Sectional extraction and purification method of active component bulk drug of traditional Chinese medicinal materials

Abstract

The invention relates to the technical field of traditional Chinese medicine active component extraction and raw material medicine purification, in particular to a staged extraction and purification method of traditional Chinese medicine active component raw material medicine. Wherein the raw material medicine of the active component of the traditional Chinese medicinal materials is a matrine raw material medicine, and the preparation method comprises the following steps: S1, carrying out contact extraction on the matrine-containing traditional Chinese medicinal materials and an acidic ethanol aqueous solution to obtain a total alkaloid extracting solution; s2, obtaining a first-time segmented extraction solution; s3, obtaining a desalted enrichment solution; s4, obtaining a matrine fumarate crystal; s5, obtaining a matrine organic phase; and S6, obtaining the matrine bulk drug. By constructing a series purification technical scheme of ethanol-phosphate aqueous two-phase segmented enrichment, nanofiltration dialysis replacement desalination, fumaric acid selective salification crystallization, free alkali release extraction and displacement anti-solvent crystallization, segmented selective enrichment and purification of matrine in the total alkaloid extracting solution are realized.

Description

A method for fractional extraction and purification of active components from traditional Chinese medicinal materials Technical Field

[0001] This invention relates to the field of extraction and purification technology of active components from Chinese medicinal materials, and particularly to a method for fractional extraction and purification of active components from Chinese medicinal materials. Background Technology

[0002] Matrine is a representative quinolone alkaloid found in legumes such as Sophora flavescens and Sophora alopecuroides, and can be used as a raw material in the preparation of related drugs. The preparation method of matrine derived from traditional Chinese medicinal materials typically employs a process combining extraction, separation, and crystallization purification.

[0003] In existing technologies, such as CN105237537B, a method for preparing matrine and sophoridine from bitter bean seeds is disclosed. This method mainly uses an acidic polar organic solvent aqueous solution containing a reducing agent to extract total alkaloids from bitter bean seeds. After alkalization, the extract is separated by adsorption using a macroporous adsorption resin. The residue and water-soluble impurities not adsorbed by the resin are then removed by washing with water. The target products are then eluted with a polar organic solvent aqueous solution to obtain crude matrine and crude sophoridine extracts. Further purification is achieved through crystallization and recrystallization to obtain refined matrine and refined sophoridine. The key aspect of this existing technology lies in the purification route that combines acidic extraction with macroporous adsorption resin separation.

[0004] The aforementioned existing technologies can achieve the extraction and purification of matrine. However, judging from their publicly available technical routes, the core of their processes still mainly relies on resin adsorption and separation after the initial extraction, as well as subsequent crystallization and recrystallization purification. For total alkaloid extracts directly derived from Chinese medicinal materials, they usually also contain other alkaloid components and impurities with properties similar to matrine. Existing technologies still have room for further improvement in the segmented selective enrichment and subsequent purification of matrine in such complex systems.

[0005] Therefore, existing technologies still require a preparation method that can selectively enrich and purify matrine in total alkaloid extracts in stages. Summary of the Invention

[0006] To overcome the aforementioned technical deficiencies, the present invention aims to provide a method for the fractional extraction and purification of active components from traditional Chinese medicinal materials. This invention achieves the fractional selective enrichment and purification of matrine in total alkaloid extracts by constructing a tandem purification technology scheme consisting of "ethanol-phosphate aqueous two-phase fractional enrichment, nanofiltration dialysis displacement desalting, selective fumaric acid salt formation crystallization, free alkali release extraction, and displacement antisolvent crystallization."

[0007] This invention discloses a method for fractional extraction and purification of active components from traditional Chinese medicine (TCM) raw materials, wherein the active component raw material is matrine. The method includes the following steps: S1, extracting the matrine-containing TCM raw material by contacting it with an acidic ethanol aqueous solution to obtain a total alkaloid extract; S2, concentrating the total alkaloid extract under reduced pressure and adjusting the ethanol volume fraction, adding a phosphate phase-forming salt to form an ethanol-phosphate aqueous two-phase system, separating the phases and collecting the upper phase enriched with matrine to obtain the first fractional extract; S3, subjecting the first fractional extract to nanofiltration dialysis to remove... Phosphate phase salts and low molecular weight impurities are separated to obtain a desalted enriched solution; S4, fumaric acid ethanol solution is added to the desalted enriched solution to cause matrine to crystallize out in the form of fumarate, and solid-liquid separation is performed to obtain matrine fumarate crystals; S5, the matrine fumarate crystals are dispersed in water and adjusted to alkalinity to convert matrine into free base, and then extracted with an acetate-based organic solvent to obtain the matrine organic phase; S6, the matrine organic phase is concentrated and an alcohol crystallization solvent is added to form a crystallization mother liquor, and then water is added as an antisolvent for displacement antisolvent crystallization to obtain matrine raw material.

[0008] Preferably, in S1, the medicinal material is the dried root of Sophora flavescens or the mature seed of Sophora alopecuroides, and the medicinal material is subjected to alkaline pre-wetting treatment before being extracted by contact with acidic ethanol aqueous solution.

[0009] Preferably, the Chinese medicinal materials are pulverized to 24-50 mesh, and the alkaline pre-wetting treatment is carried out using an ammonium bicarbonate aqueous solution with a mass fraction of 0.2%-0.8% for 0.5-2 hours; the acidic ethanol aqueous solution is an ethanol aqueous solution with a volume fraction of 55%-72% and contains formic acid with a mass fraction of 0.15%-0.60%.

[0010] Preferably, in step S2, the total alkaloid extract is concentrated under reduced pressure to an ethanol volume fraction of 18%–28%, and then one or both of dipotassium hydrogen phosphate and potassium dihydrogen phosphate are added to make the total phosphate mass fraction in the ethanol-phosphate aqueous two-phase system 12%–20%. After standing and phase separation, the upper phase is collected.

[0011] Preferably, after readjusting the ethanol volume fraction and total phosphate mass fraction of the lower phase obtained from the S2 phase separation, a second phase separation operation is performed, and the upper phase obtained from the second phase separation is incorporated into the first fractionated extract.

[0012] Preferably, in S3, the nanofiltration dialysis replacement is performed using an alcohol-resistant nanofiltration membrane with a molecular weight cutoff of 150–300 Da.

[0013] Preferably, in step S3, the pH of the first fractionated extract is adjusted to 7.2–8.2 before nanofiltration dialysis replacement is performed. Deionized water with a volume of 2–6 times that of the feed solution entering the nanofiltration dialysis replacement step is used for dialysis replacement. The nanofiltration dialysis replacement ends when the conductivity of the permeate drops below 2.0 mS / cm.

[0014] Preferably, in S4, the volume fraction of ethanol in the fumaric acid ethanol solution is 85% to 100%, and the amount of fumaric acid added is 0.45 to 0.55 times the molar amount of matrine in the desalting enrichment solution.

[0015] Preferably, in step S4, the desalting enrichment solution is heated to 35–50°C, fumaric acid ethanol solution is added dropwise, and the solution is kept at this temperature for 10–40 minutes, then cooled to 5–15°C to crystallize the salt.

[0016] Preferably, the matrine fumarate crystals obtained in S4 are washed with a mixed washing solution composed of anhydrous ethanol and ethyl acetate, wherein the volume ratio of anhydrous ethanol to ethyl acetate is 1:(1-4).

[0017] Preferably, in step S5, the pH of the dispersion of matrine fumarate crystals in water is adjusted to 9.5–10.5 using ammonia water, and extraction is performed using one or both of ethyl acetate and isopropyl acetate.

[0018] Preferably, in step S5, after extraction, the matrine organic phase is washed with water with a conductivity not exceeding 50 μS / cm until the pH of the washing solution is 7.0–8.0.

[0019] Preferably, in step S6, the alcohol crystallization solvent is isopropanol, the antisolvent is water, and after concentrating the organic phase of matrine to obtain a concentrated solution, isopropanol is added to the concentrated solution, and then water is added dropwise.

[0020] Preferably, in step S6, when visible turbidity appears in the system after the addition of the antisolvent and does not disappear after stirring for 5 to 10 minutes, 0.5% to 2.0% of matrine seed crystals by mass of the concentrated solution are added, and the temperature is lowered to 0 to 10°C at a cooling rate of 0.2 to 0.8°C / min.

[0021] Preferably, the crystals obtained in S6 are centrifuged and then dried at 40–55°C and 20–60 kPa absolute pressure to obtain matrine raw material.

[0022] Compared with existing technologies, the above-mentioned technical solution has the following advantages: 1. This invention does not rely solely on a single extraction or recrystallization method, but rather employs a series of processes including ethanol-phosphate aqueous phase separation, nanofiltration dialysis replacement, fumaric acid salt crystallization, free alkali release and acetate-based organic solvent extraction, and displacement antisolvent crystallization. This allows matrine to be gradually separated from strongly polar impurities, inorganic salt impurities, and homologous alkaloid impurities at different stages. Since each separation unit targets different types of impurities, it effectively increases the enrichment of matrine in the final product, resulting in a higher content of the target component in the obtained matrine raw material; 2. Matrine shares certain similarities with components such as sophoridine and oxymatrine in terms of source, structure, and physicochemical properties. Conventional single extraction or ordinary crystallization methods often fail to achieve both separation efficiency and yield. This invention enhances the utilization of subtle differences between matrine and its homologues by combining fumaric acid salt crystallization with subsequent displacement antisolvent crystallization. This helps reduce the residue of impurities such as sophoridine and oxymatrine in the final product, improving the purification effect and quality consistency of the active pharmaceutical ingredient. 3. This invention incorporates a nanofiltration dialysis replacement step after aqueous phase separation, effectively removing phosphate-based phase-forming salts and some low-molecular-weight inorganic impurities introduced in the preceding phase separation, thus preventing high-salt systems from directly entering the subsequent crystallization step. This not only reduces crystal entrainment and mother liquor residue but also lowers the amount of inorganic salt residue in the final product, improving the cleanliness and overall quality of the final product. 4. In existing technologies, purity is often improved by increasing the number of crystallization cycles or increasing recrystallization intensity, but this often leads to increased loss of the target component and a decrease in overall yield. This invention combines pre-stage fractional enrichment with post-stage targeted purification, ensuring that matrine is significantly enriched and desalted before entering the final crystallization step. Therefore, high purity can be obtained without relying on multiple recrystallizations, thus improving both the purity of the final product and maintaining a high yield. 5. This invention sequentially links extraction, liquid-phase separation, membrane desalting, salt formation crystallization, free alkali release, liquid-liquid extraction, and displacement antisolvent crystallization, forming a continuous purification pathway from crude to refined. The functional division between each step is clear, and the intermediate products obtained in previous steps provide more suitable processing targets for subsequent steps; therefore, the overall process has good logical consistency and implementation stability.Meanwhile, as can be seen from the embodiments in the specification, the present invention can achieve the preparation of the target product under the conditions of the lower limit, middle value and upper limit of the parameter range, indicating that the present invention has good parameter adaptability; 6. Compared with the traditional route mainly based on acid extraction, resin adsorption, elution and ordinary crystallization, the present invention introduces ethanol-phosphate aqueous two-phase separation in the front stage, nanofiltration dialysis replacement in the middle stage, and fumaric acid salt crystallization and displacement antisolvent crystallization in the back stage, so that the entire process is no longer limited to a single separation mechanism, but through the synergistic effect of multiple separation mechanisms, the target component enrichment ability, impurity removal ability and final product quality control ability in the preparation process of matrine raw material are improved. Attached Figure Description

[0023] Figure 1 is a schematic diagram of the steps of a fractional extraction and purification method for active components of traditional Chinese medicine raw materials according to the present invention; Figure 2 is a schematic diagram of ethanol-phosphate aqueous two-phase phase separation and desalting enrichment; Figure 3 is a schematic diagram of salt formation crystallization, free alkali release and displacement antisolvent crystallization; Figure 4 is a schematic diagram of the comparison curve of matrine content; Figure 5 is a schematic diagram of the comparison curve of total yield; Figure 6 is a schematic diagram of the comparison of the content of major impurities; Figure 7 is a schematic diagram of the comparison curve of inorganic salt residue. Detailed Implementation

[0024] The advantages of the present invention will be further illustrated below with reference to the accompanying drawings and specific embodiments.

[0025] Exemplary embodiments will now be described in detail, examples of which are illustrated in the accompanying drawings. When the following description relates to the drawings, unless otherwise indicated, the same numerals in different drawings denote the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this disclosure. Rather, they are merely examples of apparatuses and methods consistent with some aspects of this disclosure as detailed in the appended claims.

[0026] The terminology used in this disclosure is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. The singular forms “a,” “the,” and “the” as used in this disclosure and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term “and / or” as used herein refers to and includes any and all possible combinations of one or more of the associated listed items.

[0027] It should be understood that although the terms first, second, third, etc., may be used in this disclosure to describe various information, such information should not be limited to these terms. These terms are used only to distinguish information of the same type from one another. For example, without departing from the scope of this disclosure, first information may also be referred to as second information, and similarly, second information may also be referred to as first information. Depending on the context, the word "if" as used herein may be interpreted as "when," "when," or "in response to determination."

[0028] In the description of this invention, it should be understood that the terms "longitudinal", "lateral", "up", "down", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0029] In the description of this invention, unless otherwise specified and limited, it should be noted that the terms "installation", "connection" and "linking" should be interpreted broadly. For example, they can refer to mechanical or electrical connections, or internal connections between two components. They can be direct connections or indirect connections through an intermediate medium. Those skilled in the art can understand the specific meaning of the above terms according to the specific circumstances.

[0030] In the following description, suffixes such as "module," "part," or "unit" used to denote elements are used only for the convenience of the description of the invention and have no specific meaning in themselves. Therefore, "module" and "part" can be used interchangeably.

[0031] This embodiment provides a method for fractional extraction and purification of active components from traditional Chinese medicine (TCM) raw materials, wherein the active component raw material is matrine. The method includes the following steps: S1, extracting the matrine-containing TCM raw material by contacting it with an acidic ethanol aqueous solution to obtain a total alkaloid extract; S2, concentrating the total alkaloid extract under reduced pressure and adjusting the ethanol volume fraction, adding a phosphate phase-forming salt to form an ethanol-phosphate aqueous two-phase system, separating the phases and collecting the upper phase enriched with matrine to obtain the first fractional extract; S3, subjecting the first fractional extract to nanofiltration dialysis to remove... After removing phosphate-forming salts and low-molecular-weight impurities, a desalted enriched solution is obtained; S4, fumaric acid ethanol solution is added to the desalted enriched solution to cause matrine to crystallize out in the form of fumarate, and solid-liquid separation is performed to obtain matrine fumarate crystals; S5, the matrine fumarate crystals are dispersed in water and adjusted to alkalinity to convert matrine into free base, and then extracted with an acetate-based organic solvent to obtain the matrine organic phase; S6, the matrine organic phase is concentrated and an alcohol crystallization solvent is added to form a crystallization mother liquor, and then water is added as an antisolvent for displacement antisolvent crystallization to obtain matrine raw material.

[0032] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are only for illustrating the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Raw materials not specifically mentioned can be obtained through conventional commercial channels, and equipment not specifically mentioned can be conventional equipment in the art. Unless otherwise specified, the conditions such as temperature, time, volume fraction, mass fraction, pressure, flow rate, and feed rate described in each embodiment are based on the actual control values ​​during implementation.

[0033] This invention relates to a method for the segmented extraction and purification of active components from traditional Chinese medicine raw materials, wherein the active component is matrine. The method comprises six stages: S1 extraction, S2 ethanol-phosphate aqueous two-phase separation, S3 nanofiltration dialysis displacement, S4 fumaric acid salt formation and crystallization, S5 free base release and extraction, and S6 displacement antisolvent crystallization. These six stages are not isolated from each other, but rather constitute a continuous chain of segmented enrichment and targeted purification processes. The initial stage involves extraction and aqueous phase separation to transfer the target component matrine from the solid matrix of the medicinal material to the liquid phase, where it further concentrates in the complex liquid system and selectively migrates to the ethanol phase. The middle stage uses nanofiltration dialysis to reduce the content of phase-forming salts and low-molecular-weight impurities. The final stage involves fumaric acid salt crystallization, free alkali release, extraction with acetate organic solvents, and displacement antisolvent crystallization to gradually separate matrine from homologous alkaloid impurities, inorganic salt residues, and mother liquor impurities in different chemical states and solvent environments, thereby obtaining matrine raw material with high purity and low inorganic salt residue.

[0034] As shown in Figure 1, the method of this invention generally includes six stages: S1 extraction, S2 ethanol-phosphate aqueous two-phase separation, S3 nanofiltration dialysis replacement, S4 fumaric acid salt formation and crystallization, S5 free base release and extraction, and S6 displacement antisolvent crystallization. As shown in Figure 2, the ethanol-phosphate aqueous two-phase separation and nanofiltration dialysis replacement together constitute the front-end segmented enrichment and desalting module; as shown in Figure 3, fumaric acid salt formation and crystallization, free base release and extraction, and displacement antisolvent crystallization together constitute the rear-end targeted purification module. Through the organic connection between the front-end segmented enrichment and the rear-end targeted purification, the segmented selective enrichment and purification capacity of matrine in the total alkaloid extract can be effectively improved.

[0035] To enable those skilled in the art to more clearly understand the scope of application of certain higher-level technical concepts in this invention, several technical terms are explained below.

[0036] The term "medicinal herbs containing matrine" as used in this invention refers to medicinal herbs whose target extraction component is matrine, and whose raw materials naturally contain matrine or whose main alkaloid is matrine. These medicinal herbs preferably meet the following conditions: first, matrine can be detected in the raw material; second, the matrine contained therein can be extracted in an acidic aqueous ethanol solution and transferred to the liquid phase; and third, after aqueous phase separation, nanofiltration dialysis displacement, and subsequent salt formation crystallization and antisolvent crystallization, a final product with matrine as the main component can be obtained. In a specific embodiment of this invention, dried roots of *Sophora flavescens* and mature seeds of *Sophora alopecuroides* are preferably used as representative raw materials. On the one hand, these two types of raw materials have a high matrine content and a stable source; on the other hand, the homologous alkaloid impurities such as sophoridine and oxymatrine contained therein are representative, fully demonstrating the technical effect of the segmented enrichment and targeted purification route described in this invention. Other medicinal herbs with matrine sources that meet the above applicable conditions can also be used as raw material sources for the method of this invention.

[0037] The term "phosphate phase-forming salt" as used in this invention refers to a phosphate-based phase-forming substance capable of forming an aqueous two-phase system with ethanol and water, and enabling matrine to exhibit usable partitioning differences within the aqueous two-phase system. This type of phosphate phase-forming salt preferably possesses the following characteristics: it can form a stable phase-separated system with water within the ethanol volume fraction range defined in this invention; it has sufficient phase-forming ability in the system to form a clear upper and lower phase interface; and it does not introduce unacceptable side reactions or adverse effects during subsequent nanofiltration dialysis replacement and salt crystallization processes. In specific embodiments of this invention, one or both of dipotassium hydrogen phosphate and potassium dihydrogen phosphate are preferably used as the phosphate phase-forming salt. Using dipotassium hydrogen phosphate alone can form a relatively stable ethanol-salt aqueous two-phase system; using potassium dihydrogen phosphate alone can obtain a clearer phase-separated interface at higher phase-forming salt concentrations; using both together is beneficial for achieving a balance between phase-forming ability, system acidity / basicity, and interphase partitioning behavior.

[0038] The "acetic acid ester organic solvent" referred to in this invention refers to an acetate ester organic medium that can effectively extract matrine free base in the free base release step and has good compatibility with the subsequent isopropanol / water shift antisolvent crystallization process. This type of organic solvent preferably meets the following requirements: good partitioning ability for matrine free base; forms a two-phase system with water, facilitating liquid-liquid separation; is easily removed during subsequent vacuum concentration and solvent switching; and does not undergo side reactions with matrine that are detrimental to purification. In specific embodiments of this invention, ethyl acetate and isopropyl acetate are preferably used as one or both of the acetate ester organic solvents. Ethyl acetate has good volatility and mature process applicability; isopropyl acetate is beneficial under certain conditions for improving the retention of the target component in the organic phase and subsequent crystallization behavior; the combined use of the two can achieve a better balance between extraction capacity and subsequent solvent switching.

[0039] The "alcohol crystallization solvent" referred to in this invention refers to an alcohol medium used in the displacement antisolvent crystallization step to form a mother liquor with the concentrated organic phase of matrine and to establish a controllable supersaturated crystallization environment together with the subsequent antisolvent water. This type of crystallization solvent preferably meets the following conditions: it has a certain solubility for matrine to form a homogeneous mother liquor; it is miscible with water or has good compatibility to form a controllable supersaturated state through continuous dropwise addition of water; and it does not affect the crystal quality of matrine during subsequent seed crystal induction and cooling crystallization processes. In a specific embodiment of this invention, isopropanol is preferably used as the alcohol crystallization solvent. The reason for choosing isopropanol is that it has good compatibility with water and can form a suitable mother liquor environment under the process conditions of this invention, thereby facilitating subsequent displacement antisolvent crystallization and crystal quality control.

[0040] To ensure comparability of results obtained from different embodiments, this embodiment employs a unified detection method for the final product. The contents of matrine, sophoridine, and oxymatrine were determined using high-performance liquid chromatography (HPLC). Specifically, an appropriate amount of the final product was accurately weighed, dissolved and diluted to volume with a methanol-water mixture, and filtered through a 0.45 μm organic filter membrane if necessary to obtain the test solution. Matrine, sophoridine, and oxymatrine reference solutions were prepared separately as standard solutions. A C18 reversed-phase column (250 mm × 4.6 mm, 5 μm particle size) was used. The mobile phase consisted of acetonitrile and potassium dihydrogen phosphate buffer solution. The detection wavelength was 220 nm; the column temperature was 30 °C; the flow rate was 1.0 mL / min; and the injection volume was 10 μL. The peak areas of the test solution and standard solutions were measured, and the mass fractions of matrine, sophoridine, and oxymatrine in the final product were calculated using the external standard method. The moisture content of the final product was determined using the Karl Fischer moisture determination method. An appropriate amount of the final product was accurately weighed and titrated in a Karl Fischer moisture analyzer. The endpoint consumption was recorded, and the water mass fraction in the sample was calculated. Inorganic salt residues in the final product were detected using ion chromatography. An appropriate amount of the final product was accurately weighed, added to ultrapure water, and dissolved or extracted using ultrasound. After filtration, the filtrate was used as the test solution, and the inorganic ion content was determined using ion chromatography. The inorganic salt residue was calculated based on the total ion content, and the result was expressed in mg / kg. The total yield was calculated based on the measured mass of matrine in the raw material. Let the raw material input mass be... The mass fraction of matrine in the raw material is The final product quality is The mass fraction of matrine in the final product is Then the total return Calculate using the following formula: in, This indicates the quality of the raw materials input. This indicates the mass fraction of matrine in the raw material. Indicates the quality of the final product. This indicates the mass fraction of matrine in the final product. This indicates the total yield.

[0041] For ease of understanding, this embodiment will be described in detail through the following specific embodiments 1 to 3.

[0042] Specific Example 1: This example is implemented using a combination of lower process parameters to demonstrate that, under milder extraction conditions, lower aqueous phase formation intensity, lower nanofiltration dialysis displacement intensity, lower salt-forming agent addition amount, and lower seed crystal induction intensity, the present invention can still achieve the segmented selective enrichment and purification of matrine in the total alkaloid extract.

[0043] S1 Extraction and Preparation of Total Alkaloid Extract: In this embodiment, dried Sophora flavescens roots were used as raw material. 10.0 kg of dried Sophora flavescens roots were weighed and manually sorted before feeding to remove visible mud, stones, moldy parts, fibrous roots, and obviously corky ineffective tissue. The sorted dried Sophora flavescens roots were then fed into a mechanical pulverizer for pulverization and sieved through a 24-mesh sieve to achieve a low particle size. Using a relatively coarse particle size setting demonstrates that this method does not rely on the energy-intensive pretreatment method of ultra-fine pulverization, and also helps to avoid excessive fine powder entering the extract and increasing filtration difficulties. After pulverization, initial component analysis of the raw material was performed, revealing a matrine mass fraction of 2.40%, a sophoridine mass fraction of 0.58%, and an oxymatrine mass fraction of 0.91%.

[0044] Subsequently, an alkaline pre-wetting treatment was performed. Specifically, 6.0 L of a 0.2% ammonium bicarbonate aqueous solution was prepared and sprayed evenly onto the surface of the pulverized dried Sophora flavescens roots, ensuring the raw material was uniformly wetted. After spraying, the wetted raw material was placed in a sealed container and allowed to stand at room temperature for 0.5 h. This alkaline pre-wetting treatment, on the one hand, moderately swells the plant tissue, thus facilitating the subsequent penetration of the extraction solvent into the raw material tissue; on the other hand, it allows some of the alkaloid components existing in salt form to transform into a state more conducive to subsequent extraction.

[0045] The extraction medium was a 55% (v / v) aqueous ethanol solution with 0.15% (w / w) formic acid added to form an acidic aqueous ethanol solution. Pre-wetting dried roots of *Sophora flavescens* were loaded into a countercurrent percolation extraction apparatus, and 60.0 L of the aforementioned acidic aqueous ethanol solution was added. Countercurrent percolation extraction was performed at 35°C. During the extraction process, the system temperature and percolation rate were kept relatively stable to avoid uneven extraction due to excessively high local temperatures or large fluctuations in flow rate. After extraction, the resulting extracts were combined and filtered through a plate and frame filter to remove plant fibers, fine powder, and insoluble suspended solids, yielding 68.4 L of total alkaloid extract. Analysis of this total alkaloid extract revealed that, in addition to matrine, it still contained sophoridine, oxymatrine, water-soluble small molecule impurities, plant-derived pigments, and a certain amount of inorganic ions.

[0046] S2. Ethanol-phosphate aqueous two-phase separation and preparation of the first fractionated extract: The total alkaloid extract obtained in step S1 was transferred to a vacuum concentration device and concentrated under reduced pressure at a temperature not exceeding 55°C, while recovering some ethanol, causing the ethanol volume fraction in the system to gradually decrease. When the ethanol volume fraction in the system was adjusted to 18% and the total volume of the liquid was approximately 24.0 L, the concentration was stopped.

[0047] Dipotassium hydrogen phosphate was added to the above concentrated solution to achieve a total phosphate mass fraction of 12% in the ethanol-phosphate aqueous two-phase system. Stirring was maintained during the addition process to ensure the dipotassium hydrogen phosphate was fully dissolved and evenly distributed throughout the liquid phase system. After the system was thoroughly mixed, stirring was stopped, and the mixture was allowed to stand at 25°C for 20 minutes. During this standing process, a distinct two-phase structure gradually formed: an upper phase enriched in ethanol and a lower phase enriched in phosphate. 11.3 L of the upper phase was collected as the first fractionated extract. Considering that some target components might still remain in the lower phase, ethanol was added back to the separated lower phase, and the ethanol volume fraction and total phosphate mass fraction were adjusted to the aforementioned levels. A second phase separation was then performed to obtain a second upper phase of 3.2 L, which was then combined with the first upper phase. After this step, matrine underwent one directional enrichment at the liquid phase level.

[0048] S3 Nanofiltration Dialysis Replacement and Preparation of Desalted Enriched Solution: The pH of the first fractionated extract obtained in step S2 was adjusted to 7.2 and fed into an alcohol-resistant nanofiltration device. The nanofiltration membrane used in this embodiment has a molecular weight cutoff of 150 Da. Dialysis replacement was performed using deionized water with a volume twice that of the feed solution entering this step, and the conductivity of the permeate was monitored in real time. When the conductivity of the permeate dropped below 2.0 mS / cm, dialysis replacement was stopped, yielding 10.6 L of desalted enriched solution. The concentrations of matrine, sophoridine, and oxymatrine in the desalted enriched solution were measured to be 18.7 g / L, 2.6 g / L, and 1.9 g / L. Simultaneously, the inorganic salt levels were significantly reduced. This result indicates that even with a lower dialysis replacement water volume and a nanofiltration membrane with a lower molecular weight cutoff, the phosphate residue introduced by the aqueous two-phase system can still be effectively controlled.

[0049] S4. Crystallization of fumaric acid to obtain matrine fumarate crystals: The desalted enrichment solution obtained in step S3 was heated to 35°C, while an 85% (v / v) fumaric acid ethanol solution was prepared. Based on the detected concentration of matrine in the desalted enrichment solution, the molar amount of matrine was calculated, and fumaric acid was added at 0.45 times this molar amount. Stirring was maintained during the dropwise addition to ensure uniform dispersion of the fumaric acid and preferential reaction with matrine. After the dropwise addition was completed, the temperature was maintained for 10 min to further promote the salt formation process. The system was then cooled to 5°C and maintained for 2 h, gradually precipitating matrine fumarate crystals. 278 g of matrine fumarate crystals were obtained after filtration and washed with a 1:1 (v / v) mixture of anhydrous ethanol and ethyl acetate to remove mother liquor and some soluble impurities from the crystal surface.

[0050] S5 Free Base Release and Acetate-Based Organic Solvent Extraction: The matrine fumarate crystals obtained in step S4 were dispersed in 2.8 L of purified water to form a homogeneous dispersion. The pH of the dispersion was then adjusted to 9.5 with ammonia and kept at this temperature for 15 min to convert matrine from the fumarate state to the free base state. In this example, ethyl acetate was used for three extractions, with 2.0 L of ethyl acetate added each time. After standing and separating the layers, the organic phases were collected and combined to obtain the matrine organic phase. The organic phase was then washed with purified water with a conductivity not exceeding 50 μS / cm until the pH of the washings reached 7.0.

[0051] S6. Shift-displacement antisolvent crystallization to obtain matrine raw material: The matrine organic phase obtained in step S5 was concentrated under reduced pressure to 2.1 L to obtain a concentrated solution. 1.8 L of isopropanol was added to this concentrated solution to form a crystallization mother liquor, followed by continuous dropwise addition of 1.2 L of purified water as an antisolvent. When visible turbidity appeared in the system and did not disappear after stirring for 5 min, matrine seed crystals at 0.5% of the mass of matrine in the concentrated solution were added, and the temperature was lowered to 0℃ at a cooling rate of 0.2℃ / min and maintained at this temperature for 4 h. Afterwards, wet crystals were obtained by centrifugation and dried at 40℃ and 20 kPa absolute pressure for 10 h to obtain 184.1 g of matrine raw material.

[0052] The obtained matrine raw material was subjected to quality testing, and the content of matrine was found to be 98.72%, sophoridine content was 0.41%, oxymatrine content was 0.28%, moisture content was 0.63%, and inorganic salt residue was 310 mg / kg. Based on a raw material weight of 10.0 kg of dried Sophora flavescens root, a matrine content of 2.40%, a final product weight of 184.1 g, and a matrine content of 98.72%, the overall yield was calculated to be 76.7% using the aforementioned formula. The above test results indicate that even with a lower combination of process parameters, this method can still obtain matrine raw material with a high matrine content, low sophoridine and oxymatrine content, and low levels of moisture and inorganic salt residue.

[0053] Specific Embodiment 2: This embodiment is implemented using a combination of intermediate process parameters to illustrate that, under preferred conditions, the present invention can achieve a better balance between purity, yield, impurity control, and inorganic salt residue control.

[0054] S1 Extraction and Preparation of Total Alkaloid Extract: In this embodiment, mature seeds of *Sophora flavescens* were used as raw material. 10.0 kg of mature *Sophora flavescens* seeds were weighed, and after removing damaged, moldy, and impurity particles, they were mechanically pulverized and passed through a 37-mesh sieve. Testing revealed that the mature *Sophora flavescens* seeds contained 2.65% matrine, 0.61% sophoridine, and 0.88% oxymatrine.

[0055] Subsequently, 7.5 L of a 0.5% ammonium bicarbonate aqueous solution was prepared and sprayed onto the crushed mature bitter bean seeds for pre-wetting, followed by standing under sealed conditions for 1.25 h. The extraction medium was a 63.5% ethanol aqueous solution with 0.375% formic acid added, for a total addition of 90.0 L. Countercurrent percolation extraction was performed at 42.5℃. After extraction, the extracts were combined and filtered to obtain 95.2 L of total alkaloid extract.

[0056] S2 Ethanol-Phosphate Aqueous Two-Phase Separation and Preparation of the First Fractional Extract: The total alkaloid extract was concentrated under reduced pressure at a temperature not exceeding 55°C, while recovering some ethanol to adjust the ethanol volume fraction to 23% and the feed volume to 26.0 L. Subsequently, dipotassium hydrogen phosphate and potassium dihydrogen phosphate were added simultaneously at a mass ratio of 1:1 to achieve a total phosphate mass fraction of 16% in the ethanol-phosphate aqueous two-phase system. After thorough stirring, the mixture was allowed to stand at 25°C for 55 min. After standing, the phases were clearly separated, and the upper phase of 13.8 L was collected as the first fractional extract. The ethanol volume fraction and total phosphate mass fraction of the lower phase were adjusted again, and the phase separation was repeated once more to obtain a second upper phase of 4.6 L, which was then combined with the first upper phase.

[0057] S3 Nanofiltration Dialysis Replacement and Preparation of Desalted Enriched Solution: The pH of the first fractionated extract was adjusted to 7.7 and fed into an alcohol-resistant nanofiltration unit. The nanofiltration membrane used had a molecular weight cutoff of 225 Da. Dialysis replacement was performed with deionized water at a volume four times that of the feed solution entering this step. The conductivity of the permeate was continuously monitored. The operation was terminated when the conductivity dropped below 2.0 mS / cm, yielding 12.4 L of desalted enriched solution. Analysis showed that the mass concentrations of matrine, sophoridine, and oxymatrine in the desalted enriched solution were 20.8 g / L, 2.1 g / L, and 1.5 g / L, respectively.

[0058] S4 fumaric acid crystallization to obtain matrine fumarate crystals: The desalted enrichment solution was heated to 42.5℃, and a fumaric acid ethanol solution with a volume fraction of 92.5% was prepared. Based on the measured content of matrine, fumaric acid was added at 0.50 times its molar amount. Stirring was maintained during the dropwise addition. After the addition was completed, the temperature was maintained for 25 min, then lowered to 10℃ and maintained for 3 h. Filtration yielded 315 g of matrine fumarate crystals, which were washed with a mixed washing solution of anhydrous ethanol and ethyl acetate at a volume ratio of 1:2.5.

[0059] S5 Free Base Release and Acetate-Based Organic Solvent Extraction: Matrine fumarate crystals were dispersed in 3.2 L of purified water. The pH of the dispersion was adjusted to 10.0 with ammonia and kept at this temperature for 20 min. Subsequently, three extractions were performed using a 1:1 volume ratio of ethyl acetate and isopropyl acetate, with 2.2 L of the mixed solvent added each time. The resulting organic phases were combined. The organic phase was then washed with purified water with a conductivity not exceeding 50 μS / cm until the pH of the washings reached 7.5.

[0060] S6 displacement antisolvent crystallization to obtain matrine raw material: The organic phase was concentrated under reduced pressure to 2.5 L, and 2.1 L of isopropanol was added to form a crystallization mother liquor. Then, 1.6 L of purified water was continuously added dropwise as an antisolvent. When visible turbidity appeared in the system and did not disappear after stirring for 7 min, matrine seed crystals at 1.25% of the mass of matrine in the concentrated solution were added, and the temperature was lowered to 5 °C at a cooling rate of 0.5 °C / min and held at this temperature for 6 h. After centrifugation, the mixture was dried at 47.5 °C and 40 kPa absolute pressure for 12 h to obtain 214.3 g of matrine raw material.

[0061] The obtained matrine raw material was subjected to quality testing, and the content of matrine was found to be 99.18%, sophoridine content was 0.23%, oxymatrine content was 0.17%, moisture content was 0.48%, and inorganic salt residue was 185 mg / kg. Based on a raw material weight of 10.0 kg of mature bitter bean seeds, a matrine content of 2.65%, a final product weight of 214.3 g, and a matrine content of 99.18% in the final product, the overall yield was calculated to be 80.8% using the aforementioned formula. The above test results indicate that under the combined intermediate process parameters, the matrine content of the final product was further increased, while the contents of sophoridine and oxymatrine were further reduced. Simultaneously, the control effect on inorganic salt residue was more significant, and the overall yield remained at a high level.

[0062] Specific Example 3: This example is implemented using a higher combination of process parameters to illustrate that under conditions of higher extraction intensity, higher aqueous phase formation intensity, higher desalting intensity, and higher crystallization induction intensity, the present invention can still maintain process stability and obtain matrine raw material that meets the requirements.

[0063] S1 Extraction and Preparation of Total Alkaloid Extract: 10.0 kg of dried Sophora flavescens root was weighed, impurities were removed manually, and then mechanically pulverized and passed through a 50-mesh sieve. High-performance liquid chromatography (HPLC) analysis showed that the raw material contained 2.40% matrine, 0.59% sophoridine, and 0.90% oxymatrine. Subsequently, the raw material was pre-wetted by spraying with 8.0 L of a 0.8% ammonium bicarbonate aqueous solution and allowed to stand for 2 hours under sealed conditions.

[0064] The extraction medium was a 72% (v / v) aqueous ethanol solution containing 0.60% formic acid, with a total addition of 120.0 L. Countercurrent percolation extraction was performed at 50 °C. After extraction, the extracts were combined and filtered through a plate and frame filter to obtain 124.8 L of total alkaloid extract.

[0065] S2 Ethanol-Phosphate Aqueous Two-Phase Separation and Preparation of the First Fractional Extract: The total alkaloid extract was concentrated under reduced pressure at a temperature not exceeding 55°C to adjust the ethanol volume fraction to 28% and the feed volume to 30.0 L. Potassium dihydrogen phosphate was then added to bring the total phosphate mass fraction in the ethanol-phosphate aqueous two-phase system to 20%. After thorough stirring, the mixture was allowed to stand at 25°C for 90 min, resulting in clear phase separation. 15.2 L of the upper phase was collected as the first fractional extract. The ethanol volume fraction and total phosphate mass fraction of the lower phase were readjusted, and the phase separation was repeated once more to obtain a second upper phase of 5.1 L, which was then combined with the first upper phase.

[0066] S3 Nanofiltration Dialysis Replacement and Preparation of Desalted Enriched Solution: The pH of the first fractionated extract was adjusted to 8.2 and fed into an alcohol-resistant nanofiltration unit. The nanofiltration membrane used had a molecular weight cutoff of 300 Da. Dialysis replacement was performed using deionized water at a volume six times that of the feed solution in this step. The conductivity of the permeate was continuously monitored. When it dropped below 2.0 mS / cm, the replacement was stopped, yielding 13.6 L of desalted enriched solution. Analysis showed that the mass concentrations of matrine, sophoridine, and oxymatrine in the desalted enriched solution were 18.9 g / L, 2.8 g / L, and 2.0 g / L, respectively.

[0067] S4. Crystallization of fumaric acid to obtain matrine fumarate crystals: The desalting enrichment solution was heated to 50°C, and an ethanol solution of fumaric acid was prepared using anhydrous ethanol as the solvent. Fumaric acid was added at 0.55 times the molar amount of matrine. After the addition was complete, the temperature was maintained for 40 min, then lowered to 15°C and maintained for 3 h to allow matrine fumarate crystals to precipitate. After filtration, 334 g of matrine fumarate crystals were obtained and washed with a mixed washing solution of anhydrous ethanol and ethyl acetate in a volume ratio of 1:4.

[0068] S5 Free Base Release and Acetate-Based Organic Solvent Extraction: Matrine fumarate crystals were dispersed in 4.0 L of purified water. The pH of the dispersion was adjusted to 10.5 with ammonia and kept at this temperature for 25 min. Subsequently, three extractions were performed using isopropyl acetate, with 2.5 L of isopropyl acetate added each time. The organic phases were then combined. The organic phase was then washed with purified water with a conductivity not exceeding 50 μS / cm until the pH of the washings reached 8.0.

[0069] S6 displacement antisolvent crystallization to obtain matrine raw material: The organic phase was concentrated under reduced pressure to 2.8 L, and 2.4 L of isopropanol was added to form a crystallization mother liquor. Subsequently, 2.0 L of purified water was continuously added dropwise as an antisolvent. When visible turbidity appeared in the system and did not disappear after stirring for 10 min, matrine seed crystals with a mass of 2.0% of the mass of matrine in the concentrated solution were added, and the temperature was lowered to 10 °C at a cooling rate of 0.8 °C / min and held at this temperature for 4 h. After centrifugation, the wet crystals were dried at 55 °C and 60 kPa absolute pressure for 8 h to obtain 188.6 g of matrine raw material.

[0070] The obtained matrine raw material was subjected to quality testing, and the content of matrine was found to be 98.93%, sophoridine content was 0.36%, oxymatrine content was 0.25%, moisture content was 0.55%, and inorganic salt residue was 235 mg / kg. Based on a raw material weight of 10.0 kg of dried Sophora flavescens root, a matrine content of 2.40%, a final product weight of 188.6 g, and a matrine content of 98.93%, the overall yield was calculated to be 78.5% using the aforementioned formula. The above test results indicate that even under higher process parameter combinations, the obtained matrine raw material still maintains a high matrine content and low levels of sophoridine, oxymatrine, and inorganic salt residue.

[0071] Summary Table 1 of Main Process Parameters and Final Product Results Table 2 Comparison of Main Results of Each Embodiment As shown in Figure 4 and Table 2, the matrine content of the final products obtained in the three specific embodiments is at a relatively high level, with the highest in Specific Embodiment 2. This indicates that under the intermediate parameter combination, a good match can be achieved between the pre-stage fractional enrichment and the post-stage targeted purification. As shown in Figure 5 and Table 2, the overall yield of the three specific embodiments is at a relatively high level, indicating that the present invention does not sacrifice yield for purity, but rather improves purity while maintaining the retention rate of the target components. As shown in Figure 6, the contents of sophoridine and oxymatrine are controlled at a low level in the three specific embodiments, indicating that the present invention has a good effect on the removal of homologous alkaloid impurities. As shown in Figure 7, the inorganic salt residues in the three specific embodiments are at a low level, indicating that the nanofiltration dialysis replacement step plays an important role in reducing inorganic salt residues.

[0072] As can be seen from the above specific embodiments, Tables 1 and 2, and Figures 4 to 7, the present invention, through a series of technical routes including ethanol-phosphate aqueous two-phase fractional enrichment, nanofiltration dialysis displacement desalting, selective fumaric acid salt formation crystallization, free alkali release extraction, and displacement antisolvent crystallization, can more effectively enrich matrine in complex total alkaloid systems and further reduce the introduction of impurities such as sophoridine, oxymatrine, and inorganic salts. The three specific embodiments, from the perspectives of lower, intermediate, and higher process parameter combinations, illustrate that the technical solutions of the present invention can achieve higher matrine content, lower homologous impurity content, and lower inorganic salt residues. This indicates that the present invention not only achieves fractional selective enrichment and purification of matrine in total alkaloid extracts but also has good process adaptability and implementation stability.

[0073] It should be noted that the embodiments of the present invention have better implementability and are not intended to limit the present invention in any way. Any person skilled in the art may use the above-disclosed technical content to change or modify it into equivalent effective embodiments. However, any modifications or equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A method for fractional extraction and purification of active components from traditional Chinese medicinal materials, wherein, The active ingredient of the traditional Chinese medicine is matrine, characterized by the following steps: S1, extracting the matrine-containing traditional Chinese medicine by contacting it with an acidic ethanol aqueous solution to obtain a total alkaloid extract; S2, concentrating the total alkaloid extract under reduced pressure and adjusting the ethanol volume fraction, adding a phosphate phase-forming salt to form an ethanol-phosphate aqueous two-phase system, separating the phases and collecting the upper phase enriched with matrine to obtain a first fractionated extract; S3, subjecting the first fractionated extract to nanofiltration dialysis to remove the phosphate phase-forming salt and low-molecular-weight impurities. S4. Add fumaric acid ethanol solution to the desalted enriched solution to allow matrine to crystallize out as fumarate, and separate the solid and liquid to obtain matrine fumarate crystals; S5. Disperse the matrine fumarate crystals in water and adjust to alkalinity to convert matrine into free base, and then extract with an acetic acid ester organic solvent to obtain the matrine organic phase; S6. Concentrate the matrine organic phase and add an alcohol crystallization solvent to form a crystallization mother liquor, and then add water as an antisolvent for displacement antisolvent crystallization to obtain matrine raw material.

2. The fractional extraction and purification method according to claim 1, characterized in that, In S1, the medicinal material is the dried root of Sophora flavescens or the mature seed of Sophora alopecuroides, and the medicinal material is subjected to alkaline pre-wetting treatment before being extracted by contact with the acidic ethanol aqueous solution.

3. The fractional extraction and purification method according to claim 2, characterized in that, The Chinese medicinal materials are pulverized to 24-50 mesh. The alkaline pre-wetting treatment is carried out using an ammonium bicarbonate aqueous solution with a mass fraction of 0.2%-0.8% for 0.5-2 hours. The acidic ethanol aqueous solution is an ethanol aqueous solution with a volume fraction of 55%-72% and contains formic acid with a mass fraction of 0.15%-0.60%.

4. The fractional extraction and purification method according to claim 1, characterized in that, In step S2, the total alkaloid extract is concentrated under reduced pressure to an ethanol volume fraction of 18%–28%, and then one or both of dipotassium hydrogen phosphate and potassium dihydrogen phosphate are added to make the total phosphate mass fraction in the ethanol-phosphate aqueous two-phase system 12%–20%. After standing and phase separation, the upper phase is collected.

5. The fractional extraction and purification method according to claim 4, characterized in that, After readjusting the ethanol volume fraction and total phosphate mass fraction of the lower phase obtained from the S2 phase separation, perform another phase separation operation, and incorporate the upper phase obtained from the second phase separation into the first fractionated extract.

6. The fractional extraction and purification method according to claim 1, characterized in that, In S3, the nanofiltration dialysis replacement is performed using an alcohol-resistant nanofiltration membrane, and the nanofiltration membrane has a molecular weight cutoff of 150–300 Da.

7. The fractional extraction and purification method according to claim 6, characterized in that, In step S3, the pH of the first fractional extraction solution is adjusted to 7.2-8.2 before nanofiltration dialysis replacement is performed. Deionized water with a volume of 2-6 times that of the feed solution entering the nanofiltration dialysis replacement step is used for dialysis replacement. The nanofiltration dialysis replacement ends when the conductivity of the permeate drops below 2.0 mS / cm.

8. The fractional extraction and purification method according to claim 1, characterized in that, In S4, the volume fraction of ethanol in the fumaric acid ethanol solution is 85% to 100%, and the amount of fumaric acid added is 0.45 to 0.55 times the molar amount of matrine in the desalting enrichment solution.

9. The fractional extraction and purification method according to claim 8, characterized in that, In step S4, the desalting enrichment solution is heated to 35–50°C, the fumaric acid ethanol solution is added dropwise, and the solution is kept at this temperature for 10–40 minutes. Then, the temperature is lowered to 5–15°C to allow for salt crystallization.

10. The fractional extraction and purification method according to claim 8, characterized in that, The matrine fumarate crystals obtained in S4 were washed with a mixed washing solution consisting of anhydrous ethanol and ethyl acetate, wherein the volume ratio of anhydrous ethanol to ethyl acetate was 1:(1-4).

Images