica gel column, eluting with a dichloromethane-methanol system, wherein the ratio of dichloromethane to methanol during the elution process is 100:0 and 95:5, respectively, collecting the eluate in sections, and detecting the collected eluate with a high performance liquid chromatography, combining the eluate with a glycyrrhizin coumarin content ≥ 20%, concentrating under reduced pressure until it contains no solvent, and then dissolving with n-hexane; S5: wet-loading the n-hexane solution of glycyrrhizin collected in S4 onto a silica gel column, eluting with a n-hexane-ethyl acetate system, wherein the ratio of n-hexane to ethyl acetate during the elution process is 80:20 and 70:30, respectively, collecting the eluate in sections, and detecting the collected eluate with a high performance liquid chromatography, combining the eluate with a glycyrrhizin content of ≥20%, concentrating under reduced pressure until it contains no solvent, and then dissolving it with methanol; S6: wet C-chromatography of the methanol solution of glycyrrhizic coumarin collected in S5 18 Column separation, elution with a methanol-water system, the ratio of methanol to water during the elution process is 50:50 and 70:30, respectively, the eluate is collected in sections, and the collected eluate is detected by high performance liquid chromatography, the eluate with a glycyrrhizic coumarin content of ≥20% is combined, concentrated under reduced pressure until it contains no solvent, and then dissolved with methanol; S7: The glycyrrhizin methanol solution collected in S6 was subjected to semi-preparative liquid chromatography and washed with a methanol-water system with a ratio of methanol to water of 70:

30. The fractions at the peak of glycyrrhizin were collected separately, combined, concentrated under reduced pressure until there was no methanol smell, and freeze-dried to obtain glycyrrhizin powder with a purity of ≥98%.

7. The method for extracting and purifying glycyrrhizic coumarin from plants according to claim 6, characterized in that: The thickness of the licorice slices in step S1 is 0.5-4 mm; in step S2, the amount of ethyl acetate is 5-20 mL / g, the extraction time is 30-120 min, the extraction temperature is 60-90° C., and the number of extractions is 2-3 times; in step S3, the amount of dichloromethane is 4-15 mL / g, and the standing time is 20-120 min.

8. The method for extracting and purifying glycyrrhizic coumarin from plants according to claim 7, characterized in that: The silica gel selected in step S4 is 300-400 mesh chromatography silica gel, and the sample loading amount of the silica gel column is 1 / 2-1 / 10 of the silica gel dosage, wherein the 100:0 dichloromethane and methanol mixture is used to wash 6-7 column volumes, and the 95:5 dichloromethane and methanol mixture is used to wash 5-6 column volumes; The silica gel selected in step S5 is 300-400 mesh chromatography silica gel, and the sample loading amount of the silica gel column is 1 / 2-1 / 10 of the silica gel dosage, wherein 16-17 column volumes are washed with 80:20 n-hexane and ethyl acetate, and 8-9 column volumes are washed with 70:30 n-hexane and ethyl acetate.

9. The method for extracting and purifying glycyrrhizic coumarin from plants according to claim 8, characterized in that: Step S6 C 18 The sample loading volume of the column is 1 / 10~1 / 20 of its packing volume, among which 50:50 methanol and water are used to wash 5~6 column volumes, and 70:30 methanol and water are used to wash another 5~6 column volumes.

10. The method for extracting and purifying glycyrrhizic coumarin from plants according to claim 9, characterized in that: The injection volume of the semi-preparative liquid chromatography in step S7 is 10-100 μL.

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