Method for extracting dauricine from rhizoma menispermi

Abstract

The invention relates to the technical field of plant active ingredient extraction, in particular to a method for extracting dauricine from rhizoma menispermi, which comprises the following steps: extracting powder obtained by crushing the rhizoma menispermi by using a first mixed solution containing a first inorganic acid and a first alcohol solvent, carrying out solid-liquid separation, and adjusting the pH value to obtain a rhizoma menispermi alkaline extracting solution; carrying out resin adsorption treatment on the rhizoma menispermi alkaline extracting solution, washing the resin by using alkali liquor, water and acid liquor in sequence, and eluting by using a second mixed solution containing second inorganic acid and a second alcohol solvent to obtain eluent; filtering the eluent with activated carbon, adjusting the eluent to be alkaline, and precipitating to obtain rhizoma menispermi total alkali; the method comprises the following steps: dissolving the rhizoma menispermi total alkali in a halogenated hydrocarbon organic solvent, sequentially extracting with alkali liquor and acid liquor to obtain extract liquor, and further precipitating to obtain dauricine. The method is short in technological process, easy and convenient to operate and environmentally friendly, the content and extraction efficiency of the target dauricine are improved, and industrial expanded production is facilitated.

Description

Technical Field

[0001] This application belongs to the field of plant active ingredient extraction technology, and in particular relates to a method for extracting puerarin from Sophora flavescens root. Background Technology

[0002] Northern Bean Root (Menispermum dauricum DC.) is the dried rhizome of the Menispermaceae family plant, mainly produced in Northeast, North, and Shaanxi provinces of China. It possesses properties of clearing heat and detoxifying, dispelling wind and relieving pain, and is clinically used to treat sore throat, enteritis and dysentery, and rheumatic pain. Northern Bean Root contains various alkaloids, with a total alkaloid content of approximately 1.7-2.5%, primarily consisting of dauricin, dauriline, dauricinol, dauricinol, and dauricinoline. These five alkaloids are all phenolic alkaloids, belonging to the tail-tail monooxybridged dibenzyltetrahydroisoquinoline class. Among them, dauricin exhibits antiarrhythmic, myocardial oxygen consumption reduction, anti-atherosclerotic, and platelet aggregation inhibition effects, showing broad clinical application prospects in the prevention and treatment of cardiovascular diseases.

[0003] The chemical formula of piperine is: C 38 H 44 N2O6; molecular weight: 624.8; structural formula as follows:

[0004]

[0005] The commonly used form of piperine is its sulfate, which is a white, fine needle-like crystal, odorless, with an extremely bitter taste. It gradually changes color upon exposure to light, and its aqueous solution is dextrorotatory. It is readily soluble in boiling water or ethanol, soluble in chloroform, slightly soluble in water, and almost insoluble in ether.

[0006] Currently, most methods for extracting cinnabar from puerarin employ single organic solvents or multi-step solvent extraction processes. These methods suffer from low extraction rates, low crude product content, and numerous impurities. Furthermore, the complex processes make it difficult to achieve large-scale production. Summary of the Invention

[0007] The purpose of this application is to provide a method for extracting puerarin from Sophora flavescens, aiming to solve the technical problem of how to better extract puerarin from Sophora flavescens.

[0008] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows:

[0009] This application provides a method for extracting puerarin from Sophora flavescens root, comprising:

[0010] The root of Sophora flavescens was crushed to obtain powder, and the powder was extracted with a first mixed solution containing a first inorganic acid and a first alcohol solvent. After solid-liquid separation, the pH value was adjusted to obtain an alkaline extract of Sophora flavescens.

[0011] The alkaline extract of Sophora flavescens was treated with resin adsorption, and then the resin was washed sequentially with alkaline solution, water and acid solution. The extract was then eluted with a second mixed solution containing a second inorganic acid and a second alcohol solvent to obtain an eluent.

[0012] The eluent was filtered with activated carbon, and the filtrate was adjusted to alkalinity to precipitate and obtain total alkali from Sophora flavescens.

[0013] The total alkaloids of the Sophora flavescens root were dissolved in a halogenated hydrocarbon organic solvent, and then extracted with alkali and acid solutions in sequence to obtain an extract. The extract was then precipitated to obtain basal alkaloids.

[0014] In some embodiments, in the first mixed solution, the first inorganic acid is selected from at least one of hydrochloric acid and sulfuric acid, and the first alcohol solvent is selected from at least one of methanol and ethanol;

[0015] And / or, in the first mixed solution, the mass concentration of the first inorganic acid is 0.5-1%, and the volume concentration of the first alcohol solvent is 60-80%.

[0016] In some embodiments, the conditions for extracting the powder with the first mixed solution include: an extraction temperature of 40-70°C, an extraction time of 6-8 hours; and / or, 2-3 extractions.

[0017] In some embodiments, the step of adjusting the pH value after solid-liquid separation to obtain an alkaline extract of Sophora flavescens includes: adjusting the pH value of the extract after solid-liquid separation to 6.5-8, then concentrating and filtering under pressure, and adding inorganic alkali to a mass concentration of 0.5-1%.

[0018] In some embodiments, in the step of washing the resin sequentially with alkali, water, and acid, the alkali is selected from an aqueous solution of sodium hydroxide or potassium hydroxide with a mass concentration of 0.5-1%, and the acid is selected from an aqueous solution of hydrochloric acid or sulfuric acid with a mass concentration of 0.15-0.5%.

[0019] In some embodiments, in the second mixed solution, the second inorganic acid is selected from at least one of hydrochloric acid and sulfuric acid, and the second alcohol solvent is selected from at least one of methanol and ethanol;

[0020] And / or, in the second mixed solution, the mass concentration of the second inorganic acid is 3-5%, and the volume concentration of the second alcohol solvent is 60-80%.

[0021] In some embodiments, the pH of the eluent is adjusted to 6.5-8 before being filtered with activated carbon.

[0022] And / or, in the step of adjusting the filtrate to alkalinity for precipitation, the pH value of the filtrate is adjusted to 8.5-9.5, and then allowed to stand for 6-10 hours;

[0023] And / or, the precipitation process further includes vacuum drying at a temperature of 50-70°C.

[0024] In some embodiments, macroporous resin column adsorption is used in the resin adsorption treatment; and / or, the halogenated hydrocarbon organic solvent includes at least one of dichloromethane and trichloromethane.

[0025] In some embodiments, the step of sequentially extracting with alkaline and acidic solutions includes: first extracting with a 0.5-5% (w / w) aqueous solution of sodium hydroxide or potassium hydroxide, then washing with water, and then extracting with a 0.15-0.5% (w / w) aqueous solution of hydrochloric acid or sulfuric acid; and / or the number of times the sequential extraction with alkaline and acidic solutions is performed is 1-4 times.

[0026] In some embodiments, precipitating the extract includes adjusting the pH of the extract to 8.5-9.5 before precipitation.

[0027] The method for extracting puerarin from Sophora flavescens root provided in this application involves first crushing the Sophora flavescens root, then performing acid-alcohol extraction using a mixed solution containing a first inorganic acid and a first alcohol solvent, and adjusting the pH of the extract to obtain an alkaline extract. This alkaline extract is then adsorbed onto resin, washed stepwise to remove impurities (i.e., washed sequentially with alkali, water, and acid), and eluted with a mixed solution containing a second inorganic acid and a second alcohol solvent. The eluent is further filtered through activated carbon, and the total alkali of the Sophora flavescens root is precipitated. Finally, the total alkali of the Sophora flavescens root is dissolved in a halogenated hydrocarbon organic solvent, and puerarin is obtained through a series of alkali-acid extractions. This method, employing acid-alcohol extraction, resin separation, activated carbon filtration, and back-extraction techniques, not only has a short process flow, simple operation, and is environmentally friendly, but also improves the content and extraction efficiency of the target compound, puerarin, which is beneficial for large-scale industrial production. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 This is the HPLC chromatogram of the total extract of Sophora flavescens obtained in Example 1 of this application, with a content of 6.80% of puerarin.

[0030] Figure 2 This is the HPLC chromatogram of total alkaloids from Sophora flavescens obtained in Example 1 of this application, with a content of 72.13% of the alkaloids.

[0031] Figure 3 This is the HPLC chromatogram of the pure puerarin obtained in Example 1 of this application, with a puerarin content of 97.27%. Detailed Implementation

[0032] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0033] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0034] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of a single item or a plurality of items.

[0035] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0036] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0037] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass described in the embodiments of this application can be a mass unit known in the chemical industry, such as μg, mg, g, or kg.

[0038] The terms "first" and "second" are used for descriptive purposes only, to distinguish objects, such as substances, from one another, and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. For example, without departing from the scope of the embodiments of this application, "first XX" may also be referred to as "second XX," and similarly, "second XX" may also be referred to as "first XX." Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of that feature.

[0039] Currently, most methods for extracting quinine from sclerotium affine employ a single organic solvent (such as benzene or ethanol) or a multi-step solvent extraction process. These methods suffer from low extraction rates, low crude product content, and a high number of impurities. For example, using benzene as a solvent, highly toxic benzene not only poses risks to the environment and operator health but also makes process control difficult, resulting in poor repeatability and stability. Furthermore, traditional methods often require multiple extraction, filtration, and precipitation steps, leading to complex processes that are difficult to scale up for production.

[0040] Based on this, this application combines acid-alcohol extraction, resin separation, activated carbon filtration, and back-extraction processes to develop an extraction method with a short process flow, simple operation, and good extraction effect of the target substance, which is environmentally friendly. The specific technical solution is as follows.

[0041] This application provides a method for extracting puerarin from Sophora flavescens root. Specifically, the method of this application includes the following steps:

[0042] S01: After crushing the root of Sophora flavescens into powder, extract the powder with a first mixed solution containing a first inorganic acid and a first alcohol solvent. After solid-liquid separation, adjust the pH value to obtain an alkaline extract of Sophora flavescens root.

[0043] S02: The alkaline extract of Sophora flavescens is treated with resin adsorption, then the resin is washed with alkali solution, water and acid solution in sequence, and then eluted with a second mixed solution containing a second inorganic acid and a second alcohol solvent to obtain the eluent.

[0044] S03: After filtering the eluent with activated carbon, the filtrate is adjusted to alkaline to precipitate and obtain the total alkali of Sophora flavescens.

[0045] S04: Dissolve the total alkaloids of Sophora flavescens in a halogenated hydrocarbon organic solvent, and then extract with alkali and acid solutions in sequence to obtain an extract. Precipitate the extract to obtain basal alkaloids.

[0046] In this embodiment, the Sophora flavescens root is first pulverized, and then the pulverized Sophora flavescens root powder is subjected to acid-alcohol extraction using a first mixed solution containing a first inorganic acid and a first alcohol solvent. The alcohol solvent can effectively dissolve the alkaloids and other active ingredients in the Sophora flavescens root, while the addition of the inorganic acid can promote the conversion of some alkaloids into salt form, increase their polarity, and make them easier to dissolve in the extract. At the same time, the inorganic acid environment can partially hydrolyze the pectin in the cell wall, making the plant cell structure loose, thereby increasing the permeability of the mixed solution to the active ingredients in the Sophora flavescens root powder, and allowing more alkaloids to dissolve.

[0047] In this embodiment, the alkaline extract of *Sophora tigrin* obtained above is adsorbed onto a resin, which allows for better separation and purification of the target product, puerarin. Specifically, the alkaline extract is directly loaded onto a column for adsorption. Different eluents are used consecutively (first, non-target impurities are washed away with alkaline solution, then pigments are washed away with water, and finally alkaline impurities are washed away with acid), followed by elution of the target product using a second mixed solution containing a second inorganic acid and a second alcohol solvent. This process not only reduces the risk of column blockage and incomplete elution due to the poor water solubility of puerarin, but also achieves high content and high recovery rate of the target product under mild conditions.

[0048] In this embodiment, the eluent is filtered through activated carbon to further remove impurities such as pigments and metals. The filtrate obtained after activated carbon filtration is adjusted to alkaline, and precipitation occurs in the alkaline environment to obtain total alkali from Sophora flavescens.

[0049] In this embodiment, the total alkaloids of Sophora flavescens are redissolved in a halogenated hydrocarbon organic solvent, and then further extracted with a series of alkali and acid solutions to obtain a high content of basal alkaloids. The halogenated hydrocarbon organic solvent has good solubility for most organic alkaloids and forms a two-phase separation with water, facilitating subsequent efficient extraction and separation.

[0050] In summary, the embodiments of this application employ acid-alcohol extraction, resin separation, activated carbon filtration, and back-extraction technologies, which not only have a short process flow, simple operation, and environmental friendliness, but also improve the content and extraction efficiency of the target compound, puerarin, which is beneficial for large-scale industrial production.

[0051] Step S01 is the crushing and acid-alcohol extraction of Sophora flavescens root.

[0052] In some embodiments, the Sophora flavescens root is pulverized to a particle size of 50-100 mesh, at which point the Sophora flavescens root powder is more readily subjected to acid-alcohol extraction. Acid-alcohol extraction involves extracting the Sophora flavescens root powder using a mixed solution containing both an inorganic acid and an alcohol solvent.

[0053] In some embodiments, the mixed solution for acid-alcohol extraction contains an organic acid and an alcohol solvent. Specifically, the powder of *Sophora tonkinensis* root is extracted using a first mixed solution containing a first inorganic acid and a first alcohol solvent.

[0054] In some embodiments, the mass concentration of the first inorganic acid in the first mixed solution is 0.5-1%. The volume concentration of the first alcohol solvent is 60-80%. Under these conditions, the total alkaloids of *Sophora japonica* root can be extracted more effectively. In the embodiments of this application, mass concentration refers to the percentage of solute mass to the total mass of the solution, and volume concentration refers to the percentage of solute (liquid) volume to the total solution volume. Unless otherwise specified, alcohol solvent concentration refers to volume concentration, and other concentrations are mass concentrations. In the first mixed solution, the mass concentration of the first inorganic acid is 0.5-1%, meaning the mass of the first inorganic acid accounts for 0.5-1% of the total mass of the first mixed solution; the volume concentration of the first alcohol solvent is 60-80%, meaning the volume of the first alcohol solvent accounts for 0.5-1% of the total volume of the first mixed solution.

[0055] In some embodiments, the first inorganic acid in the first mixed solution may be selected from at least one of hydrochloric acid and sulfuric acid. The first alcohol solvent is selected from at least one of methanol and ethanol. These alcohol solvents are volatile, have very low toxicity, are environmentally friendly, meet the requirements of green chemistry, and are safer to operate.

[0056] In some embodiments, the conditions for extracting *Sophora tonkinensis* root powder with the first mixed solution include: an extraction temperature of 40-70°C and an extraction time of 6-8 hours. Under these conditions, the total alkaloids of *Sophora tonkinensis* root powder can be extracted effectively. Specifically, extraction can be performed 2-3 times under the above conditions.

[0057] In some embodiments, the step of adjusting the pH value of the extracted liquid after solid-liquid separation to obtain an alkaline extract of *Sophora flavescens* includes: adjusting the pH value of the solid-liquid separated extract to 6.5-8, then concentrating and filtering under pressure, and adding an inorganic alkali to a mass concentration of 0.5-1% to obtain the alkaline extract of *Sophora flavescens*. Specifically, ammonia water can be used to adjust the pH value of the extract to 6.5-8; after adjusting the pH value of the extract to 6.5-8, it is concentrated under pressure, at which point the alcohol solvent can be recovered. After filtration, the added inorganic alkali can be potassium hydroxide or sodium hydroxide, and the mass concentration of the added inorganic alkali is 0.5-1%, that is, the mass of the added inorganic alkali in the alkaline extract of *Sophora flavescens* accounts for 0.5-1% of the total mass of the extract. After adding the inorganic alkali, insoluble matter can be further filtered, increasing the content of purslane in the alkaline extract of *Sophora flavescens*, which is beneficial to subsequent resin adsorption and separation.

[0058] Step S02 is the resin adsorption and separation step.

[0059] In some embodiments, macroporous resin column adsorption is used in the resin adsorption process; specifically, the macroporous resin used in the resin column is one of D101, HPD-826, DM130, and LX-38. By employing macroporous resin column adsorption and utilizing the unique molecular sieve selectivity of macroporous resin, the efficient separation and purification of the target product, piperine, can be effectively achieved.

[0060] In some embodiments, after the alkaline extract of Sophora flavescens is treated with resin adsorption, the adsorbed resin can be washed sequentially with alkaline solution, water, and acid solution; wherein the alkaline solution is selected from an aqueous solution of sodium hydroxide or potassium hydroxide with a mass concentration of 0.5-1%, and the acid solution is selected from an aqueous solution of hydrochloric acid or sulfuric acid with a mass concentration of 0.15-0.5%.

[0061] The sodium hydroxide and potassium hydroxide aqueous solutions of the above concentrations can effectively remove non-target impurities, while the hydrochloric acid or sulfuric acid aqueous solutions of the above concentrations can effectively remove alkaline impurities. After washing the resin with alkaline, water, and acid solutions, the resin is then eluted with a second mixed solution containing a second inorganic acid and a second alcohol solvent.

[0062] In some embodiments, the mass concentration of the second inorganic acid in the second mixed solution is 3-5%, meaning the mass of the second inorganic acid accounts for 3-5% of the total mass of the second mixed solution; the volume concentration of the second alcohol solvent is 60-80%, meaning the volume of the second alcohol solvent accounts for 60-80% of the total volume of the second mixed solution. Under these conditions, the total alkali of Sophora flavescens root can be better eluted from the resin.

[0063] In some embodiments, in the second mixed solution, the second inorganic acid is selected from at least one of hydrochloric acid and sulfuric acid, and the second alcohol solvent is selected from at least one of methanol and ethanol.

[0064] Step S03 is the activated carbon adsorption and precipitation step.

[0065] In some embodiments, the pH of the eluent is adjusted to 6.5-8 before filtration with activated carbon. For example, the pH of the eluent is adjusted to 6.5-8 with ammonia, then the solvent is recovered by vacuum concentration, filtered with activated carbon, and then adjusted to alkalinity with an inorganic alkaline solution. The total alkali of *Sophora benjamina* var. *chinensis* precipitates out in the alkaline environment. The inorganic alkaline solution can be a 3-5% (w / w) aqueous solution of potassium hydroxide or sodium hydroxide.

[0066] In some embodiments, in the step of adjusting the filtrate to alkalinity for precipitation, the pH of the filtrate is adjusted to 8.5-9.5, and then allowed to stand for 6-10 hours. This condition can better precipitate the total alkali from Sophora flavescens.

[0067] In some embodiments, after precipitation, the process further includes: collecting and washing the precipitate, and vacuum drying to obtain total alkaloids from Sophora flavescens. Vacuum drying can be performed at a temperature of 50-70°C to obtain dried powder of total alkaloids from Sophora flavescens.

[0068] Step S04 is the resolution and back-extraction of halogenated hydrocarbons in organic solvents.

[0069] In some embodiments, the halogenated hydrocarbon organic solvent includes at least one of dichloromethane and trichloromethane. The aforementioned halogenated hydrocarbon organic solvents exhibit excellent solubility for organic alkaloids and can form a more pronounced two-phase separation with water, facilitating subsequent efficient extraction and separation using a separatory funnel.

[0070] In some embodiments, the step of dissolving the total alkali of *Sophora flavescens* in a halogenated hydrocarbon organic solvent and then extracting it sequentially with an alkaline solution and an acid solution includes: first extracting with a 0.5-5% (w / w) aqueous solution of sodium hydroxide or potassium hydroxide, then washing with water, and finally extracting with a 0.15-0.5% (w / w) aqueous solution of hydrochloric acid or sulfuric acid. Specifically, the alkaline solution used in the extraction is a 0.5-5% (w / w) aqueous solution of sodium hydroxide or potassium hydroxide, and the acid solution used in the extraction is a 0.15-0.5% (w / w) aqueous solution of hydrochloric acid or sulfuric acid.

[0071] In some embodiments, the extraction is performed sequentially with alkali and acid solutions 1-4 times, that is, the number of cycles of extraction with alkali and acid solutions can be 1-4 times. In each cycle, alkali extraction can be performed independently once or more (e.g., 2-4 times), and acid extraction can be performed independently once or more (e.g., 2-4 times).

[0072] For example, dissolve the total alkaloids of *Sophora flavescens* in a halogenated hydrocarbon organic solvent, filter through filter paper, and then pour into a separatory funnel. Extract 1-4 times with the aforementioned alkali solution, and then wash 1-4 times with purified water. Finally, extract 1-2 times with acid, collect the acidic aqueous layer as the extract, and then precipitate it out.

[0073] In some embodiments, precipitation of the extract includes adjusting the pH of the extract to 8.5-9.5 before precipitation. The pH can be adjusted using an inorganic alkaline solution, such as a 0.5-1% aqueous solution of potassium hydroxide or sodium hydroxide.

[0074] Through multiple cyclic extractions, the crude puerarin obtained by separation can be dissolved and recovered using a halogenated hydrocarbon organic solvent. Further purification is carried out through steps such as extraction, washing, pH adjustment and precipitation to remove impurities and obtain a high-content puerarin product.

[0075] In some embodiments, the method for extracting puerarin from Sophora flavescens in this application includes the following steps:

[0076] Step 1, Extraction:

[0077] The pulverized Sophora flavescens powder was extracted with a first mixed solution containing a first inorganic acid and a first alcohol solvent. The solution was filtered, the pH was adjusted, the solution was concentrated under reduced pressure, filtered again, and an inorganic base was added to the extract to adjust the pH. The solution was then filtered again to obtain an alkaline extract of Sophora flavescens.

[0078] Step 2, resin column separation:

[0079] The alkaline extract of Sophora flavescens was adsorbed using a macroporous resin column. The resin was washed sequentially with alkaline solution, water, and acid solution. Finally, it was eluted with a second mixed solution containing a second inorganic acid and a second alcohol solvent, and the eluent was collected.

[0080] Step 3, enrichment of total alkaloids in Sophora flavescens:

[0081] The pH of the eluent was adjusted, the concentration was reduced under reduced pressure, and after filtration with activated carbon, it was adjusted to alkaline with an inorganic alkaline solution. The total alkali of Sophora flavescens precipitated out, and after filtration, washing, and vacuum drying, the total alkali of Sophora flavescens was obtained.

[0082] Step 4, Isolation of puerarin:

[0083] The total alkaloids of Sophora flavescens were dissolved in a halogenated hydrocarbon organic solvent, filtered, and then back-extracted successively with alkali, water, and acid. The inorganic acid extract was collected and the pH was adjusted. A white precipitate was formed, filtered, washed with water, and dried to obtain crude Sophora flavescens alkaloids.

[0084] Step 5, Refining of Ficus pumila alkaloids:

[0085] The crude purpurein was dissolved again in a halogenated hydrocarbon organic solvent, and then back-extracted successively with alkali, water, and acid. The inorganic acid extract was collected and the pH was adjusted. A white precipitate was formed, filtered, washed with water until neutral, and dried to obtain pure purpurein.

[0086] In summary, the embodiments of this application employ an acid-alcohol extraction combined with resin adsorption separation, activated carbon filtration, and back-extraction process. This process is not only short, easy to operate, and environmentally friendly, but also efficiently yields high purity of puerarin, with the final product content reaching over 97.00%. This is of great significance for the development of new active ingredients and new drugs.

[0087] The following description is based on specific embodiments.

[0088] Example 1

[0089] A method for extracting puerarin from Sophora flavescens root includes the following steps:

[0090] Step 1, Extraction:

[0091] The roots of *Radix Sophorae Flavescentis* were pulverized to a 50-mesh powder. 11 kg of the powder was placed in a 100L acid-resistant extraction vessel, and 79.5L of a first mixed solution (containing 0.5% hydrochloric acid and 75% methanol) was added. The mixture was heated to 50℃ and stirred for 6 hours. After filtration, the extract was collected. This process was repeated once, and the extracts were combined. Ammonia was added to adjust the pH to 6.5, and methanol was recovered under reduced pressure to 40L. The extract was then cooled and filtered. Sodium hydroxide was added to bring the inorganic alkali concentration in the extract to 1%. Insoluble matter was filtered out to obtain an alkaline extract of *Radix Sophorae Flavescentis*. Chromatographic results are shown below. Figure 1 .

[0092] Step 2, resin column separation:

[0093] The alkaline extract of *Sophora tigrinosa* was adsorbed onto a macroporous resin column (LX-38). The resin was washed with 30 L of 0.5% sodium hydroxide aqueous solution, followed by 30 L of purified water, and then 30 L of 0.2% hydrochloric acid aqueous solution. Finally, the solution was eluted with 50 L of a second mixed solution (containing 3% hydrochloric acid and 75% methanol), and the eluent was collected.

[0094] Step 3, enrichment of total alkaloids in Sophora flavescens:

[0095] The eluent was adjusted to pH 7 with ammonia, and the volume was reduced to 20 L under reduced pressure. 30 g of activated carbon was added, and the mixture was filtered. The pH was then adjusted to pH 9 with a 5% sodium hydroxide aqueous solution, and the mixture was allowed to stand for 8 hours until complete precipitation. The precipitate was collected, washed, and finally dried under vacuum at 60 °C to obtain 283 g of total alkaloids from *Sophora bean* root. Chromatographic results are shown below. Figure 2 .

[0096] Step 4, Isolation of puerarin:

[0097] 283g of total alkaloids from *Sophora flavescens* root was dissolved in 2L of dichloromethane, filtered through filter paper, and then poured into a separatory funnel. 8L of 3% sodium hydroxide aqueous solution was added, and the mixture was extracted four times, followed by washing four times with 8L of purified water. Finally, it was extracted twice with 0.4% hydrochloric acid aqueous solution, and the acidic aqueous layer was collected as the extract. The pH was adjusted to 9, and the mixture was allowed to stand until a white precipitate had completely formed. The precipitate was collected and dried to obtain 198g of crude *Sophora flavescens* alkaloids.

[0098] Step 5, purification of puerarin:

[0099] The crude datura oleifera alkaloid was dissolved again in 1 L of dichloromethane, filtered, and the solution was transferred to a separatory funnel. It was extracted four times with 4 L of 0.5% sodium hydroxide aqueous solution, and then washed four times with pure water. Extraction was then performed with 0.4% hydrochloric acid aqueous solution, and the acidic aqueous layer was collected as the extract. The pH was adjusted to 9 with 0.5% sodium hydroxide aqueous solution, and the mixture was allowed to stand until a white precipitate had completely formed. The precipitate was collected and washed until neutral. Finally, 176 g of pure datura oleifera alkaloid with a purity of 97.27% was obtained by drying. (See chromatographic results for details). Figure 3 .

[0100] Example 2

[0101] A method for extracting puerarin from Sophora flavescens root includes the following steps:

[0102] Step 1, Extraction:

[0103] The roots of *Sophora flavescens* were pulverized to a 100-mesh powder. 11 kg of the powder was placed in a 100L acid-resistant extraction tank, and 79.5L of a first mixed solution (containing 1% hydrochloric acid and 80% methanol) was added. The mixture was heated to 70℃ and stirred for 8 hours. After filtration, the extract was collected. This process was repeated twice, and the extracts were combined. Ammonia was added to adjust the pH to 7, and methanol was recovered under reduced pressure to 40L. The mixture was then cooled and filtered. Sodium hydroxide was added to bring the inorganic alkali concentration in the extract to 1%. Insoluble matter was filtered out to obtain an alkaline extract of *Sophora flavescens*.

[0104] Step 2, resin column separation:

[0105] The alkaline extract of *Sophora tigrinosa* was adsorbed onto a macroporous resin column (LX-38). The resin was washed with 30 L of 0.7% sodium hydroxide aqueous solution, followed by 30 L of purified water, and then 30 L of 0.3% hydrochloric acid aqueous solution. Finally, the solution was eluted with 80 L of a second mixed solution (containing 4% hydrochloric acid and 80% methanol), and the eluent was collected.

[0106] Step 3, enrichment of total alkaloids in Sophora flavescens:

[0107] The eluent was adjusted to pH 7.2 with ammonia, and the volume was reduced to 20 L under reduced pressure. 30 g of activated carbon was added, and the mixture was filtered. The pH was then adjusted to 9.2 with a 4.5% sodium hydroxide aqueous solution, and the mixture was allowed to stand for 7 hours until the precipitate was fully formed. The precipitate was collected, washed, and finally dried under vacuum at 60 °C to obtain 298 g of total alkaloids from *Sophora bean* root.

[0108] Step 4, Isolation of puerarin:

[0109] 298g of total alkaloids from *Sophora bean* root was dissolved in 2L of dichloromethane, filtered through filter paper, and then poured into a separatory funnel. 8L of 3.5% sodium hydroxide aqueous solution was added, and the mixture was extracted three times, followed by washing four times with 8L of purified water. Finally, the mixture was extracted three times with 0.5% hydrochloric acid aqueous solution, and the acidic aqueous layer was collected as the extract. The pH was adjusted to 9, and the mixture was allowed to stand until a white precipitate had completely formed. The precipitate was collected and dried to obtain 217g of crude *Sophora bean* alkaloids.

[0110] Step 5, purification of puerarin:

[0111] The crude purpuricine was dissolved again in 1 L of dichloromethane, filtered, and the solution was transferred to a separatory funnel. It was extracted three times with 4 L of 1% sodium hydroxide aqueous solution, and then washed four times with pure water. The extract was then extracted three times with a 0.5% hydrochloric acid aqueous solution, and the acidic aqueous layer was collected as the extract. The pH was adjusted to 9.5 with a 0.5% sodium hydroxide aqueous solution, and the mixture was allowed to stand until a white precipitate had completely formed. The precipitate was collected and washed until neutral. Finally, 199 g of pure purpuricine with a purity of 97.98% was obtained by drying.

[0112] Example 3

[0113] A method for extracting puerarin from Sophora flavescens root includes the following steps:

[0114] Step 1, Extraction:

[0115] The roots of *Sophora flavescens* were pulverized to a 50-mesh powder. 11 kg of the powder was placed in a 100L acid-resistant extraction tank, and 79.5L of a first mixed solution (containing 1% hydrochloric acid and 65% methanol) was added. The mixture was heated to 45℃ and stirred for 7 hours. After filtration, the extract was collected. This process was repeated once, and the extracts were combined. Ammonia was added to adjust the pH to 8, and methanol was recovered under reduced pressure to 40L. The mixture was then cooled and filtered. Sodium hydroxide was added to bring the inorganic alkali concentration in the extract to 0.5%. Insoluble matter was filtered out to obtain an alkaline extract of *Sophora flavescens*.

[0116] Step 2, resin column separation:

[0117] The alkaline extract of *Sophora tigrinosa* was adsorbed onto a macroporous resin column (LX-38). The resin was washed with 30 L of 0.5% sodium hydroxide aqueous solution, followed by 30 L of purified water, and then 30 L of 0.2% hydrochloric acid aqueous solution. Finally, the solution was eluted with 50 L of a second mixed solution (containing 3% hydrochloric acid and 75% methanol), and the eluent was collected.

[0118] Step 3, enrichment of total alkaloids in Sophora flavescens:

[0119] The eluent was adjusted to pH 7.5 with ammonia, and the volume was reduced to 20 L under reduced pressure. 30 g of activated carbon was added, and the mixture was filtered. The pH was then adjusted to 9 with a 4% sodium hydroxide aqueous solution, and the mixture was allowed to stand for 8 hours until the precipitate was fully formed. The precipitate was collected, washed, and finally dried under vacuum at 60 °C to obtain 291 g of total alkaloids from *Sophora bean* root.

[0120] Step 4, Isolation of puerarin:

[0121] 291g of total alkaloids from *Sophora bean* root was dissolved in 2L of dichloromethane, filtered through filter paper, and then poured into a separatory funnel. 8L of 5% sodium hydroxide aqueous solution was added, and the mixture was extracted four times, followed by washing four times with 8L of purified water. Finally, four extractions were performed with 0.2% hydrochloric acid aqueous solution. The acidic aqueous layer was collected as the extract, the pH was adjusted to 8.7, and the mixture was allowed to stand until a white precipitate had completely formed. The precipitate was collected and dried to obtain 206g of crude *Sophora bean* alkaloids.

[0122] Step 5, purification of puerarin:

[0123] The crude purpureusine was dissolved again in 1 L of dichloromethane, filtered, and the solution was transferred to a separatory funnel. It was extracted twice with 4 L of 0.8% sodium hydroxide aqueous solution, and then washed twice with pure water. Extraction was then performed with 0.2% hydrochloric acid aqueous solution, and the acidic aqueous layer was collected as the extract. The pH was adjusted to 8.8 with 0.5% sodium hydroxide aqueous solution, and the mixture was allowed to stand until a white precipitate had completely formed. The precipitate was collected and washed until neutral. Finally, 187 g of pure purpureusine with a purity of 97.43% was obtained by drying.

[0124] Comparative Example 1

[0125] A method for extracting puerarin from Sophora flavescens root includes the following steps:

[0126] Step 1, Extraction:

[0127] The roots of *Sophora flavescens* were pulverized to a 100-mesh powder. 11 kg of the powder was placed in a 100L acid-resistant extraction tank, and 8000 mL of 65% methanol aqueous solution was added. The mixture was heated to 45℃ and stirred for 7 hours. After filtration, the extract was collected. This process was repeated once, and the extracts were combined. The methanol was recovered under reduced pressure to 40L, and the mixture was cooled and filtered. The recovered methanol extract was then acidified by slowly adding pre-prepared dilute sulfuric acid to the 40L recovered extract to adjust the pH to 3, resulting in an acidic extract.

[0128] Step 2, Impurity Removal:

[0129] The acidic extract was extracted with 4 L of chloroform. After separation, the organic layer was removed. This process was repeated twice to remove fat-soluble impurities, and the acidic aqueous phase was collected.

[0130] Step 3, Isolation of puerarin:

[0131] Acidic aqueous phase was added to ammonia solution to adjust the pH to 10. The mixture was then extracted twice with 4 L of chloroform, and the chloroform extracts were combined and concentrated to 2 L. The concentrate was then extracted twice with 3% sodium hydroxide solution. The chloroform solution was washed with water until neutral and dehydrated with sodium sulfate, then recovered to dryness. The solids were dissolved in 1 L of sulfuric acid, and the pH was adjusted to 10 with ammonia. The mixture was extracted twice with 1 L of diethyl ether, followed by twice with 1 L of 3% sodium hydroxide solution. The alkaline solutions were combined. Ammonia was added to adjust the pH to 9, and the mixture was extracted twice with 1 L of diethyl ether. The combined ether solutions were recrystallized to obtain 50.9 g of piperine.

[0132] Comparative Example 2

[0133] A method for extracting puerarin from Sophora flavescens root includes the following steps:

[0134] Step 1, Extraction:

[0135] The roots of *Sophora flavescens* were pulverized to a 100-mesh powder. 11 kg of the powder was placed in a 100L acid-resistant extraction tank, and 8000 mL of 65% methanol aqueous solution was added. The mixture was heated to 45℃ and stirred for 7 hours. After filtration, the extract was collected. This process was repeated once, and the extracts were combined. The methanol was recovered under reduced pressure to 40L, and the mixture was cooled and filtered. The recovered methanol extract was then acidified by slowly adding pre-prepared dilute sulfuric acid to the 40L recovered extract to adjust the pH to 3, resulting in an acidic extract.

[0136] Step 2, Resin Separation:

[0137] The acidic extract was added to a cation exchange resin at a rate of 2 BV / h, followed by washing with deionized water until the color lightened. Elution was then performed using a 10% hydrochloric acid aqueous solution at a flow rate of 4 BV / h. The pH was adjusted to 7 with sodium hydroxide, and the solution was loaded onto a macroporous adsorption resin. Elution was then performed sequentially with water, 50% methanol solution, and 70% ethanol solution. The eluent was collected, recovered under reduced pressure, and dissolved under reflux in petroleum ether-acetone (volume ratio 4:1). The solution was repeatedly recrystallized and dried to obtain 95 g of piperine.

[0138] Comparative analysis shows that in Comparative Example 1, the method of first extracting with alcohol followed by acid dissolution may result in a lower total alkaloid recovery rate due to the possibility of some alkaloids binding with the plant matrix, local degradation, or incomplete dissolution during the acid dissolution stage. In contrast, the present application uses a mixed acid-alcohol extraction method, which fully utilizes the advantage of acid promoting partial hydrolysis of cell walls, resulting in higher product extraction rate and content. In Comparative Example 2, the method of first applying an acid solution to a cation exchange resin and then an alkaline solution to a macroporous adsorption resin suffers from column blockage and incomplete elution due to the poor water solubility of purslane, leading to a low subsequent product yield. In contrast, the present application uses an alkaline extract for direct adsorption onto the macroporous resin column, followed by washing with alkaline solution, pure water, and acid, and then eluting with acid and alcohol, ultimately achieving a high target product recovery rate and fewer impurities. Furthermore, if toluene is used as a solvent for recovery, its dissolving power and phase separation effect are inferior to dichloromethane, resulting in even lower recovery and separation rates, and incomplete extraction of purslane. Therefore, Examples 1-3 of this application have better results than Comparative Examples 1-2.

[0139] Furthermore, the high-performance liquid chromatography (HPLC) used in Example 1 of this application employs the detection method under the entry for Sophora flavescens root extract in the Chinese Pharmacopoeia, namely, the determination of puerarin by high-performance liquid chromatography (General Rule 0512). The specific details are as follows:

[0140] (1) Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile-0.05% triethylamine solution (45:55) was used as the mobile phase; the detection wavelength was 284 nm. The theoretical plate number, calculated based on the piracetam alkaloid peak, should not be less than 6000.

[0141] (2) Preparation of reference solution: Take an appropriate amount of piperine reference standard, weigh it accurately, place it in a brown volumetric flask, add methanol to prepare a solution containing 0.2 mg of piperine per ml, and it is ready (this product should be prepared immediately and stored away from light).

[0142] (3) Preparation of test solution: Take this product, grind it into a fine powder, take about 30 mg, weigh it accurately, place it in a stoppered conical flask, add 25 ml of methanol accurately, stopper tightly, weigh it, sonicate (power 140 W, frequency 42 kHz) for 30 minutes, take it out, let it cool, weigh it again, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0143] (4) Determination method: Accurately pipette 10 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0144] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for extracting puerarin from Sophora flavescens root, characterized in that, include: The root of Sophora flavescens was crushed to obtain powder, and the powder was extracted with a first mixed solution containing a first inorganic acid and a first alcohol solvent. After solid-liquid separation, the pH value was adjusted to obtain an alkaline extract of Sophora flavescens. The alkaline extract of Sophora flavescens was treated with resin adsorption, and then the resin was washed sequentially with alkaline solution, water and acid solution. The extract was then eluted with a second mixed solution containing a second inorganic acid and a second alcohol solvent to obtain an eluent. The eluent was filtered with activated carbon, and the filtrate was adjusted to alkalinity to precipitate and obtain total alkali from Sophora flavescens. The total alkaloids of the Sophora flavescens root were dissolved in a halogenated hydrocarbon organic solvent, and then extracted with alkali and acid solutions in sequence to obtain an extract. The extract was then precipitated to obtain basal alkaloids.

2. The method as described in claim 1, characterized in that, In the first mixed solution, the first inorganic acid is selected from at least one of hydrochloric acid and sulfuric acid, and the first alcohol solvent is selected from at least one of methanol and ethanol; And / or, in the first mixed solution, the mass concentration of the first inorganic acid is 0.5-1%, and the volume concentration of the first alcohol solvent is 60-80%.

3. The method as described in claim 1, characterized in that, The conditions for extracting the powder with the first mixed solution include: an extraction temperature of 40-70℃, an extraction time of 6-8h; and / or, 2-3 extractions.

4. The method as described in claim 1, characterized in that, The steps for adjusting the pH value after solid-liquid separation to obtain the alkaline extract of Sophora flavescens include: adjusting the pH value of the extract after solid-liquid separation to 6.5-8, then concentrating and filtering under pressure, and adding inorganic alkali to a mass concentration of 0.5-1%.

5. The method as described in claim 1, characterized in that, In the step of washing the resin sequentially with alkali, water, and acid, the alkali is selected from an aqueous solution of sodium hydroxide or potassium hydroxide with a mass concentration of 0.5-1%, and the acid is selected from an aqueous solution of hydrochloric acid or sulfuric acid with a mass concentration of 0.15-0.5%.

6. The method as described in claim 1, characterized in that, In the second mixed solution, the second inorganic acid is selected from at least one of hydrochloric acid and sulfuric acid, and the second alcohol solvent is selected from at least one of methanol and ethanol; And / or, in the second mixed solution, the mass concentration of the second inorganic acid is 3-5%, and the volume concentration of the second alcohol solvent is 60-80%.

7. The method according to any one of claims 1-6, characterized in that, Before filtering the eluent with activated carbon, the pH value of the eluent is adjusted to 6.5-8. And / or, in the step of adjusting the filtrate to alkalinity for precipitation, the pH value of the filtrate is adjusted to 8.5-9.5, and then allowed to stand for 6-10 hours; And / or, the precipitation process further includes vacuum drying at a temperature of 50-70°C.

8. The method according to any one of claims 1-6, characterized in that, The resin adsorption treatment employs macroporous resin column adsorption; and / or, the halogenated hydrocarbon organic solvent includes at least one of dichloromethane and trichloromethane.

9. The method according to any one of claims 1-6, characterized in that, The step of sequentially extracting with alkaline and acidic solutions includes: first extracting with a 0.5-5% sodium hydroxide or potassium hydroxide aqueous solution, then washing with water, and then extracting with a 0.15-0.5% hydrochloric acid or sulfuric acid aqueous solution; and / or the sequential extraction with alkaline and acidic solutions is performed 1-4 times.

10. The method according to any one of claims 1-6, characterized in that, Precipitation of the extract includes adjusting the pH of the extract to 8.5-9.5 before precipitation.

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