A method for preparing silymarin by ethanol switching of a deep eutectic solvent extract and resin enrichment

Abstract

The invention discloses a low-moisture composite nutritional tablet containing lycopene microencapsulated powder and its preparation. The process includes raw material weighing, excipient pretreatment, mineral premixing, water-soluble vitamin premixing, lycopene premixing, low-moisture final mixing, and tableting. Based on two tablets daily, it contains vitamins C, E, D3, B1, B2, B6, B12, nicotinamide, pantothenic acid, folic acid, zinc gluconate, selenium yeast, lycopene powder, pumpkin seed extract, and taurine. The lycopene content in the powder is 9%–11%. The moisture of both the final mixture and the tablets is controlled at 2.5%–3.8%. The tablets exhibit high retention rates of lycopene, vitamins C and B1 under accelerated conditions, excellent content uniformity for zinc, selenium, and lycopene, and minimal appearance changes.

Description

[0001] DESCRIPTION

[0002] A Method for Preparing Silymarin by Ethanol Switching of a Deep Eutectic Solvent Extract and Resin Enrichment

[0003] Technical Field

[0004] The present invention relates to the technical field of extraction and purification of plant active ingredients, and in particular to a method for preparing silymarin by ethanol switching of a deep eutectic solvent extract and resin enrichment.

[0005] Background Art

[0006] Silymarin is a flavonolignan mixture extracted from mature fruits of milk thistle and is a commonly used hepatoprotective active ingredient widely applied in the fields of medicines, health products, and functional foods. Current industrial production mostly uses organic solvents such as ethanol and ethyl acetate to extract milk thistle seeds or defatted cakes, and then purification is completed through resin adsorption, extraction, crystallization, and other processes. As a class of green extraction media, deep eutectic solvents have adjustable polarity and strong penetration ability into plant tissues, and show high dissolution efficiency for flavonoid components. In recent years, they have gradually been applied to the extraction of plant active ingredients and can be used for processing cake / meal by-products from pressing milk thistle seed oil.

[0007] A deep eutectic solvent itself has relatively high viscosity, and during extraction, hydrophilic impurities such as sugars, proteins, and gums in the raw material are co-dissolved. When the obtained extract is directly introduced into macroporous resin for adsorption purification, mass transfer resistance is increased, resin adsorption capacity is reduced, the stability of the elution process is poor, and residue of deep eutectic components is also likely to occur, which affects product quality and shortens the service life of the resin. Existing purification methods for deep eutectic solvent extracts mostly use liquid-liquid extraction, which still consumes a large amount of organic solvent, has limited separation efficiency, provides poor linkage between extraction and purificationDESCRIPTION

[0008] processes, and is difficult to adapt to industrial continuous production. Conventional organic- solvent extraction processes have problems of obvious co-extraction of impurities, low extraction yield, and large fluctuations in product purity.

[0009] Summary of the Invention

[0010] The object of the present invention is to make up for deficiencies in the prior art and provide a method for preparing silymarin by ethanol switching of a deep eutectic solvent extract and resin enrichment. By means of a pretreatment mode in which ethanol transfer-dissolution solvent switching is combined with dilution-induced impurity precipitation, the present invention solves the problem that, after silymarin is extracted with a deep eutectic solvent, the extract has high viscosity and cannot be directly adapted to macroporous resin adsorption purification. In the process, low-temperature pressed defatted milk thistle seed cake / meal is used as the raw material, a betaine-lactic acid-water ternary deep eutectic solvent is used for extraction, impurities are precipitated by adjusting ethanol concentration, enrichment is performed with AB-8 macroporous resin, and a finished product is obtained by water-ethanol antisolvent crystallization. The process can reduce the viscosity of the column-loading solution to 2.0-3.0 mPa • s, effectively reduce residue of the deep eutectic solvent, and produce a product with a total silymarin content of 82%-84% and a combined content of silybin A and B of 39%-41%. The parameters of the entire process are controllable, the process steps are stably connected, and the process is suitable for industrial production.

[0011] To solve the above technical problems, the present invention provides the following technical solution: a method for preparing silymarin by ethanol switching of a deep eutectic solvent extract and resin enrichment, comprising the following steps:

[0012] (1) raw material pretreatment: mature dried milk thistle seeds are defatted by low-temperature pressing at a temperature not higher than 45 ° C, so as to avoidDESCRIPTION

[0013] thermal degradation of silymarin and reduce interference of oil with subsequent extraction and adsorption processes, thereby obtaining a defatted milk thistle seed cake / meal having an oil content not higher than 8% and a moisture content of 5%-9%; the defatted milk thistle seed cake / meal is pulverized to 50-70 mesh so that the raw material particle size is adapted to permeation of the deep eutectic solvent and dissolution of active ingredients;

[0014] (2) preparation of a deep eutectic solvent: betaine, lactic acid, and water are mixed at a mass ratio of 1:2:0.6, stirred at 55-65 ° C until transparent and homogeneous, and cooled to room temperature to obtain the deep eutectic solvent, thereby forming a stable hydrogen-bonded eutectic system having good dissolving ability for flavonolignan components;

[0015] (3) extraction with the deep eutectic solvent: the defatted milk thistle seed cake / meal is mixed with the deep eutectic solvent at a mass ratio of 1:10 and extracted at 50 °C for 90 minutes under nitrogen protection, whereby oxygen is excluded to prevent oxidative degradation of silymarin and ensure structural stability of active ingredients; solid-liquid separation is then performed to obtain a deep eutectic solvent extract;

[0016] (4) ethanol transfer-dissolution solvent switching: anhydrous ethanol is added to the deep eutectic solvent extract for solvent-switching treatment so that the ethanol volume fraction of the system is 68%, thereby breaking the hydrogen-bond network structure of the original deep eutectic solvent while precipitating macromolecular impurities such as gums and polysaccharides and converting the solvent phase into an ethanol-water system; after stirring, filtration is performed to obtain an ethanol-converted solution;

[0017] (5) dilution-induced impurity precipitation: the ethanol-converted solution is diluted with water to an ethanol volume fraction of 30%, thereby further precipitating hydrophilic impurities, matching the polarity of the column-loading solution with the adsorption characteristics of AB-8 resin, and reducing the viscosity of the system; the solution is allowed to stand for impurity precipitationDESCRIPTION

[0018] and filtered to obtain a column-loading solution;

[0019] (6) resin enrichment: the column-loading solution is passed through AB-8 weakly polar macroporous adsorption resin and eluted sequentially with water and an ethanol solution having a volume fraction of 60%; water washing removes residual deep eutectic solvent components and water-soluble impurities, and 60% ethanol can efficiently desorb silymarin components; an ethanol eluate is collected;

[0020] (7) concentration and crystallization: the ethanol eluate is concentrated under reduced pressure at 45°C to a relative density of 1.10-1.15, purified water is added as an antisolvent at a concentrate-to-purified water volume ratio of 1:3, and crystallization is performed by standing at 8 °C for 12 hours; the water-ethanol antisolvent system further removes fat-soluble impurities and small-molecule pigments to improve product purity; solid-liquid separation and vacuum drying are performed to obtain a silymarin product.

[0021] Further, in step (1), the defatted milk thistle seed cake / meal has an oil content of 4.5%-6.5% and a moisture content of 6%-8%; the oil content and moisture range can ensure the permeation efficiency of the deep eutectic solvent and the stability of the extraction process.

[0022] Further, in step (2), during preparation of the deep eutectic solvent, the stirring temperature is 60 °C and the stirring time is 30-60 minutes. Under these conditions, a homogeneous and stable deep eutectic solvent system can be formed, with no component precipitating during storage at room temperature.

[0023] Further, in step (3), the nitrogen protection comprises introducing nitrogen for replacement for 5-10 minutes before extraction starts and maintaining a nitrogen atmosphere in the extraction vessel during extraction, so as to fully replace the air in the vessel and avoid oxidative browning of active ingredients during extraction.

[0024] Further, in step (4), the volume ratio of the deep eutectic solvent extract to anhydrous ethanol is 1:2.2, and the stirring time is 30 minutes. At this ratio, theDESCRIPTION

[0025] ethanol volume fraction of the system can be accurately controlled to 68%, thereby ensuring impurity precipitation and solvent-switching efficiency.

[0026] Further, in step (5), the standing time for impurity precipitation is 1.5-2.5 hours, and the obtained column-loading solution has a viscosity of 2.0-3.0 mPa s at 25 ° C. This viscosity range is adapted to mass-transfer requirements of macroporous resin and can ensure the adsorption capacity of the resin and the stability of the elution process.

[0027] Further, in step (6), the loading flow rate of the AB-8 weakly polar macroporous adsorption resin is 1-2 BV / h, and the loading amount is 2-4 BV. Under these parameter conditions, resin adsorption is sufficient, leakage of target components is low, and adsorption efficiency is stable.

[0028] Further, in step (6), the water-washing volume is 2-3 BV, the 60% ethanol elution volume is 3-5 BV, and the elution flow rate is 1-2 BV / h. This can sufficiently wash away impurities and completely desorb target components, with a concentrated elution peak and stable target-component yield.

[0029] Further, in step (7), the vacuum drying temperature is 40-50 ° C. This temperature range can avoid thermal decomposition of silymarin while ensuring drying efficiency and product appearance.

[0030] Compared with the prior art, the method for preparing silymarin by ethanol switching of a deep eutectic solvent extract and resin enrichment has the following beneficial effects:

[0031] First, by means of a pretreatment process in which ethanol transfer-dissolution solvent switching is combined with dilution-induced impurity precipitation, the present invention solves the problem that a deep eutectic solvent extract has high viscosity and is difficult to directly adapt to resin adsorption. Anhydrous ethanol is first added to the extract so that the ethanol volume fraction of the system reaches 68%, thereby precipitating macromolecular impurities suchDESCRIPTION

[0032] as gums and polysaccharides and breaking the hydrogen-bond network structure of the deep eutectic solvent, and thus reducing the viscosity of the system. Then water is added to dilute the system to an ethanol volume fraction of 30% to further precipitate impurities, so that the polarity of the column-loading solution matches the adsorption characteristics of AB-8 resin. The viscosity of the column-loading solution at 25 ° C can be reduced to 2.0-3.0 mPa • s, thereby alleviating resin mass-transfer resistance and adsorption load while reducing the residual amount of deep eutectic solvent components in the product.

[0033] Second, the present invention realizes stable preparation of silymarin through a combined process of deep eutectic solvent extraction, macroporous resin enrichment, and water-ethanol antisolvent crystallization. The betaine-lactic acid-water ternary deep eutectic solvent is used to process low-temperature pressed defatted cake / meal, thereby improving the extraction yield of silymarin components and reducing the amount of traditional organic solvent used. The resin enrichment step and the crystallization step act synergistically in purification, so that a product having a total silymarin content of 82%-84% and a combined content of silybin A and B of 39%-41 % can be stably obtained. The parameters of the whole process are fixed and controllable, all process steps are smoothly connected, and industrial scale-up production can be realized without special equipment.

[0034] Other advantages, objectives, and features of the present invention will be set forth to some extent in the following description, and to some extent will be apparent to those skilled in the art upon examination of the following description, or may be learned from practice of the present invention.

[0035] Brief Description of the Drawings

[0036] To more clearly describe the embodiments of the present invention or the technical solutions in the prior art, the drawings required for describing theDESCRIPTION

[0037] embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention, and a person of ordinary skill in the art may obtain other drawings based on these drawings without creative effort.

[0038] FIG. 1 is an overall process flow chart of the method for preparing silymarin by ethanol switching of a deep eutectic solvent extract and resin enrichment according to the present invention;

[0039] FIG. 2 is a process flow chart of the steps of deep eutectic solvent extraction, ethanol transfer-dissolution, and dilution-induced impurity precipitation in the present invention;

[0040] FIG. 3 is a process flow chart of the steps of macroporous adsorption resin enrichment and antisolvent crystallization in the present invention.

[0041] Detailed Description

[0042] To further illustrate the technical means and effects adopted by the present invention for achieving the intended purpose of the invention, the specific embodiments, structures, features, and effects of the present invention are described in detail below in conjunction with the accompanying drawings and preferred embodiments.

[0043] The raw materials and equipment used in the examples are conventional and readily available in the plant extraction industry: mature dried milk thistle seeds are commercially available bulk goods; betaine and lactic acid are food-grade industrial raw materials; anhydrous ethanol is industrial first-grade product; AB-8 weakly polar macroporous adsorption resin is a commercially available industrial-grade product; and purified water meets the standards for industrial production water. The low-temperature press, plate-and-frame filter press, stirred batching tank, resin adsorption column, reduced-pressure concentration tank,DESCRIPTION

[0044] crystallization tank, and vacuum drying oven involved are all general-purpose production equipment in this field.

[0045] Pretreatment method for the AB-8 macroporous adsorption resin: dry resin is taken, ethanol with a volume fraction of 95% is added so that the liquid level is 20 cm higher than the resin layer, and the resin is sealed and soaked for 24 hours to allow full swelling. Wet packing is adopted; the inner diameter of the resin column is 12 cm, the resin packing height is 96 cm, the packed column volume is about 10 L, and the diameter-to-height ratio is 1:8. After column packing is completed, forward rinsing is continued with 95% ethanol at a flow rate of 1 BV / h until the effluent, when mixed with an equal volume of purified water, shows no turbidity; then purified water is used for flushing at a flow rate of 2 BV / h until the effluent has no obvious ethanol odor. Subsequently, 2 B V each of a hydrochloric acid solution with a mass fraction of 5% and a sodium hydroxide solution with a mass fraction of 5% are introduced in sequence, each soaked for 2 hours, and the waste liquid is drained. Finally, continuous rinsing with purified water is performed until the pH of the effluent is neutral, and the resin is ready for use.

[0046] Example 1

[0047] Twenty kilograms of mature dried milk thistle seeds were placed into a low-temperature press, and the pressing temperature was controlled not to exceed 45°C for cold pressing and defatting to obtain defatted milk thistle seed cake / meal. The cake / meal was placed into a pulverizer, pulverized, and passed through a 60-mesh sieve. Sampling and testing showed that the cake / meal had an oil content of 5.6% and a moisture content of 7.1%. The overall process flow chart of theDESCRIPTION

[0048] method of the present invention is shown in FIG. 1.

[0049] Betaine, lactic acid, and purified water were weighed and placed into a stirred batching tank at a mass ratio of 1:2: 0.6. Stirring was started at a rotation speed controlled at 80 r / min, the temperature was raised to 60°C, and stirring was maintained for 45 minutes until the liquid in the tank was transparent and homogeneous. Heating was stopped, and stirring was continued while cooling to room temperature, thereby preparing the deep eutectic solvent.

[0050] Five kilograms of the above defatted milk thistle seed cake / meal was weighed and placed into an extraction tank, and 50 kg of the prepared deep eutectic solvent was added. Stirring was started to mix the materials uniformly. Before extraction, nitrogen was introduced into the tank to replace the air therein for 8 minutes; during extraction, a trace amount of nitrogen was continuously introduced to maintain a slight positive pressure in the tank, with the pressure being about 0.02 MPa, so as to isolate the air. The extraction temperature was controlled at 50°C, and extraction was performed with stirring for 90 minutes at a stirring speed of 60 r / min. After extraction, the materials in the tank were transferred to a plate-and-frame filter press and subjected to pressure filtration for solid-liquid separation at a pressure of 0.3 MPa. The clarified filtrate was collected to obtain a deep eutectic solvent extract. As shown in FIG. 2, the extract was subsequently subjected to ethanol transfer-dissolution and dilution-induced impurity precipitation.

[0051] Anhydrous ethanol was slowly added to the obtained deep eutectic solvent extract, with the volume ratio of the extract to anhydrous ethanol controlled at 1:2.2. After addition, stirring was continued for 30 minutes at a rotation speed ofDESCRIPTION

[0052] 50 r / min, and the ethanol volume fraction of the system was detected to be 68%. Stirring was stopped, the system was allowed to stand for 15 minutes, and then filtration was performed to remove precipitated gums and insoluble impurities, thereby obtaining an ethanol-converted solution.

[0053] Purified water was added to the ethanol-converted solution for dilution, and after uniform stirring and mixing, the ethanol volume fraction of the system was detected to be 30%. Stirring was stopped, and the system was allowed to stand for 2 hours to sufficiently precipitate impurities therein. After standing, filtration was performed to remove bottom precipitates, thereby obtaining a clarified column-loading solution.

[0054] The column-loading solution was introduced into the pretreated AB-8 macroporous adsorption resin column, with the loading flow rate controlled at 1.5 BV / h and the cumulative loading amount being 3 BV. After loading, purified water was first introduced for elution; the water-washing volume was 2.5 BV and the elution flow rate was 2 BV / h, so as to wash away residual water-soluble impurities and deep eutectic solvent components. After water washing, an ethanol solution having a volume fraction of 60% was used for elution; the elution flow rate was 1.5 BV / h and the cumulative elution volume was 4 BV. The eluate was collected in portions, and the target elution fractions were combined to obtain an ethanol eluate. As shown in FIG. 3, the eluate was subjected to antisolvent crystallization after reduced-pressure concentration.

[0055] The ethanol eluate was transferred into a reduced-pressure concentration tank. The water-bath temperature was controlled at 45 °C, and the vacuum degree in the tank was -0.08 MPa for reduced-pressure concentration. DuringDESCRIPTION

[0056] concentration, samples were taken periodically, and the relative density was determined while hot. When the relative density of the concentrated solution reached 1.12 (60 °C), concentration was stopped, and the concentrated solution was discharged.

[0057] The concentrated solution was transferred into a crystallization tank. Purified water, as an antisolvent, was slowly added at a volume ratio of concentrated solution to purified water of 1:3 while stirring at 30 r / min. After the addition, stirring was continued for 10 minutes to make the system uniformly mixed. A cooling program was started, and the temperature was slowly lowered to 8 °C at a rate of 2 ° C per hour. Stirring was then stopped, and the system was allowed to stand for crystallization for 12 hours. After crystallization, the crystal slurry was discharged and centrifuged with a centrifuge to collect crystals. The crystals were spread on trays and placed in a vacuum drying oven; the temperature was controlled at 45 ° C and the vacuum degree at -0.09 MPa, and drying was performed to constant weight to obtain 280 g of silymarin product.

[0058] Example 2

[0059] Twenty kilograms of mature dried milk thistle seeds were placed into a low-temperature press, and the pressing temperature was controlled not to exceed 45°C for cold pressing and defatting to obtain defatted milk thistle seed cake / meal. The cake / meal was pulverized and passed through a 50-mesh sieve. Sampling and testing showed that the cake / meal had an oil content of 6.2% and a moisture content of 7.5%.

[0060] Betaine, lactic acid, and purified water were weighed and placed into a stirred batching tank at a mass ratio of 1 :2:0.6. Stirring was started at 80 r / min, theDESCRIPTION

[0061] temperature was raised to 55 °C, and stirring was maintained for 60 minutes until the liquid was transparent and homogeneous. The liquid was cooled to room temperature to prepare the deep eutectic solvent.

[0062] Five kilograms of the above defatted milk thistle seed cake / meal was weighed and placed into an extraction tank, and 50 kg of the deep eutectic solvent was added and mixed uniformly. Before extraction, nitrogen was introduced for replacement for 5 minutes; during extraction, a slight positive-pressure nitrogen atmosphere was maintained in the tank. The temperature was controlled at 50 °C, and extraction was performed with stirring for 90 minutes. After extraction, plate-and-frame pressure filtration was performed for solid-liquid separation, and the filtrate was collected to obtain a deep eutectic solvent extract.

[0063] Anhydrous ethanol was added to the extract at a volume ratio of extract to anhydrous ethanol of 1:2.2, and stirring was performed for 30 minutes. The ethanol volume fraction of the system was 68%. Filtration was performed to remove precipitated impurities, thereby obtaining an ethanol-converted solution.

[0064] Purified water was added to the ethanol-converted solution to dilute the system to an ethanol volume fraction of 30%. The solution was allowed to stand for impurity precipitation for 1.5 hours and then filtered to obtain a column-loading solution.

[0065] The column-loading solution was introduced into the AB-8 macroporous adsorption resin column, with a loading flow rate of 1.0 BV / h and a loading amount of 3 BV. After water washing with 2.5 BV, an ethanol solution having a volume fraction of 60% was used for elution at an elution flow rate of 1.0 BV / h and an elution volume of 4 BV, and the ethanol eluate was collected.DESCRIPTION

[0066] The eluate was concentrated under reduced pressure at 45 °C and -0.08 MPa to a relative density of 1.12 (60 °C). Purified water was added at a volume ratio of 1:3, and after mixing uniformly, the system was cooled to 8 °C and allowed to stand for crystallization for 12 hours. After centrifugal separation, crystals were collected and vacuum-dried at 45 ° C to constant weight to obtain 265 g of silymarin product.

[0067] Example 3

[0068] Twenty kilograms of mature dried milk thistle seeds were placed into a low-temperature press, and the pressing temperature was controlled not to exceed 45°C for cold pressing and defatting to obtain defatted milk thistle seed cake / meal. The cake / meal was pulverized and passed through a 70-mesh sieve. Sampling and testing showed that the cake / meal had an oil content of 4.9% and a moisture content of 6.8%.

[0069] Betaine, lactic acid, and purified water were weighed and placed into a stirred batching tank at a mass ratio of 1 :2:0.6. Stirring was started at 80 r / min, the temperature was raised to 65 °C, and stirring was maintained for 30 minutes until the liquid was transparent and homogeneous. The liquid was cooled to room temperature to prepare the deep eutectic solvent.

[0070] Five kilograms of the above defatted milk thistle seed cake / meal was weighed and placed into an extraction tank, and 50 kg of the deep eutectic solvent was added and mixed uniformly. Before extraction, nitrogen was introduced for replacement for 10 minutes; during extraction, a slight positive-pressure nitrogen atmosphere was maintained in the tank. The temperature was controlled at 50 °C, and extraction was performed with stirring for 90 minutes. After extraction,DESCRIPTION

[0071] plate-and-frame pressure filtration was performed for solid-liquid separation, and the filtrate was collected to obtain a deep eutectic solvent extract.

[0072] Anhydrous ethanol was added to the extract at a volume ratio of extract to anhydrous ethanol of 1:2.2, and stirring was performed for 30 minutes. The ethanol volume fraction of the system was 68%. Filtration was performed to remove precipitated impurities, thereby obtaining an ethanol-converted solution.

[0073] Purified water was added to the ethanol-converted solution to dilute the system to an ethanol volume fraction of 30%. The solution was allowed to stand for impurity precipitation for 2.5 hours and then filtered to obtain a column-loading solution.

[0074] The column-loading solution was introduced into the AB-8 macroporous adsorption resin column, with a loading flow rate of 1.5 BV / h and a loading amount of 3 BV. After water washing with 2.5 BV, an ethanol solution having a volume fraction of 60% was used for elution at an elution flow rate of 1.5 BV / h and an elution volume of 4 BV, and the ethanol eluate was collected.

[0075] The eluate was concentrated under reduced pressure at 45 °C and -0.08 MPa to a relative density of 1.15 (60 °C). Purified water was added at a volume ratio of 1:3, and after mixing uniformly, the system was cooled to 8 °C and allowed to stand for crystallization for 12 hours. After centrifugal separation, crystals were collected and vacuum-dried at 42 ° C to constant weight to obtain 270 g of silymarin product.

[0076] Comparative Example 1

[0077] An ethanol-water solution having a volume fraction of 70% was used as the extraction medium in place of the deep eutectic solvent, and the remainingDESCRIPTION

[0078] operating steps and parameter settings for raw material pretreatment, resin enrichment, concentration, and crystallization were kept consistent with those in Example 1, thereby preparing 230 g of silymarin product.

[0079] Comparative Example 2

[0080] The same deep eutectic solvent extraction process as in Example 1 was adopted. After a deep eutectic solvent extract was obtained, ethanol transfer-dissolution solvent-switching treatment was not performed. Purified water was directly added to the extract to dilute the total liquid volume to be the same as the total volume of the column-loading solution in Example 1. After uniform mixing, the liquid was directly introduced into the AB -8 macroporous adsorption resin column. The subsequent operating steps and parameter settings for elution, concentration, and crystallization were kept consistent with those in Example 1, thereby preparing 255 g of silymarin product.

[0081] Comparative Example 3

[0082] The same extraction, ethanol transfer-dissolution solvent switching, and dilution-induced impurity precipitation steps as in Example 1 were adopted. After a solution after impurity precipitation was obtained, AB-8 macroporous adsorption resin enrichment was not performed; instead, the solution was directly transferred into a reduced-pressure concentration tank for concentration, and subsequent water-ethanol antisolvent crystallization and drying were performed under the same conditions to prepare 265 g of silymarin product.

[0083] Comparative Example 4

[0084] The same extraction, ethanol transfer-dissolution solvent switching, dilution-induced impurity precipitation, and resin enrichment steps as in ExampleDESCRIPTION

[0085] 1 were adopted. After an ethanol eluate was obtained, water-ethanol antisolvent crystallization was not performed; instead, the eluate was concentrated under reduced pressure to an extract-like state and then directly vacuum-dried to constant weight to obtain 270 g of silymarin product.

[0086] The intermediate materials and final products of each example and comparative example were tested for indicators, and the test methods were as follows:

[0087] Extraction yield: calculated by a gravimetric method, wherein extraction yield = dry weight of final silymarin product / dry weight of defatted milk thistle seed cake / meal charged x 100%.

[0088] Viscosity of column-loading solution: detected using a rotational viscometer, with the test temperature kept constant at 25 ° C, and the average value of three parallel determinations was taken.

[0089] Contents of total silymarin, silybin A, and silybin B: determined by high-performance liquid chromatography. Chromatographic conditions: Cl 8 chromatographic column (250 mm x 4.6 mm, 5 p m); mobile phase of methanol-water-glacial acetic acid (volume ratio 48:52:0.5); detection wavelength 288 nm; column temperature 30°C; flow rate 0.8 mL / min; injection volume 20 p L. Appropriate amounts of silybin A and silybin B reference substances were accurately weighed, and methanol was added to prepare a mixed reference solution containing 0.1 mg of each per 1 mL. The contents of silybin A and silybin B were calculated by an external standard method; the remaining silymarin-related chromatographic peaks were converted according to the response coefficient of silybin, and the contents of all components were summedDESCRIPTION

[0090] to obtain the total silymarin content.

[0091] Moisture content: determined by Karl Fischer titration.

[0092] Residual deep eutectic solvent: the betaine content was determined by high-performance liquid chromatography, and the lactic acid content was determined by acid-base titration; the sum of the two values was taken as the total residual amount of the deep eutectic solvent.

[0093] The test results of the examples and comparative examples are shown in Table 1 below:

[0094] ITEM EX. 1 EX.2 EX.3 COMP. EX. 1 COMP. EX.2 COMP. EX.3 COMP. EX.4 YIELD 5.6% 5.3% 5.4% 4.6% 5.1% 5.3% 5.4% vise.

[0095] 2.4 mPa s 2.6 mPa s 2.5 mPa s 1.9 mPa s 8.8 mPa s 2.5 mPa s 2.4 mPa s (25°C)

[0096] TOTAL

[0097] 83.4% 82.1% 82.8% 75.8% 77.1% 68.4% 73.8% SILYM.

[0098] SILYBIN

[0099] 40.6% 39.4% 39.9% 34.0% 34.9% 31.2% 35.1% A+B

[0100] MOIST. 3.0% 3.3% 3.2% 3.7% 4.0% 4.5% 4.1% DES NOT

[0101] 0.27% 0.31% 0.30% 0.68% 0.42% 0.38% RES. DETECTED

[0102]

[0103] TABLE 1

[0104] It can be seen from the test data that the products prepared in the three examples had total silymarin contents between 82.1% and 83.4% and combined contents of silybin A and silybin B between 39.4% and 40.6%. Due to differences in raw material batches and normal fluctuations in operating conditions, the product indicators can stably fluctuate within the ranges of 82%-84% and 39%-41 %, indicating good process reproducibility.

[0105] Compared with Comparative Example 1 , the deep eutectic solvent extraction method of the present process provides an extraction yield higher than that ofDESCRIPTION

[0106] conventional 70% ethanol extraction, and the product purity after subsequent purification is also higher. Compared with Comparative Example 2, after ethanol transfer-dissolution solvent switching and dilution-induced impurity precipitation treatment, the viscosity of the column-loading solution decreased from 8.8 mPa s to 2.4-2.6 mPa s, the total silymarin content of the product increased by about 6 percentage points, and the residual amount of the deep eutectic solvent decreased by more than half, indicating that the solvent-switching step can effectively overcome the disadvantages of directly loading a deep eutectic extract onto the column. From the data comparison of Comparative Examples 3 and 4 with Example 1 , it can be seen that the two purification steps of resin enrichment and antisolvent crystallization cooperate with each other to jointly improve product purity; if either step is omitted, both the total product content and the silybin proportion decrease significantly.

[0107] The above descriptions are merely preferred embodiments of the present invention and do not limit the present invention in any form. Although the present invention has been disclosed above by preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make some changes or modifications to the technical contents disclosed above within the scope of the technical solution of the present invention to form equivalent embodiments with equivalent changes, provided that such changes or modifications do not depart from the contents of the technical solution of the present invention. Any simple modifications, equivalent changes, and modifications made to the above embodiments according to the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

CLAIMS1. A method for preparing silymarin by ethanol switching of a deep eutectic solvent extract and resin enrichment, characterized in that the method comprises the following steps:(1) raw material pretreatment: mature dried milk thistle seeds are defatted by low-temperature pressing at a temperature not higher than 45°C to obtain a defatted milk thistle seed cake / meal having an oil content not higher than 8% and a moisture content of 5%-9%; the defatted milk thistle seed cake / meal is pulverized to 50-70 mesh;(2) preparation of a deep eutectic solvent: betaine, lactic acid, and water are mixed at a mass ratio of 1:2:0.6, stirred at 55-65 ° C until transparent and homogeneous, and cooled to room temperature to obtain the deep eutectic solvent;(3) extraction with the deep eutectic solvent: the defatted milk thistle seed cake / meal is mixed with the deep eutectic solvent at a mass ratio of 1:10, extracted at 50 ° C for 90 minutes under nitrogen protection, and subjected to solid-liquid separation to obtain a deep eutectic solvent extract;(4) ethanol transfer-dissolution solvent switching: anhydrous ethanol is added to the deep eutectic solvent extract for solvent-switching treatment so that the ethanol volume fraction of the system is 68%, and after stirring, filtration is performed to obtain an ethanol-converted solution;(5) dilution-induced impurity precipitation: the ethanol-converted solution is diluted with water to an ethanol volume fraction of 30%, allowed to stand for impurity precipitation, and filtered to obtain a column-loading solution;(6) resin enrichment: the column-loading solution is passed through AB-8 weakly polar macroporous adsorption resin, eluted sequentially with water and an ethanol solution having a volume fraction of 60%, and an ethanol eluate is collected;(7) concentration and crystallization: the ethanol eluate is concentrated under reduced pressure at 45°C to a relative density of 1.10-1.15, purified water is added as an antisolvent at a concentrate-to-purified water volume ratio of 1:3,CLAIMScrystallization is performed by standing at 8 ° C for 12 hours, and solid-liquid separation and vacuum drying are performed to obtain a silymarin product.

2. The method for preparing silymarin by ethanol switching of a deep eutectic solvent extract and resin enrichment according to claim 1, wherein in step (1), the defatted milk thistle seed cake / meal has an oil content of 4.5%-6.5% and a moisture content of 6%-8%.

3. The method for preparing silymarin by ethanol switching of a deep eutectic solvent extract and resin enrichment according to claim 1, wherein in step (2), during preparation of the deep eutectic solvent, the stirring temperature is 60°C and the stirring time is 30-60 minutes.

4. The method for preparing silymarin by ethanol switching of a deep eutectic solvent extract and resin enrichment according to claim 1, wherein in step (3), the nitrogen protection comprises introducing nitrogen for replacement for 5-10 minutes before extraction starts and maintaining a nitrogen atmosphere in the extraction vessel during the extraction.

5. The method for preparing silymarin by ethanol switching of a deep eutectic solvent extract and resin enrichment according to claim 1 , wherein in step (4), the volume ratio of the deep eutectic solvent extract to anhydrous ethanol is 1:2.2, and the stirring time is 30 minutes.

6. The method for preparing silymarin by ethanol switching of a deep eutectic solvent extract and resin enrichment according to claim 1, wherein in step (5), the standing time for impurity precipitation is 1.5-2.5 hours, and the obtained column-loading solution has a viscosity of 2.0-3.0 mPa s at 25°C.

7. The method for preparing silymarin by ethanol switching of a deep eutectic solvent extract and resin enrichment according to claim 1 , wherein in step (6), the loading flow rate of the AB-8 weakly polar macroporous adsorption resin is 1-2 BV / h, and the loading amount is 2-4 BV.

8. The method for preparing silymarin by ethanol switching of a deep eutectic solvent extract and resin enrichment according to claim 1 , wherein in step (6), theCLAIMSwater-washing volume is 2-3 BV, the 60% ethanol elution volume is 3-5 BV, and the elution flow rate is 1-2 BV / h.

9. The method for preparing silymarin by ethanol switching of a deep eutectic solvent extract and resin enrichment according to claim 1, wherein in step (7), the vacuum drying temperature is 40-50°C.

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