Method for extracting tilianin from dracocephalum moldavica

Abstract

The invention provides a method for extracting tilianin from dracocephalum moldavica, and relates to the technical field of medicine extraction. The method comprises the following steps: mixing dracocephalum moldavica or dracocephalum moldavica products with an ethanol water solution, extracting, and concentrating the obtained extracting solution to obtain a concentrated solution; mixing the concentrated solution with petroleum ether, carrying out first extraction, and collecting a water phase; mixing the water phase with ethyl acetate for second extraction, collecting an ethyl acetate phase, and removing the solvent in the ethyl acetate phase to obtain an ethyl acetate extract; dissolving the ethyl acetate extract with dimethyl sulfoxide, and mixing the obtained extract solution with water for precipitation to obtain a precipitate; and recrystallizing the precipitate to obtain tilianin. According to the method disclosed by the invention, the restriction of column chromatography is avoided, and the high-purity monomer extraction of tilianin in dracocephalum moldavica is realized by adopting an extraction method, an anti-solvent method and a recrystallization method. The method provided by the invention is simple in process, the purity of the finished product is high (93% or above and can reach 97%), and batch production is easy to realize.

Description

Technical Field

[0001] The present invention relates to the technical field of drug extraction, and particularly to a method for extracting silymarin from Dracocephalum moldavica. Background Art

[0002] Dracocephalum moldavica L. is a characteristic Uyghur medicine native to Xinjiang. Its Uyghur name is Badiranjbuya. It is an annual herbaceous plant of the Lamiaceae family. It is often used as medicine with the whole above-ground part. Its medicinal material standard is included in both "Drug Standards of the Ministry of Health of the People's Republic of China·Uyghur Medicine Volume" and "Uyghur Medicine (Part I)". The medicinal materials of Dracocephalum moldavica mainly contain components such as flavonoids, volatile oils, phenylpropanoids, polysaccharides, amino acids, proteins, and trace elements. As one of the main components in the effective part of total flavonoids of Dracocephalum moldavica, silymarin has pharmacological effects such as treating atherosclerosis, protecting myocardial ischemia-reperfusion injury, anti-inflammatory, and antioxidant. It has certain curative effects on various diseases, especially has a significant therapeutic effect on cardiovascular diseases, and has strong application research value.

[0003] At present, there is little research on the extraction and purification of silymarin at home and abroad. It mainly uses alcohol extraction followed by separation and purification using traditional column chromatography. For example, total flavonoids are obtained from Dracocephalum moldavica using a macroporous resin adsorption chromatography column, and then silymarin is obtained by silica gel column chromatography of the total flavonoids. This method has high costs, long time consumption, low purity, and low yield due to the restriction of column chromatography in the process, making it difficult to meet the requirements of batch preparation. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a method for extracting silymarin from Dracocephalum moldavica. The present invention avoids the restriction of column chromatography and provides a method for extracting silymarin with simple process, low cost, and high product purity.

[0005] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides a method for extracting silymarin from Dracocephalum moldavica, including the following steps:

[0007] Mix Dracocephalum moldavica or Dracocephalum moldavica products with an ethanol aqueous solution for extraction, and concentrate the obtained extract to obtain a concentrated solution;

[0008] Mix the concentrated solution with petroleum ether for the first extraction, and collect the aqueous phase;

[0009] Mix the aqueous phase with ethyl acetate for the second extraction, and collect the ethyl acetate phase; Remove the solvent in the ethyl acetate phase to obtain an ethyl acetate extract;

[0010] Dissolve the ethyl acetate extract with dimethyl sulfoxide, mix the resulting extract solution with water for precipitation to obtain a precipitate;

[0011] Recrystallize the precipitate to obtain silymarin; the solvent used for recrystallization is an aqueous alcohol solution.

[0012] Preferably, the volume fraction of ethanol in the aqueous ethanol solution is 35-75%, and the dosage ratio of Dracocephalum moldavica or Dracocephalum moldavica product to the aqueous ethanol solution is 1 g:(10-50) mL.

[0013] Preferably, the extraction temperature is 70-90 °C, the number of extractions is 1-3 times, and the single extraction time is 1-3 h.

[0014] Preferably, the density of the concentrated solution is 0.93-0.98 g / cm 3 。

[0015] Preferably, the volume ratio of the concentrated solution to petroleum ether is 1:(1-3).

[0016] Preferably, the volume ratio of the aqueous phase to ethyl acetate is 1:(1-3).

[0017] Preferably, the concentration of the extract solution is 50-200 mg / mL, and the volume ratio of the extract solution to water is 1:10-1:50.

[0018] Preferably, the mixing of the extract solution and water is to add water to the extract solution, and the addition rate of water is 0.5-6 mL / min.

[0019] Preferably, the alcohol in the aqueous alcohol solution is methanol or ethanol, the volume fraction of alcohol in the aqueous alcohol solution is 10-70%, and the aqueous alcohol solution is alkaline.

[0020] Preferably, the recrystallization includes: dissolving the precipitate in the aqueous alcohol solution until supersaturated, then performing solid-liquid separation, cooling the obtained liquid phase for crystallization to obtain a crystalline substance; successively performing acid washing, water washing and drying on the crystalline substance; the dissolution temperature is 20-60 °C, and the crystallization temperature is 2-8 °C.

[0021] The present invention provides a method for extracting silybin from Dracocephalum moldavica, comprising the following steps: mixing Dracocephalum moldavica or a Dracocephalum moldavica product with an aqueous ethanol solution for extraction, concentrating the obtained extract to obtain a concentrated solution; mixing the concentrated solution with petroleum ether for first extraction and collecting the aqueous phase; mixing the aqueous phase with ethyl acetate for second extraction and collecting the ethyl acetate phase; removing the solvent in the ethyl acetate phase to obtain an ethyl acetate extract; dissolving the ethyl acetate extract with dimethyl sulfoxide, mixing the obtained extract solution with water for precipitation to obtain a precipitate; and recrystallizing the precipitate to obtain silybin. The present invention avoids the limitation of column chromatography, and adopts extraction, anti-solvent method and recrystallization method to realize the extraction of high-purity monomer of silybin from Dracocephalum moldavica. The method provided by the present invention has simple process, low cost, high product purity (above 93%, up to 97%), and is easy to realize batch production. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is a process flow chart for extracting silybin from Dracocephalum moldavica in an embodiment of the present invention;

[0023] Figure 2 is the influence of ethanol concentration on the yield of silybin in Example 1;

[0024] Figure 3 is the influence of extraction temperature on the yield of silybin in Example 1;

[0025] Figure 4 is the influence of solid-liquid ratio on the yield of silybin in Example 1;

[0026] Figure 5 is the influence of extraction time on the yield of silybin in Example 1;

[0027] Figure 6 is the influence of extraction times on the yield of silybin in Example 1;

[0028] Figure 7 is the high performance liquid chromatography (HPLC) of the ethanol extract in Example 2;

[0029] Figure 8 is the high performance liquid chromatography (HPLC) of the petroleum ether layer in Example 2;

[0030] Figure 9 is the high performance liquid chromatography (HPLC) of the ethyl acetate layer in Example 2;

[0031] Figure 10 is the high performance liquid chromatography (HPLC) of the water layer in Example 2;

[0032] Figure 11 is the high performance liquid chromatography (HPLC) of different concentrations of the drug by the anti-solvent method in Example 3;

[0033] Figure 12 It is the high performance liquid chromatography (HPLC) of the silybin product in Example 4. Figure 12 In (a) is the high performance liquid chromatography of the silybin product, and in (b) is the enlarged view of the inset (ultraviolet spectrogram) in (a). Detailed implementation mode

[0034] The present invention provides a method for extracting silybin from Dracocephalum moldavica, comprising the following steps:

[0035] Mix Dracocephalum moldavica or Dracocephalum moldavica products with an ethanol aqueous solution for extraction, and concentrate the obtained extract to obtain a concentrated solution;

[0036] Mix the concentrated solution with petroleum ether for the first extraction, and collect the aqueous phase;

[0037] Mix the aqueous phase with ethyl acetate for the second extraction, and collect the ethyl acetate phase; Remove the solvent in the ethyl acetate phase to obtain an ethyl acetate extract;

[0038] Dissolve the ethyl acetate extract with dimethyl sulfoxide, mix the obtained extract solution with water for precipitation to obtain a precipitate;

[0039] Recrystallize the precipitate to obtain silybin; The solvent used for the recrystallization is an alcohol aqueous solution.

[0040] In the present invention, unless otherwise specified, the raw materials involved are well-known commercially available products in the art.

[0041] Figure 1 It is the process flow chart for extracting silybin from Dracocephalum moldavica in the embodiment of the present invention. The following is combined with Figure 1 for detailed description.

[0042] The present invention mixes Dracocephalum moldavica or Dracocephalum moldavica products with an ethanol aqueous solution for extraction, and concentrates the obtained extract to obtain a concentrated solution.

[0043] In the present invention, the Dracocephalum moldavica or the Dracocephalum moldavica product is preferably in powder form. The Dracocephalum moldavica or the Dracocephalum moldavica product can be pulverized and sieved to obtain powder, and the present invention has no particular requirement for the particle size of the powder; in the embodiments of the present invention, the Dracocephalum moldavica product is specifically Dracocephalum moldavica cut pieces. In the present invention, the volume fraction of ethanol in the ethanol aqueous solution is preferably 35-75%, and can be 35%, 50%, 55%, 60%, 65%, 70% or 75%, more preferably 55%; the dosage ratio of the Dracocephalum moldavica or the Dracocephalum moldavica product to the ethanol aqueous solution (i.e., the solid-liquid ratio) is preferably 1 g:(10-50) mL, and can be 1 g:10 mL, 1 g:20 mL, 1 g:30 mL, 1 g:40 mL or 1 g:50 mL, more preferably 1 g:30 mL, and the mass of the Dracocephalum moldavica or the Dracocephalum moldavica product is calculated based on dry weight. In the present invention, the extraction temperature is preferably 70-90°C, and can be 70, 75, 80, 85 or 90°C, more preferably 80°C, and the extraction temperature is preferably achieved by water bath heating; the number of extractions is preferably 1-3 times, and can be 1, 2 or 3 times, more preferably 2 times, and the single extraction time is preferably 1-3 h, and can be 1, 1.5, 2, 2.5 or 3 h, more preferably 1.5 h. By controlling the above extraction conditions in the present invention, a higher yield of silybin can be obtained.

[0044] In the present invention, the specific operation of the extraction is preferably as follows: adding the ethanol aqueous solution to the Dracocephalum moldavica or the Dracocephalum moldavica product, and performing extraction under the condition that the water bath temperature is 70-90°C, and filtering to obtain an extract (i.e., an ethanol extract); when the number of extractions is greater than 1 time, the single extracts obtained by filtration after each extraction are combined as the extract.

[0045] In the present invention, the concentration is preferably vacuum concentration. The density of the concentrated solution is preferably 0.93-0.98 g / cm 3 and can be 0.93, 0.94, 0.95, 0.96, 0.97 or 0.98 g / cm 3 . By controlling the density of the concentrated solution within the above range in the present invention, a small amount of ethanol remains in the concentrated solution, which is beneficial to improving the enrichment effect of silybin in the subsequent ethyl acetate extraction stage and increasing the transfer rate of silybin; when the density of the concentrated solution is too large, the subsequent ethyl acetate extraction effect is poor, and silybin cannot be effectively enriched, resulting in a decrease in the transfer rate; when the density of the concentrated solution is too small, more ethanol remains, and in the subsequent ethyl acetate extraction stage, it will be miscible with ethyl acetate, resulting in abnormal extraction and enrichment.

[0046] After obtaining the concentrated solution, the present invention mixes the concentrated solution with petroleum ether for the first extraction, collects the aqueous phase (denoted as the first aqueous phase), and the rest is the petroleum ether phase.

[0047] In the present invention, the volume ratio of the concentrated solution to petroleum ether (PE) is preferably 1:(1 - 3), and can be 1:1, 1:2 or 1:3. When the addition amount of the petroleum ether is too small, the extraction of pigments, fat-soluble components and small polar substances is incomplete, affecting the purity of the final product. When the addition amount of the petroleum ether is too large, the production cost increases.

[0048] In the present invention, the specific operation method of the first extraction is preferably as follows: placing the concentrated solution in a separating funnel, adding petroleum ether thereto, shaking well, standing for stratification, to obtain a petroleum ether phase (upper layer, also referred to as the petroleum ether layer in the examples) and a first aqueous phase (lower layer, also referred to as the first aqueous layer in the examples). In the present invention, the function of the first extraction is to remove pigments, fat-soluble components and small polar substances in the concentrated solution. The above substances are present in the petroleum ether layer, and the petroleum ether layer does not dissolve silymarin; the first aqueous phase is the product phase, and a small amount of ethanol remaining in the concentrated solution is also present in the first aqueous phase.

[0049] After obtaining the first aqueous phase, in the present invention, the first aqueous phase is mixed with ethyl acetate for a second extraction, and the ethyl acetate phase is collected, and the rest is the aqueous phase (denoted as the second aqueous phase).

[0050] In the present invention, the volume ratio of the first aqueous phase to ethyl acetate is preferably 1:(1 - 3), and can be 1:1, 1:2 or 1:3. When the addition amount of the ethyl acetate is too small, the extraction of silymarin is incomplete, resulting in a decrease in the yield. When the addition amount of the ethyl acetate is too large, the production cost increases, the extraction impurities increase, affecting the purity of the final product.

[0051] In the present invention, the specific operation of the second extraction is preferably as follows: placing the first aqueous phase in a separating funnel, adding ethyl acetate thereto, shaking well, standing for stratification, to obtain an ethyl acetate phase (upper layer, also referred to as the ethyl acetate layer in the examples) and a second aqueous phase (lower layer, also referred to as the second aqueous layer in the examples).

[0052] In the present invention, since the solubility of silymarin in ethyl acetate is much greater than that in water, most of it is transferred to the ethyl acetate layer; the main components of the second aqueous phase are substances with relatively large polarity and a small amount of silymarin.

[0053] After obtaining the ethyl acetate phase, in the present invention, the solvent in the ethyl acetate phase is removed to obtain an ethyl acetate extract.

[0054] The present invention preferably removes the solvent in the ethyl acetate phase by means of vacuum drying. The solvent is mainly ethyl acetate and also includes a small amount of ethanol.

[0055] Through two-step extraction in the present invention, silymarin is enriched, the purity is improved, and each step of extraction has a high transfer rate of silymarin.

[0056] After obtaining the ethyl acetate extract, the present invention dissolves the ethyl acetate extract with dimethyl sulfoxide (DMSO), mixes the extract solution with water for precipitation, and obtains a precipitate.

[0057] In the present invention, the concentration of the extract solution is preferably 50-200 mg / mL, and can be 50, 100, 150 or 200 mg / mL, more preferably 50 mg / mL. In the present invention, the water is preferably deionized water, and the volume ratio of the extract solution to water is preferably 1:10-1:50, and can be 1:10, 1:20, 1:30, 1:40 or 1:50, more preferably 1:20; the mixing of the extract solution and water is preferably adding water to the extract solution, the addition rate of the water is preferably 0.5-6 mL / min, and can be 0.5, 1, 2, 3, 4, 5 or 6 mL / min, more preferably 4 mL / min, and the addition method of the water is preferably dropwise addition. By controlling the above conditions in the present invention, it is beneficial to improve the purity of silymarin while ensuring a high transfer rate of silymarin.

[0058] In the present invention, the specific operation of the precipitation is preferably: adding water to the extract solution, stirring, standing for precipitation, centrifuging the obtained suspension, and washing the obtained solid phase (removing dimethyl sulfoxide) and drying in sequence to obtain the precipitate. In the present invention, the standing time is subject to sufficient precipitation and can be 24 h.

[0059] The present invention purifies the enriched silymarin by the antisolvent method. The antisolvent method is to first dissolve the poorly soluble drug in an organic solvent miscible with water, mix it with water (antisolvent) under certain conditions, and a supersaturation phenomenon occurs when the two phases are mixed, so that the poorly soluble drug precipitates from the solvent system to form crystal nuclei, and the crystals further grow, and finally purification is obtained. The antisolvent method involves process conditions such as the type of good solvent, the initial concentration of the drug, the volume ratio of the good solvent-antisolvent, and the injection speed.

[0060] In the present invention, dimethyl sulfoxide shows good solubility in the ethyl acetate extract, and dimethyl sulfoxide belongs to Class 3 solvents (solvents with low potential toxicity) in the ICH residual solvent classification. Therefore, dimethyl sulfoxide is used as the good solvent; since dimethyl sulfoxide is miscible with water and the solubility of silymarin in pure water is only 1.57 μg / mL, water is selected as the antisolvent, and obvious precipitates appear after adding water.

[0061] After obtaining the precipitate, the present invention recrystallizes the precipitate to obtain silymarin.

[0062] In the present invention, the solvent used for recrystallization is preferably an aqueous alcohol solution. The alcohol in the aqueous alcohol solution is preferably methanol or ethanol. Since ethanol belongs to Class 3 solvents of ICH, it is more preferably ethanol. The volume fraction of alcohol in the aqueous alcohol solution is preferably 10-70%, and can be 10%, 20%, 30%, 40%, 50%, 60% or 70%, more preferably 30%. The aqueous alcohol solution is preferably alkaline, and the pH value is preferably greater than 7 and less than or equal to 12, and can be 8, 9, 10, 11 or 12, more preferably 12. In the present invention, saturated lime water is preferably added to the aqueous alcohol solution to adjust the aqueous alcohol solution to be alkaline. By controlling the above recrystallization conditions, the present invention is beneficial to improving the purity of silymarin.

[0063] In the present invention, the recrystallization preferably includes: dissolving the precipitate in the aqueous alcohol solution until supersaturated, followed by solid-liquid separation, cooling the obtained liquid phase for crystallization to obtain a crystalline product; sequentially performing pickling, water washing and drying on the crystalline product to obtain pure silymarin.

[0064] In the present invention, the temperature for dissolution is preferably 20-60°C, and can be 30, 40 or 50°C. The temperature for dissolution can be achieved by performing water bath heating on the aqueous alcohol solution. In the present invention, the precipitate is preferably added to the aqueous alcohol solution at 20-60°C, and stirred until supersaturated. In the present invention, the method of solid-liquid separation can be centrifugation or filtration. After centrifugation, the supernatant is taken, and after filtration, the continuous filtrate is taken to obtain the liquid phase. In the present invention, the temperature for crystallization is preferably 2-8°C, and can be 2, 3, 4, 5, 6, 7 or 8°C; the crystallization is preferably carried out under static conditions, and the crystallization time is based on sufficient crystallization, and can be 24 h; the crystalline product can be obtained by centrifugal separation.

[0065] In the present invention, the reagent used for pickling (also known as acidified washing) is preferably hydrochloric acid; the specific operation of pickling is preferably: after drying the crystalline product, it is mixed with water to obtain a suspension; the pH value of the suspension is adjusted to 5-6 with hydrochloric acid, and filtered to obtain a pickled product; the solid-liquid ratio of the crystalline product to water can be 1 mg:4 mL. In the present invention, the reason for pickling is that since an alkaline aqueous alcohol solution is used during recrystallization, flavonoid compounds may undergo chemical reactions under alkaline conditions and turn yellow; since saturated lime water is used to adjust the pH of the aqueous alcohol solution, non-acidification will result in the presence of calcium salts in the product, which are difficult to remove and affect the product content. In the present invention, the water washing is specifically to wash the pickled product to neutral.

[0066] To further illustrate the present invention, the method for extracting silymarin from Dracocephalum moldavica provided by the present invention will be described in detail below with reference to examples, but they should not be construed as limiting the protection scope of the present invention.

[0067] Example 1

[0068] Alcohol extraction process of silybin

[0069] Taking the yield of silybin as the evaluation index, the effects of ethanol concentration, extraction temperature, solid-liquid ratio, extraction time, and extraction times on the extraction of silybin were investigated.

[0070] (1) Ethanol concentration

[0071] Take 10 g of the powder of the medicinal material (Dracocephalum moldavica L. slices), weigh it precisely, add the solvent according to the solid-liquid ratio of 1:10 (w / v, g / mL), keep the water bath temperature at 75 °C, extract once, and the extraction time is 1 h. Investigate the effects of different concentrations of ethanol aqueous solutions 15%, 35%, 55%, 75%, 95% (v / v) on the yield of silybin extraction. Conduct 3 parallel experiments, measure the yield of silybin, and take the average value. The results are as Figure 2 shown in Table 1; among them, the calculation formula for the yield of silybin is: yield of silybin (mg / g) = mass of silybin (mg) / mass of medicinal material (g). The content of silybin is determined by liquid chromatography. Chromatographic conditions: SunFire-C18 column (5 μm, 4.6×150 mm); acetonitrile-0.1% formic acid aqueous solution, gradient elution: 0 - 10 min, 20 - 25% acetonitrile; 10 - 15 min, 25 - 30% acetonitrile; 15 - 25 min, 30 - 50% acetonitrile; 25 - 30 min, 50% acetonitrile; detection wavelength 330 nm; flow rate 1.0 mL / min; column temperature 35 °C, injection volume 10 μL.

[0072] Table 1 Yield of silybin extracted by ethanol aqueous solutions with different concentrations

[0073]

[0074] The yield of silybin first increases and then decreases with the concentration of the ethanol aqueous solution, and reaches the maximum value of 1.98 mg / g when the concentration of the ethanol aqueous solution is 55%. When the concentration of the ethanol aqueous solution is less than 55%, increasing the ethanol concentration will reduce the polarity of the solution. According to the "similar compatibility" principle, silybin with similar polarity is more easily dissolved. The increase in the concentration of the ethanol aqueous solution will damage the plant cell wall and accelerate the dissolution of flavonoids from the cells. However, when the concentration of the ethanol aqueous solution exceeds 55%, the yield of silybin begins to decrease, which may be due to the excessive ethanol concentration leading to the dissolution of other impurities and the too low polarity of the solution, which is not conducive to the dissolution of silybin. Therefore, 55% ethanol is more suitable.

[0075] (2) Extraction temperature

[0076] Take 10g of the powder of the medicinal material (Xiangqinglan decoction piece), accurately weigh it, put it into 55% (v / v) ethanol aqueous solution according to the solid-liquid ratio of 1:10 (w / v, g / mL), extract it once, and the extraction time is 1h. The effect of different extraction temperatures of 50, 60, 70, 80, and 90℃ on the extraction rate of cynarin was investigated. The experiment was carried out in parallel for 3 times to determine the cynarin yield and take the average value. The results are as follows Figure 3 And as shown in Table 2.

[0077] Table 2 Yield of cynaroside extracted at different temperatures

[0078]

[0079] The yield of cynaroside showed a trend of increasing first and then gradually flattening with the increase of extraction temperature. When the extraction temperature was in the range of 50-80℃, due to the increase of extraction temperature, on the one hand, the molecular movement accelerated, and the dissolution of cynaroside in the cells of the fragrant blue orchid was accelerated. On the other hand, the increase in temperature would reduce the viscosity of the ethanol solution, and the resistance of cynaroside molecules to diffuse into the solution through the solid-liquid interface would be reduced, which would be beneficial to the dissolution of cynaroside in the fragrant blue orchid. When the extraction temperature was in the range of 80-90℃, there was no obvious change in the dissolution of cynaroside in the fragrant blue orchid, which may be because the dissolution of cynaroside in the fragrant blue orchid was relatively complete, and there was no obvious change when the temperature was increased again. Therefore, the extraction temperature was selected to be 80℃.

[0080] (3) Solid-liquid ratio

[0081] Take 10g of powdered medicinal material (Xiangqinglan decoction piece), extract once at 80℃, extract for 1h, and investigate the effect of different solid-liquid ratios of 1:10, 1:20, 1:30, 1:40, 1:50 (w / v, g / mL) of ethanol-water solution on the extraction yield of cynaroside. The concentration of ethanol-water solution is 55% (v / v). Three parallel experiments were performed to determine the yield of cynaroside and take the average value. The results are shown in Figure 4 And as shown in Table 3.

[0082] Table 3 Yield of cynaroside extracted with different solid-liquid ratios

[0083]

[0084] The yield of cynarin first increased and then decreased with the increase of solid-liquid ratio. When the solid-liquid ratio was in the range of 1:10-1:30 (g / mL), the yield of cynarin increased significantly, which may be the result of the increase in contact area. When the solid-liquid ratio was 1:30-1:50 (g / mL), the yield of cynarin tended to be stable, which may be due to the fact that the dissolution of cynarin was almost complete, and excessive use of solvent would lead to waste and excessive energy consumption in the later concentration process, so the use of a large amount of solvent was not cost-effective. Therefore, a solid-liquid ratio of 1:30 (g / mL) was selected.

[0085] (4) Extraction time

[0086] Take 10 g of the medicinal material powder, accurately weigh it, and put it into 55% (v / v) ethanol aqueous solution according to the solid-liquid ratio of 1:30 (w / v, g / mL). Extract once at the extraction temperature of 80°C. Examine the effects of different extraction times of 1.0, 1.5, 2.0, 2.5, and 3.0 h on the yield of silymarin. Conduct 3 parallel experiments, measure the yield of silymarin, and take the average value. The results are as Figure 5 shown in Table 4 and Table 5 below.

[0087] Table 4 Yields of silymarin at different extraction times

[0088]

[0089]

[0090] The effect of extraction time on the yield of silymarin shows a trend of increasing first and then leveling off. Because the increase in extraction time will lead to an increase in the degree of damage to the cell membrane of Dracocephalum moldavica, a concentration gradient difference will be formed inside and outside the cell membrane, which is conducive to the diffusion of silymarin into the ethanol solution, so the yield will increase. When the extraction time is 1.5 h, the yield of silymarin in Dracocephalum moldavica is relatively high and the yield tends to be stable. Therefore, the extraction time of 1.5 h is selected.

[0091] (5) Extraction times

[0092] Take 10 g of the medicinal material powder, accurately weigh it, and put it into 55% (v / v) ethanol aqueous solution according to the solid-liquid ratio of 1:30 (w / v, g / mL). Extract once at the extraction temperature of 80°C and the extraction time of 1.5 h. Examine the effects of extraction times (1, 2, and 3 times) on the yield of silymarin. Conduct 3 parallel experiments, measure the yield of silymarin, and take the average value. The results are as Figure 6 shown in Table 5 and Table 6 below.

[0093] Table 5 Yields of silymarin at different extraction times

[0094]

[0095] With the increase in the number of extraction times, the yield of silymarin gradually decreases. The mass of silymarin extracted in 1 time is 71.20% of the total mass extracted in 3 times, the mass of silymarin extracted in 2 times is 87.38% of the total mass extracted in 3 times, and the mass extracted in the 3rd time only accounts for 12.62% of the total mass extracted in 3 times. Considering economic factors, 2 extractions are selected as the process parameter.

[0096] Example 2

[0097] Extraction process

[0098] The Dracocephalum moldavica L. medicinal materials were subjected to the following process: ethanol aqueous solution concentration 55% (v / v), solid-liquid ratio 1:30 (w / v, g / mL), extraction time 1.5 h, extraction temperature 80 °C, extraction twice, with heating under reflux to obtain an ethanol extract. The HPLC chromatogram is as shown in Figure 7 shown, with the purity of silybin being 13.60%. After the ethanol extract was concentrated under reduced pressure, the following extraction steps were carried out.

[0099] (1) Petroleum ether (PE) extraction

[0100] The ethanol extract of Dracocephalum moldavica L. was concentrated under reduced pressure to half of its volume (density 0.943 g / cm 3 , containing 64.9 mg of silybin) and placed in a separating funnel. Petroleum ether was added in a volume ratio of 1:1, shaken well, and after standing for layer separation, the petroleum ether layer (upper layer) and the water layer (lower layer) were obtained. The upper layer was dried under reduced pressure, dissolved in a 70% (v / v) methanol aqueous solution, and detected by HPLC. The HPLC chromatogram of the petroleum ether layer is as shown in Figure 8 shown.

[0101] It can be concluded through Figure 8 that silybin was not dissolved in the petroleum ether layer (containing 0 mg of silybin). According to the analysis of the extraction situation, pigments, fat-soluble components, and small polar substances were removed.

[0102] (2) Ethyl acetate (EA) extraction

[0103] The solution after petroleum ether extraction (i.e., the water layer) was placed in a separating funnel. Ethyl acetate was added in a volume ratio of 1:1, shaken well, and after standing for layer separation, the upper layer (ethyl acetate layer) was dried under reduced pressure to obtain an ethyl acetate extract, which was dissolved in a 70% (v / v) methanol aqueous solution and detected by HPLC. The HPLC chromatogram of the ethyl acetate layer is as shown in Figure 9 shown.

[0104] When the extract was mixed with EA, since the solubility of silybin in ethyl acetate is much greater than that in water, most of it was transferred to the ethyl acetate layer (containing 50.44 mg of silybin). The purity of silybin increased from 13.60% in the ethanol extract to 35.27%, and the transfer rate was 77.7% (the calculation formula for the transfer rate is: transfer rate % = mass of recovered silybin / mg ÷ mass of input silybin / mg * 100%).

[0105] The HPLC chromatogram of the water layer (containing 14.46 mg of silybin) is as shown in Figure 10, with the purity of silybin being 10.46% and the transfer rate being 22.3%.

[0106] (3) Experimental summary

[0107] Through two-step extraction, silybin in the extract was enriched, the purity increased, and each step of extraction had a relatively high transfer rate.

[0108] Purity of silymarin in ethanol extract: 13.60%;

[0109] Purity of silymarin in petroleum ether layer: 0%, transfer rate 0%;

[0110] Purity of silymarin in ethyl acetate layer: 35.27%, transfer rate: 77.7%;

[0111] Purity of silymarin in water layer: 10.46%, transfer rate 22.3%.

[0112] (4) Comparative example

[0113] The ethanol extract of Dracocephalum moldavica in step (1) of Example 2 was concentrated under reduced pressure to one-third of the original volume (density > 0.98 g / cm 3 ), and then petroleum ether extraction and ethyl acetate extraction were carried out successively according to steps (1) and (2) of Example 2. As a result, the transfer rate of silymarin in the ethyl acetate layer decreased to 43.23%.

[0114] Example 3

[0115] Purification by antisolvent method

[0116] The antisolvent method is to first dissolve the poorly soluble drug in an organic solvent that is miscible with the antisolvent. Under certain conditions, the antisolvent is injected. When the two phases are mixed, supersaturation occurs, causing the poorly soluble drug to precipitate from the solvent system to form crystal nuclei, and the crystals further grow to finally obtain purification.

[0117] The antisolvent method process involves process conditions such as the selection of the type of good solvent, the initial concentration of the drug, the volume ratio of the good solvent - antisolvent, and the injection rate.

[0118] (1) Solvent screening

[0119] An appropriate amount of ethyl acetate extract was dissolved in 5 solvents, namely water, methanol, ethanol, ethyl acetate, and dimethyl sulfoxide (DMSO), to reach saturation. Ethanol, water, water, ethanol, and water were added as the antisolvent system according to a volume ratio of 1:10, and they were shaken well and observed after standing.

[0120] Since the ethyl acetate extract had poor solubility in methanol, ethanol, and ethyl acetate, and there was no obvious precipitation after adding the antisolvent, the above three solvents could not be used. Dimethyl sulfoxide showed better solubility, and obvious precipitates appeared after adding pure water. DMSO belongs to Class 3 solvents (solvents with low potential toxicity) in the ICH residual solvent classification and is preferably used. Since DMSO is miscible with pure water and the solubility of silymarin in pure water is only 1.57 μg / mL, pure water was selected as the antisolvent.

[0121] (2) Drug concentration

[0122] Weigh a certain amount of ethyl acetate extract respectively, dissolve it fully in dimethyl sulfoxide, and prepare extract solutions with concentrations of 50, 100, 200, and 400 mg / mL. Add deionized water dropwise to them according to the ratio of 1:20 (v / v), stir, let it stand for 24 h, centrifuge the suspension, discard the supernatant, and wash the precipitate repeatedly with deionized water to remove dimethyl sulfoxide. After centrifuging and discarding the supernatant, dry the precipitate in an oven and weigh it. Dissolve it in a 70% methanol aqueous solution by volume and detect it by high-performance liquid chromatography to calculate the purity and transfer rate of silymarin. The HPLC chromatograms of the anti-solvent method at different concentrations are as Figure 11 shown, and the results are shown in Table 6 (the input amount of silymarin in Table 6 is obtained by quantitative detection of the ethyl acetate extract by HPLC and calculation).

[0123] Table 6 Purification results of the anti-solvent method at different concentrations

[0124] Concentration / mg / mL 50 100 200 Silymarin content / mg 4.15 8.30 16.60 Recovery content / mg 2.89 6.47 13.02 Transfer rate % 69.63 77.95 78.43 Purity % 78.53 69.90 61.55

[0125] It can be seen from Table 6 that the purity of silymarin shows a gradually increasing trend with the decrease of the concentration of the ethyl acetate extract. When the concentration of the ethyl acetate extract decreases from 200 mg / mL to 50 mg / mL, the increase in the purity of silymarin is more obvious. The purity of silymarin increases from 61.55% to 78.53%. The reason may be that as the concentration of the ethyl acetate extract continuously decreases, the impurity content is small and it is not easy to precipitate in this system, while the content of silymarin is relatively large. With the addition of the anti-solvent, the supersaturation also continuously increases, and more and more silymarin precipitates. Therefore, the purity of the precipitated silymarin increases with the decrease of the concentration of the ethyl acetate extract. Therefore, the dissolution concentration of the ethyl acetate extract is selected as 50 mg / mL.

[0126] (3) Volume ratio of good solvent to anti-solvent

[0127] Weigh the ethyl acetate extract, dissolve it fully in dimethyl sulfoxide, and prepare four extract solutions with a concentration of 50 mg / mL. Add different amounts of deionized water to them respectively to investigate the influence of different volume ratios of good solvent to anti-solvent, namely 1:10, 1:20, 1:30, and 1:40, on the purification effect. Stir, let it stand for 24 h, centrifuge the suspension, discard the supernatant, and wash the precipitate repeatedly with deionized water to remove dimethyl sulfoxide. Then centrifuge and discard the supernatant. Dry the precipitate in an oven and weigh it. Dissolve it in a 70% methanol aqueous solution by volume and detect it by high-performance liquid chromatography to calculate the purity and transfer rate of silymarin. The results are shown in Table 7.

[0128] Table 7 Purification results of different volume ratios of good solvent to anti-solvent

[0129]

[0130] Note: In Table 7, the comprehensive score = purity × 50% + transfer rate × 50%.

[0131] As can be seen from the data in Table 7, the transfer rate is the highest when the volume ratio of the good solvent to the anti-solvent is 1:20, and the purity is the highest when it is 1:30. However, the difference between the two is small. After conversion by the comprehensive score, 1:20 is selected as the optimal ratio.

[0132] Through further analysis of the data, it can be concluded that when the volume ratio of the good solvent to the anti-solvent is 1:10, the amount of the poor solvent added is insufficient, and the solubility of silymarin in the solvent is large, which affects its crystallization and precipitation; when the volume ratio of the good solvent to the anti-solvent is 1:20, the proportion of the poor solvent can play a better dilution role, and the solubility of silymarin in the solvent decreases, facilitating its crystallization and precipitation; when the volume ratio of the good solvent to the anti-solvent is 1:30, the proportion of the poor solvent can also play a better dilution role, but since more poor solvent is added than at 1:20, the dissolution of silymarin increases, resulting in a decrease in the transfer rate, but the purity will increase slightly; when the volume ratio of the good solvent to the anti-solvent is 1:40, the proportion of the poor solvent exceeds the optimal ratio. Due to the addition of a large amount of poor solvent, silymarin is dissolved, and its transfer rate will decrease significantly. The purity is not much different from that at 1:20 and 1:30, which can prove that the optimal purity of the single anti-solvent crystallization method is within this range.

[0133] (4) Anti-solvent addition rate

[0134] Weigh a certain amount of ethyl acetate extract respectively, dissolve it fully in dimethyl sulfoxide to prepare an extract solution with a concentration of 50 mg / mL. Take 5 equal-volume 50 mg / mL extract solutions respectively, and add deionized water to them at a flow rate of 0.5, 2.0, 3.0, 4.0, and 6.0 mL / min according to the ratio of 1:20 (v / v). Stir and let stand for 24 h. Centrifuge the suspension, discard the supernatant, and wash the precipitate repeatedly with deionized water to remove dimethyl sulfoxide. Then centrifuge again to discard the supernatant. The precipitate is dried in an oven and weighed, dissolved in a 70% methanol aqueous solution by volume fraction, and the purity and transfer rate of silymarin are detected and calculated by high performance liquid chromatography. The results are shown in Table 8.

[0135] Table 8 Purification results at different anti-solvent addition rates

[0136]

[0137] Note: In Table 8, the comprehensive score = purity × 50% + transfer rate × 50%.

[0138] Further analysis of the data shows that when the anti-solvent flow rate is 6 mL / min, the purity is the highest, but the overall purity difference is not obvious. When the flow rate is 0.5 mL / min, the transfer rate is the highest. Through comprehensive scoring calculation, when the flow rate is 4 mL / min, the comprehensive score is the best. Therefore, a flow rate of 4 mL / min is selected for the subsequent purification steps.

[0139] (5) Optimal process verification

[0140] Dissolve the ethyl acetate extract in DMSO to prepare a solution with a concentration of 50 mg / mL. Add pure water to it at a volume ratio of 1:20, with an addition rate of 4 mL / min. Let it stand for 24 h, centrifuge, discard the supernatant, and repeatedly wash the precipitate with deionized water to remove DMSO. Then centrifuge again to discard the supernatant. Dry the precipitate in an oven, detect it by HPLC, and calculate the purity and transfer rate of silymarin. The results are shown in Table 9.

[0141] Table 9 Optimal process verification experiment of the anti-solvent method

[0142]

[0143] According to the results in Table 9, it can be concluded that when the sample concentration is 50 mg / mL, the solvent-anti-solvent volume ratio is 1:20, and the anti-solvent flow rate is 4 mL / min, the purity of silymarin by the single anti-solvent method increases from 35.27% in the ethyl acetate extract to 74.29%. 186.75 mg of silymarin is put in, and finally 136.88 mg is recovered, with a transfer rate of 73.3%.

[0144] Example 4

[0145] Recrystallization purification

[0146] (1) Solvent concentration optimization

[0147] Water bath heat different concentrations of solvents (methanol aqueous solution or ethanol aqueous solution, volume percentages of 10%, 30%, 50%, 70%) equally to 30 °C. Add the product prepared by the anti-solvent method under the optimal process to it. Stir while adding until it is supersaturated. Turn off the water bath heating, filter, place the filtrate in an environment of 4 °C and let it stand for 24 h. Centrifuge to obtain the precipitate and dry it. Add deionized water according to the ratio of 1 mg:4 mL, adjust the pH = 5 with hydrochloric acid for acid washing, filter, wash with deionized water until neutral, and dry; dissolve it with a 70% methanol aqueous solution by volume, and detect it by HPLC. The results are shown in Table 10.

[0148] Table 10 Comparison of methanol / ethanol crystallization purity

[0149]

[0150] According to the experimental results in Table 10, when performing cooling crystallization operation with 30% methanol (v / v) solvent in the methanol system, the purity of silymarin is the highest; in the ethanol system, the purity of silymarin is the highest when cooled and crystallized with 30% ethanol. According to the experimental results, the purity of the 30% methanol system is slightly higher than that of the 30% ethanol system, but ethanol belongs to Class 3 solvents of ICH, and it is preferred to select it for process system development.

[0151] (2) Optimization of solvent pH

[0152] Water bath heat equal amounts of aqueous ethanol solutions with a concentration of 30% (v / v) and different pH values (pH = 1, 4, 7, 10, 12) to 30 °C. Add the product prepared by the antisolvent method to it, and stir while adding until it becomes supersaturated. Turn off the water bath heating, filter, place the filtrate in an environment at 4 °C and let it stand for 24 h. Centrifuge at 1000 g for 15 min to obtain the precipitate. After drying, add deionized water according to the ratio of 1 mg:4 mL, adjust the pH = 5 with hydrochloric acid for acid washing, filter, wash with deionized water until neutral, and dry; add a 70% methanol aqueous solution by volume to completely dissolve it, and detect by HPLC. The results are shown in Table 11.

[0153] Table 11 Comparison of crystallization purity of aqueous ethanol solutions with different pH values

[0154] pH 1 4 7 10 12 Purity % 84.30 84.80 82.70 93.60 97.30

[0155] Under acidic conditions, the result of the increase in silymarin purity is basically the same as that under neutral conditions, the types of impurities are basically the same, and the polarity is slightly smaller than that of silymarin. Under alkaline conditions, the result of the increase in purity is better than that under neutral and acidic conditions, the impurity content decreases, the purity increases, and as the pH increases, the purity also shows an increasing trend. Therefore, pH = 12 is selected as the process parameter.

[0156] (3) Optimization of crystallization time:

[0157] Water bath heat an ethanol solution with a volume fraction of 30% (v / v) and pH = 12 to 30 °C. Add the product prepared by the antisolvent method to it, stir until it is saturated, filter to obtain the filtrate, divide it into 5 equal parts, place them at 4 °C and let them stand for 3, 6, 12, 24, 48 h respectively, filter to obtain the precipitate, after drying, add deionized water according to the ratio of 1 mg:4 mL to make a suspension, adjust the pH of the solution = 5 with hydrochloric acid, filter, wash with deionized water, and dry to obtain the crystalline product. The crystalline product is detected by HPLC to determine the purity and transfer rate. The results are shown in Table 12.

[0158] Table 12 Optimization of crystallization time

[0159]

[0160]

[0161] According to the experimental results, with the increase of crystallization time, the transfer rate of cynaroside within 24 hours increased significantly. When the crystallization time was greater than 24 hours, the transfer rate did not change significantly, reaching the optimal value of the process.

[0162] (4) Optimal process

[0163] Heat 30% ethanol aqueous solution (pH=12) to 30°C, add anti-solvent to prepare the product, stir until the solution reaches supersaturation, filter the filtrate, place it in a 4°C environment and let it stand for 24 hours, filter and dry, add deionized water in a ratio of 1mg:4mL to make a suspension, adjust the solution pH to 5 with hydrochloric acid, filter, wash with deionized water, and dry to obtain a crystalline product. The crystalline product was tested by HPLC to determine its purity. The chromatogram is as shown below. Figure 12 After purification by crystallization, the purity of cynaroside increased to 97.2%, 14.00 mg of cynaroside was added, 6.80 mg was recovered, and the transfer rate was 48.57%.

[0164] Example 5

[0165] Take the powder of medicinal material (Canthus chinensis slices), use 55% ethanol aqueous solution with a solid-liquid ratio of 1g:30mL, extraction time of 1.5h, extraction temperature of 80°C, and extraction times of 2 times to extract the silybin component in Canthus chinensis, and concentrate the obtained extract under reduced pressure to half the volume (density of 0.943g / cm3) to obtain a concentrated solution;

[0166] Add an equal volume of petroleum ether to the concentrate, shake thoroughly, stand to separate layers, and take the water layer; add an equal volume of ethyl acetate to the water layer, shake thoroughly, stand to separate layers, take the ethyl acetate layer, and dry under reduced pressure to obtain an ethyl acetate extract;

[0167] The ethyl acetate extract was dissolved in DMSO at 50 mg / mL, and pure water was added at a volume ratio of 1:20 (water drop rate of 4 mL / min), and the precipitate was precipitated for 24 hours. The precipitate was repeatedly washed with deionized water to remove dimethyl sulfoxide, and dried. The dried precipitate was dissolved in a 30% ethanol-water (v / v) solution at 30°C (pH = 12, obtained by adding saturated lime water) until supersaturated, and the filtrate was filtered and the crystalline product was allowed to stand at 4°C for 24 hours, and then dried. The crystalline product was added to pure water at a solid-liquid ratio of 1 mg: 4 mL to prepare a suspension, and the pH was adjusted to 5 with hydrochloric acid, and the acidified product was filtered to obtain the acidified product. The acidified product was washed with an equal volume of pure water again, filtered, and dried to obtain the final product. The purity of cynaroside in the final product was 97%.

[0168] Example 6

[0169] The powder of the medicinal material (Xiangqinglan decoction piece) was taken, and the 55% ethanol aqueous solution was used at a solid-liquid ratio of 1g:30mL, extraction time of 1.5h, extraction temperature of 80℃, and extraction times of 2 times to extract the silybin component in Xiangqinglan. The obtained extract was concentrated under reduced pressure to half the volume (density of 0.943g / cm 3 ), to obtain a concentrated solution;

[0170] Add an equal volume of petroleum ether to the concentrate, shake thoroughly, stand to separate layers, and take the water layer; add an equal volume of ethyl acetate to the water layer, shake thoroughly, stand to separate layers, take the ethyl acetate layer, and dry under reduced pressure to obtain an ethyl acetate extract;

[0171] The ethyl acetate extract was dissolved in DMSO at 200 mg / mL, and pure water was added at a volume ratio of 1:30 (water drop rate of 4 mL / min), and the precipitate was precipitated for 24 hours. The precipitate was repeatedly washed with deionized water to remove dimethyl sulfoxide, and dried. The dried precipitate was dissolved in a 30% ethanol-water (v / v) solution at 30°C (pH = 12, obtained by adding saturated lime water) until supersaturated, and the filtrate was filtered and allowed to stand at 4°C for 24 hours to obtain a crystalline product, which was dried. The crystalline product was added to pure water at a solid-liquid ratio of 1 mg: 4 mL to prepare a suspension, and the pH was adjusted to 5 with hydrochloric acid, and the acidified product was filtered to obtain the acidified product. The acidified product was washed with an equal volume of pure water again, filtered, and dried to obtain the final product. The purity of cynaroside in the final product was 93%.

[0172] Example 7

[0173] Take the powder of medicinal material (Canthus chinensis slices), use 55% ethanol aqueous solution with a solid-liquid ratio of 1g:30mL, extraction time of 1.5h, extraction temperature of 80°C, and extraction times of 2 times to extract the silybin component in Canthus chinensis, and concentrate the obtained extract under reduced pressure to half the volume (density of 0.943g / cm3) to obtain a concentrated solution;

[0174] Add an equal volume of petroleum ether to the concentrate, shake thoroughly, stand to separate layers, and take the water layer; add an equal volume of ethyl acetate to the water layer, shake thoroughly, stand to separate layers, take the ethyl acetate layer, and dry under reduced pressure to obtain an ethyl acetate extract;

[0175] The ethyl acetate extract was dissolved in DMSO at a concentration of 100 mg / mL, and pure water was added at a volume ratio of 1:40 (the dropping rate of water was 4 mL / min). After standing for 24 h, a precipitate was obtained. The precipitate was washed repeatedly with deionized water to remove DMSO and then dried. The dried precipitate was dissolved in a 30% ethanol-water (v / v) solution (pH = 12, adjusted by adding saturated lime water) at 30 °C until supersaturated. The filtrate was obtained by filtration and allowed to stand at 4 °C for 24 h to obtain a crystalline product, which was then dried. The crystalline product was made into a suspension by adding pure water at a solid-liquid ratio of 1 mg:4 mL, and the pH was adjusted to 5. The acidified product was obtained by filtration, and the acidified product was added to an equal volume of pure water for washing, filtration, and drying to obtain the final product. The purity of silymarin in the final product was 94%.

[0176] The above is only the preferred embodiment of the present invention and does not impose any formal restrictions on the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for extracting silibinin from Dracocephalum moldavica, characterized in that, It includes the following steps: Mix Dracocephalum moldavica or Dracocephalum moldavica products with an ethanol aqueous solution for extraction, and concentrate the obtained extract to obtain a concentrated solution; Mix the concentrated solution with petroleum ether for the first extraction, and collect the aqueous phase; Mix the aqueous phase with ethyl acetate for the second extraction, and collect the ethyl acetate phase; remove the solvent in the ethyl acetate phase to obtain an ethyl acetate extract; Dissolve the ethyl acetate extract with dimethyl sulfoxide, mix the obtained extract solution with water for precipitation to obtain a precipitate; Recrystallize the precipitate to obtain silymarin; the solvent used for the recrystallization is an alcohol aqueous solution.

2. The method according to claim 1, wherein The volume fraction of ethanol in the ethanol aqueous solution is 35-75%, and the dosage ratio of Dracocephalum moldavica or Dracocephalum moldavica products to the ethanol aqueous solution is 1 g:(10-50) mL.

3. The method according to claim 1 or 2, characterized in that, The temperature of the extraction is 70-90 °C, the number of times is 1-3 times, and the single extraction time is 1-3 h.

4. The method according to claim 1, wherein The density of the concentrated liquid is 0.93 to 0.98 g / cm 3 .

5. The method according to claim 4, wherein The volume ratio of the concentrated solution to petroleum ether is 1:(1-3).

6. The method according to claim 1, characterized in that, The volume ratio of the aqueous phase to ethyl acetate is 1:(1-3).

7. The method according to claim 1, characterized in that, The concentration of the extract solution is 50-200 mg / mL, and the volume ratio of the extract solution to water is 1:10-1:

50.

8. The method according to claim 1 or 7, characterized in that, Mixing the extract solution with water means adding water to the extract solution, and the addition rate of water is 0.5-6 mL / min.

9. The method according to claim 1, characterized in that, The alcohol in the alcohol aqueous solution is methanol or ethanol, the volume fraction of the alcohol in the alcohol aqueous solution is 10-70%, and the alcohol aqueous solution is alkaline.

10. The method according to claim 9, characterized in that, The recrystallization includes: dissolving the precipitate in the alcohol aqueous solution until supersaturated and then performing solid-liquid separation, cooling the obtained liquid phase for crystallization to obtain a crystalline substance; sequentially performing acid washing, water washing and drying on the crystalline substance; the dissolution temperature is 20-60 °C, and the crystallization temperature is 2-8 °C.

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