A preparation method of verbascoside

Abstract

The invention belongs to the technical field of traditional Chinese medicine, and specifically relates to a preparation method of verbascoside, which comprises: extracting with acidic ethanol for the first time under reflux to obtain a first filtrate and a first filter residue; extracting the first filter residue under reflux for the second time to obtain a second filtrate; concentrating, standing, and filtering after combining the first filtrate and the second filtrate to obtain a third filtrate; adding an acidic aqueous solution containing chitosan to the third filtrate, mixing evenly, standing overnight, and filtering to obtain a fourth filtrate; purifying the fourth filtrate with a D101 macroporous resin column, eluting, collecting the eluted fraction, concentrating, standing overnight, and obtaining a supernatant; purifying the supernatant with an SP825 macroporous resin column, eluting, collecting the eluted fraction, concentrating, and drying to obtain a crude extract, crushing, adding acetone, stirring, and filtering to obtain a fifth filtrate, concentrating, and vacuum drying to obtain an extract, crushing, adding methanol and dichloromethane for recrystallization, and drying under reduced pressure to obtain verbascoside. The method has high extraction purity and good reproducibility, and is suitable for industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a preparation method of verbascoside. Background Art

[0002] Verbascoside is a phenylethanoid glycoside derivative composed of caffeic acid, hydroxytyrosol, glucose, and rhamnose. It is widely found in various natural medicinal plants and exhibits a variety of biological activities, including anti-inflammatory, antibacterial, antiviral, anti-tumor, antioxidant, analgesic, neuroprotective, immunomodulatory, and cell memory improvement. However, plant-derived verbbascosides suffer from low content and environmental pollution, making large-scale production difficult.

[0003] Chinese patent application 202310829807.5 discloses a method for extracting effective active ingredients from Buddleja buddhistata and its application. The method is to extract the crushed Buddleja buddhistata with ethanol reflux and concentrate it. The obtained Buddleja buddhistata alcohol extract is then extracted with petroleum ether, ethyl acetate and n-butanol respectively. The remaining aqueous phase is concentrated under reduced pressure and purified by D101 macroporous resin column. The target product obtained after drying is structurally identified and determined to be a verbascoside monomer compound.

[0004] Chinese patent application 202110558109.7 discloses a method for preparing verbascoside from Rehmannia glutinosa, which includes extraction, drying, purification, fraction collection and concentration steps. The Rehmannia glutinosa extract is directly separated and purified using a liquid chromatography column. The operation is simple and the transfer rate is high. High-purity verbascoside can be obtained by simply collecting the eluate at the corresponding time.

[0005] Currently, most verbascoside extraction processes in the field are limited to laboratory extraction and are unsuitable for industrial extraction. For example, Chinese patent application 202110558109.7 extracts verbascoside from only 100g of Rehmannia root. Furthermore, most verbascosides are prepared solely from the medicinal parts of Rehmannia root, failing to fully utilize the medicinal value of non-essential medicinal parts.

[0006] Therefore, there is a need in the art for a process suitable for industrial, large-scale preparation of verbascoside. Summary of the Invention

[0007] In order to achieve the above technical objectives, the present invention uses the traditional non-main medicinal part of Rehmannia glutinosa, Rehmannia glutinosa leaves, to prepare verbascoside, and hereby proposes the following technical solutions:

[0008] In one aspect, the present invention provides a method for preparing verbascoside, comprising the following steps:

[0009] (1) performing a first reflux extraction on the verbascoside raw material with acidic ethanol to obtain a first filtrate and a first filter residue;

[0010] (2) subjecting the first filter residue to a second reflux extraction with acidic ethanol to obtain a second filtrate and a second filter residue;

[0011] (3) combining the first filtrate and the second filtrate and concentrating the mixture until no alcohol tastes, then allowing the mixture to stand and filtering to obtain a third filtrate;

[0012] (4) adding an acidic aqueous solution containing chitosan to the third filtrate, mixing well, letting it stand overnight, and filtering to obtain a fourth filtrate;

[0013] (5) taking the fifth filtrate and adding it to a D101 macroporous resin column for purification, and after purification, performing a first elution with a first eluent, and collecting the first elution fraction;

[0014] (6) concentrating the first eluted fraction until there is no alcohol smell and then allowing it to stand overnight to obtain a supernatant;

[0015] (7) adding the supernatant to an SP825 macroporous resin column for purification, performing a second elution with a second eluent after purification, and collecting the second elution fraction;

[0016] (8) concentrating the second elution fraction until there is no alcohol smell and then vacuum drying to obtain a crude extract of verbascoside;

[0017] (9) grinding the crude extract of verbascoside, adding acetone, stirring, and filtering to obtain a fifth filtrate;

[0018] (10) concentrating and vacuum drying the fifth filtrate to obtain an acetone extract;

[0019] (11) Grinding the acetone extract into fine powder, adding methanol to dissolve it, and then slowly adding dichloromethane until the solution becomes turbid to obtain a mixed solution;

[0020] (12) The mixed solution is recrystallized and then dried under reduced pressure to obtain verbascoside.

[0021] In some embodiments, the verbascoside raw material in step (1) can be Rehmannia root leaves.

[0022] In some embodiments, the acidic ethanol in steps (1) and (2) is a 60%-80% ethanol solution containing 0.5% glacial acetic acid, preferably an 80% ethanol solution containing 0.5% glacial acetic acid.

[0023] The percentages are by volume and the volumes are in mL.

[0024] In some embodiments, the mass-to-volume ratio of the acidic ethanol to the verbascoside raw material in steps (1) and (2) is 8:1, wherein the volume of the acidic ethanol is measured in L, and the mass of the verbascoside raw material is measured in kg.

[0025] In some embodiments, the mass volume ratio of the verbascoside raw material in step (1) to the D101 macroporous resin in the D101 macroporous resin column in step (5) is 1:1.

[0026] In some embodiments, the mass-to-volume ratio of the verbascoside raw material in step (1) to the SP825 macroporous resin of the SP825 macroporous resin column in step (5) is 1:1, wherein the mass of the verbascoside raw material is measured in kg and the volume of the SP825 macroporous resin is measured in L.

[0027] In some embodiments, the temperature of the third filtrate in step (4) is 30-40°C.

[0028] In some embodiments, the acidic aqueous solution in step (4) contains 1% chitosan, wherein the percentage is a mass-to-volume ratio, with mass in g and volume in mL.

[0029] In some embodiments, the mass volume ratio of the acidic aqueous solution in step (4) to the verbascoside raw material in step (1) is 0.3-0.4:1, preferably 0.3:1.

[0030] In some embodiments, the acidic aqueous solution in step (4) is an aqueous solution containing 0.5% glacial acetic acid, wherein the percentage is a volume ratio.

[0031] In some embodiments, the first eluent in step (5) comprises 15% ethanol and 40% ethanol, wherein the percentages are by volume.

[0032] In some embodiments, the first elution in step (5) comprises:

[0033] The solution was eluted with 15% ethanol for 5 column volumes and finally eluted with 40% ethanol for 5 column volumes.

[0034] In some embodiments, the first elution component in step (5) is an eluate of 40% ethanol.

[0035] In some embodiments, the second eluent in step (7) comprises water and 95% ethanol, wherein the percentages are by volume.

[0036] In some embodiments, the second elution in step (7) comprises:

[0037] After elution with 5 column volumes of water, the solution was eluted with 3 column volumes of 95% ethanol.

[0038] In some embodiments, the second elution component in step (7) is a 95% ethanol eluate.

[0039] In some embodiments, the mass-to-volume ratio of the acetone in step (9) to the crude verbascoside extract in step (8) is 20:1, wherein the volume of the acetone is measured in mL and the mass of the crude verbascoside extract is measured in g.

[0040] In some embodiments, the stirring time in step (9) is 60-70 minutes.

[0041] In some embodiments, step (9) further comprises:

[0042] Add the activated carbon after adding the acetone.

[0043] In some embodiments, the mass volume ratio of the fine powder, methanol and dichloromethane in step (11) is 1:3:9, wherein the mass of the fine powder is measured in g, the volume of the methanol is measured in mL, and the volume of the dichloromethane is measured in mL.

[0044] In some embodiments, the number of recrystallizations in step (12) is 2-3 times, preferably 2 times.

[0045] In another aspect, the present invention provides verbascoside obtained by any of the aforementioned preparation methods.

[0046] In another aspect, the present invention provides the use of the verbascoside in the preparation of a medicament for preventing and / or treating chronic nephritis glomeruli.

[0047] To ensure the quality of verbascoside and guarantee the reliability, reproducibility, and rationality of the extraction, separation, and purification processes, the present invention explored the extraction, separation, and purification processes for verbascoside, ultimately determining a verbascoside extraction process that is reproducible, has a high extraction yield, and is suitable for industrial production. By utilizing a non-essential medicinal part of Rehmannia root, significant cost savings are achieved, fully realizing the medicinal value of Rehmannia root leaves, and implementing the concepts of turning waste into treasure and protecting the environment. DETAILED DESCRIPTION

[0048] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0049] As used herein, the articles "a," "an," and "an" include plural referents unless the context clearly dictates otherwise.

[0050] Materials and instruments involved in the present invention:

[0051] Rehmannia leaves are picked in autumn, removed of impurities, chopped, and dehydrated.

[0052] Chitosan, manufacturer: Shandong Huantai County Jinhu Crust Products Co., Ltd., production batch number: 0701-1.

[0053] JA503-1 / 1000 class electronic balance (Shanghai Sunny Hengping Instrument Co., Ltd.);

[0054] Mix-3000 oscillating mixer (Hangzhou Miou Instrument Co., Ltd.);

[0055] The test method involved in the present invention is:

[0056] HPLC detection of verbascoside content:

[0057] Determine with reference to the high performance liquid chromatography method (Appendix D of Part I of the Pharmacopoeia of the People's Republic of China 2005 edition).

[0058] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as the packing material; methanol-glacial acetic acid-water (32:1:67) was used as the mobile phase, with a flow rate of 1 mL / min, a detection wavelength of 330 nm, and a column temperature of 35°C. The number of theoretical plates, calculated based on the verbascoside peak, should be no less than 8500.

[0059] Preparation of Reference Solution: Accurately weigh approximately 8 mg of verbascoside reference substance into a 50 mL volumetric flask. Dissolve and dilute to the mark with a mixture of methanol, glacial acetic acid, and water (32:1:67). Shake well to prepare this concentrated reference solution. Accurately measure 2 mL of this solution into a 10 mL volumetric flask, dilute to the mark with the above-mentioned mixed solvent, and shake well to prepare this reference solution.

[0060] Preparation of the test solution: Accurately weigh approximately 0.5 g of the fine powder of this product and place it in a 50 mL stoppered conical flask. Accurately add 25 mL of methanol containing 1% glacial acetic acid and weigh the weight. Heat under reflux for 1 hour, then cool to room temperature. Weigh the weight and replace any lost solvent, shake well, let stand, and filter. Accurately measure 2 mL of the filtrate and place it in a 10 mL volumetric flask. Dilute to the mark with the above mixed solution and shake well. Filter through a microporous filter (0.45 μm). The filtrate is used as the test solution.

[0061] Determination method: Accurately aspirate 10 μL of reference solution and test solution respectively, inject into high performance liquid chromatography, determine the peak area, and calculate by external standard method.

[0062] HPLC detection of ACT related substances:

[0063] The SP liquid chromatograph was equipped with an 8810 pump, an SP-100 UV detector, and a JS-3030 chromatography workstation. The chromatographic column was a WGY C18 bonded silica column, 5 μm, 4.60 × 250 mm. The detection wavelength was 330 nm. The column temperature was 35°C. The mobile phase was water-methanol-glacial acetic acid (67:32:1). The flow rate was 1 mL / min. The injection volume was 20 L. Octadecylsilane bonded silica was used as the filler.

[0064] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with the examples. In the examples, if no specific conditions are specified, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If all reagents or instruments are not specified by the manufacturer, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention. Such structures and technologies are also described in the publication of many publications.

[0065] Example 1

[0066] 80 kg of dried Rehmannia root leaves were subjected to reflux extraction twice, first with 8 times the amount (640 L) and then with 8 times the amount (640 L) of acidic 80% ethanol (containing 0.5% glacial acetic acid), with each reflux period of 60 minutes. After reflux, the two extracts were combined and concentrated until the extract had no alcohol flavor (i.e., the concentrated liquid volume was 65-80 L, with a relative density of rd 1.04-1.05 (45° C.)). After standing for 30 minutes, water was added to 110-120 L, the extract was allowed to stand, cooled, and filtered to obtain a filtrate. 10 L of an acidic aqueous solution (containing 1% chitosan, i.e., 100 g) was added to the filtrate (temperature around 35° C.), wherein the acidic aqueous solution was an aqueous solution containing 0.5% glacial acetic acid, with stirring while adding, and the extract was refrigerated overnight. The next day, the supernatant was decanted, filtered, and the filtrate applied to a D101 macroporous resin column. Elution was performed with 5 BV (column volume) of 10% ethanol, followed by 4 BV of 40% ethanol. The 40% ethanol eluate was collected and concentrated until the alcohol-free odor (i.e., the concentrated volume was approximately 100 L). Refrigerated and allowed to stand overnight to obtain the supernatant. The next day, the supernatant was applied to a pre-treated SP825 macroporous resin column and eluted with water (3 BV tap water, 2 BV distilled water), followed by 3 BV of 95% ethanol. The 95% ethanol eluate was collected and the ethanol was recovered until the alcohol-free odor was eliminated. The 95% ethanol concentrate was dried under reduced pressure (temperature 55-60°C, vacuum greater than 0.08 MPa) to obtain a dry crude extract. The crude extract was pulverized and acetone was added at a mass-to-volume ratio of 20:1. The mixture was stirred at a low temperature (35-40°C) for 60 minutes and filtered. Add activated carbon powder (30% by weight of the crude extract) to the filtrate, decolorize for 60 minutes, and filter. Concentrate the filtrate to a small volume and dry under vacuum (temperature 55-60°C, vacuum greater than 0.08 MPa) to obtain an acetone extract. Grind the acetone extract and pass it through an 80-mesh sieve. Dissolve the fine powder in methanol and slowly add dichloromethane (in a weight-to-volume ratio of 1:3:9) until the solution becomes turbid. Refrigerate until a large amount of crystals precipitate, filter, and dry under reduced pressure (temperature 55-60°C, vacuum greater than 0.08 MPa). Repeat the crystallization process 2-3 times to obtain verbascoside.

[0067] Example 2-Example 5

[0068] The experimental conditions are the same as those in Example 1.

[0069] Various parameters of verbascoside extracted from Examples 1-5 are shown in Table 1. The results showed that the verbascoside content in the verbascoside extracts from Examples 1-5 was above 93%.

[0070] Table 1

[0071]

[0072]

[0073] Experimental Example 1 Orthogonal experimental study of extraction process

[0074] The structure of verbascosides in Rehmannia glutinosa leaves belongs to the phenylethanoid glycoside class, which is readily soluble in dilute alcohol and water but poorly soluble in weakly polar organic solvents such as benzene and dichloromethane. Previous studies have shown that these components are stable under slightly acidic conditions, and the selected extraction solvents all contain 0.5% glacial acetic acid to maintain the acidic state of the solution. To effectively extract the active verbascosides, the present invention employed an orthogonal experiment to investigate the effects of four factors—extraction solvent, solvent dosage, extraction time, and number of extractions—on the extraction of the active ingredient.

[0075] Weigh 10g of Rehmannia glutinosa leaves and select L9(3) 4 Orthogonal table for orthogonal test, see Table 2. The dry paste yield of the extract (take 10mL of the extract and determine it according to the method of Appendix X.60 of the 2005 edition of the Chinese Pharmacopoeia) and the amount of verbascoside extracted were used as indicators.

[0076] Table 2

[0077]

[0078] The orthogonal experimental results are shown in Table 3. The most relevant factor affecting the extraction amount of verbascoside is the solvent, followed by the amount of extraction solvent. The optimal condition for the highest extraction amount of verbascoside is A2B2C2D2. The optimal condition for the lowest dry paste yield is A3B3C2D2. Taking into account the optimal conditions for the two extraction rates and suitability for industrial production, A2B2C2D2 is the best optimized process. That is, using 80% ethanol (containing 0.5% glacial acetic acid) as the solvent, the amount is 8 times the amount of the crude drug, and the process is refluxed twice, each time for 60 minutes. The process results using 80% ethanol (containing 0.5% glacial acetic acid) as the solvent, the amount is 8 times the amount of the crude drug, and the process is refluxed twice, each time for 60 minutes, are shown in Table 4.

[0079] Table 3

[0080]

[0081]

[0082] Table 4

[0083]

[0084] Experimental Example 2 Experimental Study on Chitosan Precipitation Process

[0085] Rehmannia leaves contain a large amount of chlorophyll. During the 80% ethanol extraction process, chlorophyll is also extracted. After the extract is concentrated, the chlorophyll remains floating in the solution and is difficult to remove by centrifugation or filtration. By adding a precipitant to the concentrate, the chlorophyll and other insoluble suspended matter floating in the solution are precipitated, and the supernatant is decanted, thereby removing impurities. Excess chitosan can be naturally removed by water elution during resin separation. Therefore, chitosan was selected as the precipitant, and a precipitation separation process using chitosan as a precipitant was established.

[0086] (1) Selection of precipitation temperature

[0087] Since the solution temperature has a great influence on the suspended solids of chitosan precipitation, different temperature ranges have a corresponding impact on the precipitation state and precipitation amount. Therefore, according to the properties of chitosan and the characteristics of this production process, we selected three temperature ranges of 35±2℃, 45±2℃, and 55±2℃ for investigation. The investigation method and results are as follows:

[0088] Method: Weigh 300 g of Rehmannia glutinosa leaves and extract and concentrate them according to the above extraction method. Divide 300 mL of the concentrate into three equal parts, 100 mL each (equivalent to 100 g of Rehmannia glutinosa leaves). When the concentrate is cooled to 55±2°C, 45±2°C, and 35±2°C, 10 mL of an acid aqueous solution containing 1% chitosan is added respectively. The mixture is allowed to stand overnight, the supernatant is poured out, the content of verbascoside is determined, the precipitate is dried, and the weight is calculated.

[0089] Table 5 Temperature investigation of solution during precipitation

[0090]

[0091] The results are shown in Table 5. When the temperature of the solution was 45±2°C when chitosan was added, the precipitate removed was in the best state, the amount of impurities removed was the largest, and the content of verbascoside in the supernatant solution was the highest.

[0092] (2) Selection of precipitant dosage

[0093] The dosage of chitosan used as a precipitant is generally 1% of the solution weight. However, the amount of suspended matter in the solution is directly related to the dosage of chitosan. Therefore, according to the properties of chitosan and the general dosage characteristics of chitosan, we selected four ratios to investigate the optimal process of chitosan dosage and precipitation removal. The investigation method and results are as follows:

[0094] Method: Weigh 400 g of Rehmannia glutinosa leaves and extract and concentrate them according to the above extraction method. Divide 400 mL of the concentrate into four equal parts, 100 mL each (equivalent to 100 g of Rehmannia glutinosa leaves). When the concentrate is cooled to 45±2°C, 10 mL, 20 mL, 30 mL, and 40 mL of an acid aqueous solution containing 1% chitosan are added, respectively. The mixture is allowed to stand overnight, and the supernatant is poured out. The content of verbascoside is determined. The precipitated part is dried and the weight is calculated.

[0095] The results are shown in Table 6.

[0096] Table 6 Investigation of precipitant dosage

[0097]

[0098] The results showed that adding 30 mL of chitosan solution had a good precipitation effect and a high extraction rate of verbascoside.

[0099] (3) Results of three repeated tests

[0100] According to the above investigation results, 100 g of Rehmannia glutinosa leaves were weighed, and 30 mL of 1% chitosan acid aqueous solution was added at a solution temperature of 45±2°C. The solution was left overnight, the supernatant was poured out, and the verbascoside content was determined. The precipitate was dried and weighed.

[0101] Table 7 Investigation of three precipitation results

[0102]

[0103] The results are shown in Table 7. The results of three investigations show that when the solution temperature is 45±2°C, adding 30 mL of an aqueous solution containing 1% chitosan acid has a good precipitation effect and a high transfer rate of verbascoside.

[0104] Experimental Example 3 Study on separation conditions of macroporous adsorption resin

[0105] In order to further purify the Rehmannia glutinosa leaf extract, the present invention selects the macroporous resin adsorption method to separate the verbascoside, and successively screens five resins, including SP825 and HP-20 produced by Mitsubishi Corporation of Japan, D101, H103, NKA-Ⅱ produced by Nankai University Chemical Plant, and D101 produced by Tianjin Pesticide Factory.

[0106] (1) Screening of macroporous adsorption resin types

[0107] Take 500g of Rehmannia glutinosa leaves, extract according to the extraction method of Example 1, chitosan precipitate, and obtain 500mL of supernatant. Measure 50mL of supernatant (equivalent to 50g of Rehmannia glutinosa leaves medicinal material), divide it into five parts, add respectively to treated resin H103, NKA-Ⅱ, D101 and SP825, HP-20 posts, the resin dosage is 200mL, respectively with water elution, every kind of resin column water elution amount is 1000mL, then with 50% ethanol elution 1000mL, finally again with 95% ethanol elution 1000mL, draw each sample solution 25mL respectively, measure the dry paste yield by Chinese Pharmacopoeia 2005 edition, Part I, Appendix X.60 page " Extraction Assay Method", take a small amount of sample liquid and filter with microporous membrane, measure the content of verbascoside in each elution solution.

[0108] The results, shown in Table 8, demonstrate that the macroporous adsorption resin SP825 exhibits strong adsorption capacity for verbascoside. Water elution removes the most impurities (2.29 g), with an impurity removal rate of approximately 4.5% (calculated based on the crude drug: 2.29 g paste per 50 g crude drug). The desorption rate is also strong, with nearly 95% of the verbascoside component being eluted using 50% ethanol. Therefore, the present invention selected the macroporous adsorption resin SP825 as the separation resin and further explored separation conditions, including resin dosage and elution solvent.

[0109] Table 8

[0110]

[0111] (2) Selection of macroporous adsorption resin dosage and elution reagent

[0112] Macroporous adsorption resin D101\SP825 has good adsorption and desorption capacity for verbascoside in water. In order to further enhance the separation effect of the resin, reduce the amount of resin and ethanol, and lower production costs, D101 resin was used as the adsorbent for the first elution and SP825 resin was used for the second elution. The effects of resin dosage and ethanol gradient elution on the separation of verbascoside were further investigated.

[0113] 50 mL of the supernatant after chitosan precipitation (equivalent to 50 g of Rehmannia glutinosa leaves) was measured and divided into four portions, and the mixture was added to 20 mL, 30 mL, 40 mL, and 50 mL D101 resin columns, respectively. The mixture was first eluted with water at 5 times the column volume, and then eluted with 15%, 40%, 65%, and 95% ethanol at 5 times the column volume. The eluates from each portion were collected and the dry paste yield was determined according to the "Determination of Extracts" on page 60 of Appendix X, Part I of the Chinese Pharmacopoeia 2005 edition. A small amount of sample solution was filtered through a microporous membrane, and the content of verbascoside in each elution solution was determined.

[0114] Table 9

[0115]

[0116] The results, as shown in Table 9, show that a 1:1 ratio of crude drug to resin is optimal. At a ratio of 50 g:50 mL, no verbascoside was detected in the water and 15% ethanol elution fractions, while nearly 50% of other impurities were removed. The 40% ethanol elution fraction removed over 90% of verbascoside. Therefore, this process employed elution with 10% ethanol (approximately 5 column volumes) followed by 40% ethanol (approximately 4 column volumes), with the 40% ethanol eluate collected.

[0117] Experimental Example 4 Study on the Second Elution Process

[0118] As shown in Table 10, during the first separation process using the macroporous adsorption resin D101, verbascoside was mainly concentrated in the 40% ethanol elution fraction. However, the 40% ethanol elution solution contained a large amount of water during the concentration process, causing the temperature of the concentrate (mainly aqueous solution) to increase (above 60° C.) in the later stage of concentration. Due to the prolonged concentration time, verbascoside was severely decomposed and partially precipitated in the alcohol-free aqueous solution.

[0119] Table 10

[0120]

[0121] The results showed that direct concentration and drying of the 40% ethanol eluate destroyed and decomposed approximately 50% of the verbascoside. To reduce the solution temperature and time during concentration, and to remove any water-soluble precipitates, this process involves concentrating the 40% ethanol eluate until it is free of alcohol. The concentrate is then passed through the resin again, eluting first with water and then with 95% ethanol. The 95% ethanol eluate is then concentrated until it is free of alcohol and dried under reduced pressure.

[0122] (1) Second separation using SP825 macroporous adsorption resin

[0123] Measure the above supernatant and directly pass it through a macroporous adsorption resin (the ratio of crude drug amount to resin amount is 100 g: 80-100 mL) with SP825. First elute with water, the elution volume is 5 times the volume of the resin, then elute with 95% ethanol, the elution volume is 5 times the volume of the resin, concentrate the 95% ethanol eluate until there is no alcohol smell, and dry it under reduced pressure. The drying temperature is less than 55°C and the vacuum pressure is greater than 0.08 MPa.

[0124] As shown in Table 11, the results show that the recovery rate of the concentrated and dried 95% ethanol elution solution is significantly improved compared to the direct concentration and drying of the 40% ethanol elution solution, reaching over 90%. The 40% ethanol eluate is concentrated until it is free of alcohol flavor, and chitosan is added to precipitate it. The 95% ethanol eluate is then treated (a second time) with a macroporous adsorption resin, and the 95% ethanol eluate is concentrated until it is free of alcohol flavor. The crude extract of verbascoside is obtained by vacuum drying under reduced pressure. The destruction loss rate of verbascoside is less than 20%. Therefore, this stage process has a high recovery rate and is feasible.

[0125] Table 11

[0126]

[0127] Experimental Example 5 Study on the Refining Process of Verbascum Glycoside

[0128] Verbascoside belongs to the class of phenylethanoid glycosides and is readily soluble in methanol, ethanol, and water, as well as soluble in acetone and n-butanol. It is insoluble in organic solvents such as dichloromethane and petroleum ether, and exhibits excellent solubility in methanol solutions. Based on years of research, we have focused on developing a refined production process for verbbascoside, taking both production costs and operational feasibility into consideration. This invention utilizes a solvent-based purification method, employing acetone extraction, activated carbon decolorization, and methanol dissolution and dichloromethane precipitation to refine the crude verbbascoside extract, ultimately achieving a verbbascoside purity exceeding 90%.

[0129] (1) Study on the acetone extraction and activated carbon decolorization process of verbascoside

[0130] Verbascoside is well soluble in acetone. Under certain temperature conditions, acetone can dissolve the verbascoside component to a maximum extent while removing acetone-insoluble matter, making it an ideal separation method for purifying verbascoside. Therefore, dissolving the crude verbascoside extract in acetone, controlling the solution temperature and stirring continuously during the dissolution process, can achieve the dissolution of more than 80% of the verbascoside. Adding an appropriate amount of activated carbon to the acetone solution can decolorize the solution, thereby ensuring the next step of the crystallization process.

[0131] Weigh 4 portions of 10 g each of a crude extract of verbascoside and add 100 mL (10 times), 150 mL (15 times), 200 mL (20 times), and 250 mL (25 times) of acetone solution, respectively. Control the solution temperature at 30-35°C and stir with a magnetic stirrer. Remove 2 mL of the acetone solution at 30, 40, 50, 60, and 70 minutes. Determine the verbascoside content. Simultaneously, evaporate the acetone solution to dryness and calculate the paste weight.

[0132] The results are shown in Table 12. When the crude extract of verbascoside was extracted with 20 times the amount of acetone at room temperature of 30-35°C and stirred for 60 minutes, the acetone extract had the highest verbascoside content of 0.52 g / g, and the transfer rate of verbascoside could reach over 85%.

[0133] Table 12

[0134]

[0135] After filtering, the acetone extract solution turns light brown. Adding an appropriate amount of activated carbon to the solution can turn the acetone extract yellow. The present inventors discovered that decolorizing the acetone extract facilitates the subsequent crystallization process. Therefore, adding appropriate activated carbon powder for decolorization has become an essential process in the production of verbascoside.

[0136] Three portions of the filtered acetone solution were measured, each with 100 mL. Activated carbon powder was added at 1%, 2%, and 3% of the solution volume, respectively. The mixture was stirred for 60 minutes, filtered, concentrated under reduced pressure, and vacuum dried to a powder. A crystallization test was performed according to the crystallization method. The crystallization state was observed. The mixture was filtered, dried, and the crystals were powdered to determine the verbascoside content.

[0137] The results are shown in Table 13. Adding 2% activated carbon powder to the acetone extract, stirring for 60 minutes, filtering, and concentrating the dried powder (acetone extract) is conducive to crystallization, and the crystal state is good, the yield is high, and the verbascoside content is high.

[0138] Table 13

[0139]

[0140] (2) Determination of the crystallization conditions of verbascoside

[0141] Based on the solubility properties of verbascoside, such as being easily soluble in methanol but insoluble in dichloromethane, after extensive research on recrystallization methods, we ultimately chose a recrystallization method that involved dissolving in methanol, adding dichloromethane, stirring while adding, and refrigerating (generally at around zero degrees), achieving satisfactory results.

[0142] Weigh 10 g of acetone extract, add 30 mL of methanol to dissolve (about 3 times the amount of acetone extract), slowly add dichloromethane, stirring while adding until the solution becomes turbid, then add about 3.5 times the amount of methanol, and put it into a cold storage (temperature at 0±5°C). After about 48 hours, a large amount of off-white powder will appear. Filter it and dry it under reduced pressure and vacuum at a temperature of less than 55°C to form a powder.

[0143] The results are shown in Table 14. After the first recrystallization, the content of verbascoside can reach more than 70%, and the yield is greater than 30%.

[0144] Table 14

[0145] Number of trials Color state Yield (g) Yield (%) content(%) 1 Off-white powder 3.2 32 74.2 2 Off-white powder 3.1 31 72.2 3 Off-white powder 3.4 34 76.1

[0146] Weigh 10 g of the first crystallization, add 40 mL of methanol to dissolve (the volume is about 4 times the amount of the first crystallization), slowly add dichloromethane, stirring while adding until the solution becomes turbid, add about 3 times the amount of methanol, and put it into a cold storage (temperature at 0±5°C). After about 24 hours, a large amount of white powder will appear. Filter it and dry it under reduced pressure and vacuum at a temperature of less than 55°C to form a powder.

[0147] The results are shown in Table 15. After the second recrystallization, the content of verbascoside can reach more than 93%, and the yield is greater than 75%.

[0148] Table 15

[0149] Number of trials Color state Yield (g) Yield (%) content(%) 1 Off-white powder 7.8 78 94.2 2 Off-white powder 8.2 82 93.7 3 Off-white powder 8.1 81 95.4

[0150] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing verbascoside, characterized in that: The preparation method comprises the following steps: (1) performing a first reflux extraction on the verbascoside raw material with acidic ethanol to obtain a first filtrate and a first filter residue; (2) subjecting the first filter residue to a second reflux extraction with acidic ethanol to obtain a second filtrate and a second filter residue; (3) combining the first filtrate and the second filtrate and concentrating the mixture until no alcohol tastes, then allowing the mixture to stand and filtering to obtain a third filtrate; (4) adding an acidic aqueous solution containing chitosan to the third filtrate, mixing well, letting it stand overnight, and filtering to obtain a fourth filtrate; (5) taking the fourth filtrate and adding it to a D101 macroporous resin column for purification, and after purification, performing a first elution with a first eluent, and collecting the first elution fraction; (6) concentrating the first eluted fraction until there is no alcohol smell and then allowing it to stand overnight to obtain a supernatant; (7) adding the supernatant to an SP825 macroporous resin column for purification, performing a second elution with a second eluent after purification, and collecting the second elution fraction; (8) concentrating the second elution fraction until there is no alcohol smell and then vacuum drying to obtain a crude extract of verbascoside; (9) grinding the crude extract of verbascoside, adding acetone, stirring, and filtering to obtain a fifth filtrate; (10) concentrating and vacuum drying the fifth filtrate to obtain an acetone extract; (11) Grinding the acetone extract into fine powder, adding methanol to dissolve it, and then slowly adding dichloromethane until the solution becomes turbid to obtain a mixed solution; (12) recrystallizing the mixed solution and drying under reduced pressure to obtain verbascoside; The acidic ethanol in steps (1) and (2) is a 60%-80% ethanol solution containing 0.5% glacial acetic acid, wherein the percentages are by volume and the volume is in mL; The acidic aqueous solution in step (4) is an aqueous solution containing 0.5% glacial acetic acid; the temperature of the third filtrate is 43-47° C.; the mass-to-volume ratio of the acidic aqueous solution to the verbascoside raw material in step (1) is 0.3-0.4:1, wherein the mass of the verbascoside raw material is measured in g, and the volume of the acidic aqueous solution containing chitosan is measured in mL.

2. The preparation method according to claim 1, characterized in that The volume-to-mass ratio of the acidic ethanol to the verbascoside raw material in steps (1) and (2) is 8:1, wherein the volume of the acidic ethanol is measured in L and the mass of the verbascoside raw material is measured in kg; and / or, the mass volume ratio of the verbascoside raw material in step (1) to the D101 macroporous resin in the D101 macroporous resin column in step (5) is 1:1; And / or, the mass-to-volume ratio of the verbascoside raw material in step (1) to the SP825 macroporous resin in the SP825 macroporous resin column in step (7) is 1:0.8-1, wherein the mass of the verbascoside raw material is measured in kg and the volume of the SP825 macroporous resin is measured in L.

3. The preparation method according to claim 1 or 2, characterized in that The acidic aqueous solution in step (4) contains 1% chitosan, wherein the percentage is a mass-to-volume ratio, with the mass being measured in kg and the volume being measured in L.

4. The preparation method according to claim 1, characterized in that The mass volume ratio of the acidic aqueous solution in step (4) to the verbascoside raw material in step (1) is 0.3:

1.

5. The preparation method according to claim 3, characterized in that In step (5), the first eluent comprises 15% ethanol and 40% ethanol, wherein the percentages are by volume; And / or, in step (5), the first elution comprises: Elution was performed with 5 column volumes of 15% ethanol and finally with 5 column volumes of 40% ethanol.

6. The preparation method according to claim 5, characterized in that In step (7), the second eluent comprises water and 95% ethanol, wherein the percentages are by volume; And / or, in step (7), the second elution comprises: After elution with 5 column volumes of water, the sample was eluted with 3 column volumes of 95% ethanol.

7. The preparation method according to claim 5 or 6, characterized in that: The mass-to-volume ratio of the acetone in step (9) to the crude verbascoside extract in step (8) is 20:1, wherein the volume of the acetone is measured in mL and the mass of the crude verbascoside extract is measured in g; And / or, the stirring time in step (9) is 60-70 min; And / or, add the activated carbon after adding the acetone.

8. The preparation method according to claim 7, characterized in that The mass volume ratio of the fine powder, methanol and dichloromethane in step (11) is 1:3:9, wherein the mass of the fine powder is measured in g, the volume of the methanol is measured in mL, and the volume of the dichloromethane is measured in mL.

9. The preparation method according to claim 1, characterized in that The number of recrystallizations in step (12) is 2-3 times.

10. The preparation method according to claim 9, characterized in that The number of recrystallizations in step (12) is 2 times.

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