Process for the preparation of songorol

Abstract

This invention relates to the field of pharmaceutical technology, and in particular to a method for preparing Songguoling. The method includes the following steps: S1, pulverizing dried Aconitum carmichaelii and adding it to an ethanol-water solution for reflux extraction, then filtering and concentrating to obtain a concentrated solution; S2, performing column chromatography on the concentrated solution using a macroporous adsorption resin column, with gradient elution using an ethanol-water solution as the eluent; S3, acidifying fraction C, then separating it using a cation exchange resin column chromatography, with gradient elution using an ammonia-ethanol solution; S4, dissolving fraction C3 in a recrystallization solvent, allowing it to stand and crystallize, then recrystallizing, washing with the recrystallization solvent, and drying to obtain Songguoling raw material. The method of this invention yields Songguoling with high yield and high purity, resulting in a product more suitable for industrial production.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a method for preparing Songguoling. Background Technology

[0002] Song Guoling is C 20 Type II diterpenoid alkaloids are one of the main active ingredients of Aconitum carmichaelii, a traditional Chinese medicine. Modern pharmacological studies have confirmed that, in addition to analgesic and anti-inflammatory activities, they also show clear potential in regulating the nervous system and immune metabolism.

[0003] Existing methods for preparing Songguoling involve complex processes such as water extraction, macroporous resin, MCI resin, acid and alkali treatment, various column chromatography methods (silica gel, reverse phase, gel) and recrystallization. These methods suffer from drawbacks such as complex processes, high production costs, low yields and purity, and incompatibility with industrial production. Summary of the Invention

[0004] This invention covers the following technical solutions: One aspect of the present invention relates to a method for preparing sinecuronium, the method comprising the following steps: S1. After pulverizing the dried Aconitum carmichaelii, add it to an ethanol-water solution for reflux extraction, filter and concentrate to obtain a concentrated solution; S2. The concentrate was separated by column chromatography using a macroporous adsorption resin column with gradient elution using ethanol-water solution as the eluent. The eluent fractions with Rf values ​​of 0.32 to 0.45 as identified by silica gel thin-layer chromatography were collected and combined to obtain fraction C. S3. After acidification, fraction C is separated by cation exchange resin column chromatography with gradient elution using ammonia ethanol solution. The eluted fractions with Rf values ​​of 0.37 to 0.40 as identified by silica gel thin-layer chromatography are collected and combined to obtain fraction C3. S4. Dissolve the fraction C3 in a recrystallization solvent, allow it to stand and crystallize, then recrystallize it, wash it with the recrystallization solvent and dry it to obtain Songguoling raw material, wherein the recrystallization solvent is one or more of ethanol, ethyl acetate and petroleum ether.

[0005] Currently, conventional extraction methods, such as those disclosed in the patent documents cited in the background art, yield only about 0.30% of sine alkaloids. However, the method of the present invention, due to the use of specific steps and the specific solvent and extraction process, especially the combination of macroporous adsorption resin enrichment and cation exchange resin specific purification and the use of recrystallization, greatly improves the yield of sine alkaloids, reaching 0.53% in some embodiments. The method of this invention employs steps commonly used in industrial production, such as solvent extraction, macroporous resin column chromatography, cation exchange resin column chromatography, recrystallization, and drying. The process is simple and practical, and because specific process conditions are used in each step, high yield and high purity of Songguoling are obtained, thus producing an effect that is more suitable for industrial production. Attached Figure Description

[0006] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0007] Figure 1 This is a high-performance liquid chromatogram of the active pharmaceutical ingredient Songguoling obtained in Example 1 of the present invention.

[0008] The assay method was as follows: Detection system: Aglient 1260; chromatographic column: Epic C18 5μm 120 Å 250 × 4.6 mm; mobile phase: 30% methanol-water (containing 0.1% trifluoroacetic acid); injection volume: 10 μL; flow rate: 1.0 mL / min; detector: DAD detector; wavelength: 210 nm; column temperature: 30℃. Detailed Implementation

[0009] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0010] Unless otherwise stated, all terms used to disclose this invention (including technical and scientific terms) should be understood as having the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of protection of this invention. Unless the context clearly defines otherwise, the scientific and technical terms used herein, as well as terms and laboratory procedures in related fields such as neurobiology, traditional Chinese medicine chemistry, natural product chemistry, drug separation and purification technology, chromatographic analysis technology, pharmaceutical engineering, and pharmaceutical formulation engineering, are all conventional terms and standard methods well-known and widely used in the art. To facilitate understanding of the technical solutions of this invention, some related terms are further defined and explained below.

[0011] The terms “containing,” “comprising,” and “including” as used in this invention are synonyms and are inclusive or open-ended, not excluding additional, uncited members, elements, or method steps.

[0012] In this invention, the numerical range represented by endpoints includes all numerical values ​​and fractions contained within that range, as well as the endpoints mentioned.

[0013] Furthermore, in describing representative embodiments of the invention, this specification may present the methods and / or processes of the invention as a specific sequence of steps. However, the method or process should not be limited to the specific order of the steps described herein, to the extent that the method or process does not depend on the specific order of the steps presented herein. As will be understood by those skilled in the art, other sequences of steps are also possible. Therefore, the specific order of steps presented in the specification should not be construed as a limitation of the claims. Additionally, the claims relating to the methods and / or processes of the invention should not be limited to the execution of their steps in the order they are written, and those skilled in the art will readily recognize that the sequence can be changed while still remaining within the spirit and scope of the invention.

[0014] This invention relates to concentration values, which include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 2% can fluctuate within ±0.1%. For larger values ​​or values ​​that do not require overly precise control, even greater fluctuations are permitted.

[0015] As used in this invention, unless otherwise stated, the singular forms of the articles “a,” “an,” and “the” include plural referents.

[0016] In this invention, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity of 2 or more.

[0017] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0018] In this invention, terms such as "preferred," "better," "more suitable," and "ideal" merely describe implementation methods or embodiments with better effects and should be understood not to limit the scope of protection of this invention. In this invention, terms such as "optionally," "optionally," and "optional" mean that something is optional, that is, selected from either "with" or "without" a parallel solution. If multiple "optional" statements appear in a technical solution, unless otherwise specified and without contradiction or mutual constraint, each "optional" statement is independent.

[0019] In this invention, "Songguoling" refers to a C derived from the traditional Chinese medicine Aconitum carmichaelii.20 Type II diterpenoid alkaloids, possessing well-defined chemical structures and stable physicochemical properties, are among the main active components of Aconitum carmichaelii. The Songguoling described in this invention includes products of different purity levels, crystal forms, or solvation states obtained by the method of this invention. As long as their chemical nature remains unchanged, they should all be understood to fall within the scope of Songguoling as defined in this invention.

[0020] In this invention, "Compound 1" refers to the pure Songguoling prepared by the process described in Example 1 and confirmed by structural characterization, with the molecular formula C. 22 H 31 NO3, its nuclear magnetic resonance (NMR) spectrum and high-resolution mass spectrometry (HMS) data are consistent with the theoretical structure of Songguoling. Therefore, compound 1 is essentially a specific embodiment of Songguoling, representing a high-purity Songguoling product prepared using the method of this invention. Unless otherwise stated, compound 1 described in the specification is consistent with Songguoling in chemical structure and pharmaceutical properties. Compound 1 can be considered a specific instance or characterization sample of Songguoling, and its relevant physicochemical parameters and detection data can be used to characterize and support the technical and implementation effects of the Songguoling described in this invention.

[0021] In this invention, "ethanol-water solution" refers to a mixed solvent system formed by mixing ethanol and water in a certain volume fraction ratio, which is used to extract and dissolve the target components in Aconitum carmichaelii. The volume fraction of ethanol can be adjusted according to process requirements.

[0022] In this invention, "macroporous adsorption resin" refers to a polymeric adsorption material with a porous structure that can selectively adsorb small organic molecules or alkaloids.

[0023] In this invention, "cation exchange resin" refers to a polymeric resin material containing acidic functional groups that can undergo ion exchange with basic compounds.

[0024] In this invention, "active pharmaceutical ingredient" refers to a high-purity Songguoling product that meets pharmaceutical quality requirements and can be used to prepare pharmaceutical formulations, including solid products obtained by the method of this invention.

[0025] In this invention, "fraction C" and "fraction C3" refer to the elution fractions containing sine-containing compounds obtained by silica gel thin-layer chromatography and screening according to a predetermined Rf value range in the corresponding separation steps; they are only used to distinguish different fractions, but do not imply the composition, content or physicochemical properties of the fractions.

[0026] This invention relates to a method for preparing sinecuronium, the method comprising the following steps: S1. After pulverizing the dried Aconitum carmichaelii, add it to an ethanol-water solution for reflux extraction, filter and concentrate to obtain a concentrated solution; S2. The concentrate was separated by column chromatography using a macroporous adsorption resin column with gradient elution using ethanol-water solution as the eluent. The eluent fractions with Rf values ​​of 0.32 to 0.45 as identified by silica gel thin-layer chromatography were collected and combined to obtain fraction C. S3. After acidification, fraction C is separated by cation exchange resin column chromatography with gradient elution using ammonia ethanol solution. The eluted fractions with Rf values ​​of 0.37 to 0.40 as identified by silica gel thin-layer chromatography are collected and combined to obtain fraction C3. S4. Dissolve the fraction C3 in a recrystallization solvent, allow it to stand and crystallize, then recrystallize it, wash it with the recrystallization solvent and dry it to obtain Songguoling raw material, wherein the recrystallization solvent is one or more of ethanol, ethyl acetate and petroleum ether.

[0027] Through the synergistic combination of the above-mentioned extraction, macroporous adsorption resin enrichment, cation exchange resin purification and recrystallization treatment, the method of the present invention can achieve efficient separation and purification of sine phosphate while ensuring process repeatability and operational stability. It not only significantly improves product purity and yield, but also simplifies the process flow and reduces production costs, and has good prospects for industrial application.

[0028] In some embodiments, in step S1, the volume fraction of ethanol in the ethanol-water solution is 80%–98%, for example, 85%, 90%, or 95%. By reasonably adjusting the volume fraction of ethanol, the co-extraction of highly polar polysaccharides, proteins, and some inorganic impurities can be effectively suppressed while ensuring sufficient dissolution of Songguoling, thereby increasing the relative content of the target component in the extract. Preferably, the volume fraction of ethanol in the ethanol-water solution is 93%–97%; this range achieves a better balance between extraction efficiency and impurity suppression, enabling Songguoling to achieve a higher dissolution rate during reflux extraction, while significantly reducing the co-extraction ratio of pigments, resins, and other accompanying components. This provides a more stable and controllable raw material basis for subsequent macroporous adsorption resin separation and cation exchange purification steps, which is beneficial to the overall process stability and consistent control of product quality.

[0029] In some embodiments, the amount of ethanol-water solution added in step S1 is 8 to 10 times the mass of Aconitum carmichaelii, and the reflux extraction is performed 2 to 4 times, with each extraction lasting 1 to 3 hours. By reasonably controlling the amount of solvent, the number of extractions, and the extraction time per extraction, the sine trioxide in the Aconitum carmichaelii tissue can be fully contacted with the extraction solvent and gradually dissolved, thereby significantly improving the overall extraction efficiency of the target component. Through the above parameter combination control, step S1 can stably obtain an extract rich in sine trioxide with a low impurity burden, providing a reliable material basis for subsequent separation and purification steps.

[0030] In some embodiments, after the reflux extraction in step S1 is completed, the extract is concentrated under reduced pressure at 50-70°C until there is no obvious ethanol odor.

[0031] By concentrating the product under reduced pressure within a suitable temperature range, residual ethanol solvent in the system can be effectively removed while avoiding thermal degradation, structural transformation, or further dissolution of impurities caused by excessively high temperatures. This helps maintain the stability and activity of the target component. When the concentration temperature is below 50℃, the ethanol evaporation efficiency is low, and the concentration time is significantly prolonged, which is not conducive to improving production efficiency. When the concentration temperature is above 70℃, local overheating is likely to occur, causing denaturation or decomposition of some heat-sensitive components, thus affecting the quality of the final product.

[0032] In some embodiments, the macroporous adsorption resin in step S2 is selected from one of D101, AB-8, ADS-8, LK1300S, HPD100 or HPD300.

[0033] In some embodiments, when eluting fraction C in step S2, the volume ratio of ethanol to water in the ethanol-water solution is 37:63 to 43:57; preferably 40:60. By adjusting the polarity of the eluent to the above range, the interaction force between the sorbin adsorbed on the resin and the resin can be moderately weakened, thereby promoting the preferential desorption of the target component under this polarity condition, while most impurities with excessively strong or weak polarity remain on the resin column or are eluted in other elution ranges, achieving selective elution and preliminary enrichment of sorbin.

[0034] In some embodiments, the cation exchange resin in step S3 is selected from Amberlite IR-120, Amberlite FPC22, Diaion SK1B, Diaion HPK25, 732, D001 or 201×7.

[0035] In some embodiments, the acidification treatment in step S3 uses a hydrochloric acid solution or sulfuric acid solution with a mass fraction of 0.1% to 1.0% (e.g., 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%). By rationally selecting the type and concentration of the acidifying reagent, the sine and other basic components contained in fraction C can be fully converted into their corresponding salt forms, thereby significantly enhancing the ion exchange interaction between them and the cation exchange resin. This is beneficial for the stable adsorption of the target components on the resin, creating favorable conditions for subsequent efficient separation. In addition, using hydrochloric acid solution or sulfuric acid solution as the acidifying reagent has the advantages of stable source, low cost, and stable chemical properties, and will not introduce difficult-to-remove organic residues into the system, which is conducive to the smooth progress of subsequent ammonia-ethanol elution and recrystallization purification operations.

[0036] In some embodiments, when eluting fraction C3 in step S3, the volume fraction of the ammonia ethanol solution is 18% to 22%; for example, 19%, 20%, or 21%. By adjusting the concentration of the ammonia ethanol solution to the above range, while ensuring sufficient desorption of sine ethanol, it is possible to effectively suppress the simultaneous elution of impurities with similar structures and physicochemical properties, thereby achieving selective desorption and enrichment of the target component.

[0037] In some embodiments, in step S4, the drying conditions are vacuum drying at 40–60°C for 4–8 hours. By performing drying treatment in a combined low-temperature and reduced-pressure environment, residual solvents on the crystal surface and between the crystal lattice can be effectively removed, while avoiding changes in the crystal form, destruction of the crystal structure, or localized thermal degradation of songguoling caused by excessively high temperatures or excessively rapid drying. This helps maintain the physicochemical stability and quality consistency of the product.

[0038] The embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that these embodiments are only used to illustrate the technical content of the present invention and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the specific experimental conditions in the following embodiments are given priority reference to the guidelines provided in this specification, or may be carried out according to generally accepted experimental manuals or conventional experimental conditions, or other experimental methods known in the art, or according to the conditions recommended by the relevant reagent or instrument manufacturers. In specific embodiments, unless otherwise specified, minor deviations within the weighing accuracy range are allowed for the measurement parameters involving raw material components; reasonable deviations due to instrument detection accuracy or operational accuracy are also allowed for parameters such as temperature and time.

[0039] Example 1 (1) 1.0 kg of dried Aconitum carmichaelii was crushed into coarse powder, and 8 L of 95% ethanol aqueous solution was added. The mixture was refluxed and extracted twice, each time for 2 hours. The extracts were combined, filtered, and concentrated under reduced pressure at 60°C until there was no alcohol taste, yielding about 2.0 L of concentrated solution.

[0040] (2) After dispersing the above concentrate with an appropriate amount of water, gradient elution was performed using HPD300 macroporous adsorption resin column chromatography to obtain fraction C; when identified by silica gel thin-layer chromatography, the Rf value corresponding to fraction C was 0.32-0.45. The mobile phase for macroporous adsorption resin column chromatography was ethanol-water with a volume ratio of (37:63)-(43:57). Preferably, the volume ratio of ethanol-water in the mobile phase was 40:60.

[0041] It should be noted that during gradient elution, ethanol-water ratios of (37:63) to (43:57) can elute fraction C. Using silica gel thin-layer chromatography, fractions with an Rf value of 0.32-0.45 are identified as fraction C. The elution efficiency is highest for an ethanol-water ratio of 40:60. The principle behind the "preferred volume ratio" described in the following gradient elution processes is the same.

[0042] Specifically, the concentrated solution was subjected to HPD300 macroporous adsorption resin column chromatography, eluted with gradients of ethanol-water solution at volume ratios of 0:100, 20:80, 40:60, 60:40, and 95:5, yielding fractions 1-5, 6-12, 13-17, 18-23, and 24-28, for a total of 28 fractions. These fractions were then identified using silica gel thin-layer chromatography, and their Rf values ​​were observed based on the brick-red spots revealed by potassium bismuth iodide reagent. The concentrations were 0.16-0.20 (fractions 1-5), 0.24-0.28 (fractions 6-12), 0.32-0.45 (fractions 13-17), 0.48-0.56 (fractions 18-23), and 0.58-0.66 (fractions 24-28). Similar fractions were combined to obtain fractions A, B, C, D, and E. Based on the brick-red spot appearance of potassium bismuth iodide in thin-layer chromatography, fraction C was selected for further separation. Fractions 13-17 were combined to obtain fraction C.

[0043] (3) Add 0.5% dilute hydrochloric acid solution to fraction C and stir to fully dissolve and disperse it to obtain an acidic aqueous solution. Perform cation exchange resin column chromatography on fraction C using an ammonia-ethanol mobile phase to obtain fraction C3; the Rf value of fraction C3 is 0.37-0.40; the mobile phase for cation exchange resin column chromatography is an ammonia-ethanol solution with a volume ratio of (18:82)-(22:78). Preferably, the volume ratio of the ammonia-ethanol mobile phase is 20:80.

[0044] The specific procedure included: fraction C3 was subjected to chromatography using a 732 cation exchange resin column, eluted with gradients of ammonia-ethanol solutions at ratios of 5:95, 10:90, 20:80, and 30:70, collecting fractions 1-6, 7-12, 13-18, and 19-24 sequentially, for a total of 24 fractions. These fractions were then identified using silica gel thin-layer chromatography. Based on the brick-red spots observed by potassium bismuth iodide reagent, the Rf values ​​were 0.22-0.27 (fractions 1-6), 0.28-0.34 (fractions 7-12), 0.37-0.40 (fractions 13-18), and 0.45-0.52 (fractions 19-24), respectively. Similar fractions were combined to obtain four fractions, C1 to C4. Fractions 13-18 were then combined to obtain fraction C3.

[0045] Select fraction C3 for the next step of separation based on the brick-red spots shown by the potassium bismuth iodide reagent.

[0046] (4) Dissolve fraction C3 in an appropriate amount of petroleum ether-ethyl acetate (2:8), allow it to stand to crystallize, wash away impurities on the crystal surface with an appropriate amount of ethanol, and dry the obtained wet crystals under vacuum at 50°C for 6 hours to obtain approximately 5.25 g of white needle-like crystals. The determination was performed by high performance liquid chromatography (column: C18, 4.6 × 250 mm, 5 μm; mobile phase: methanol). The purity of Songguoling was ≥99.3% (using a 0.1% trifluoroacetic acid aqueous solution with gradient elution; detection wavelength: 210 nm). Based on dried Aconitum carmichaelii raw material, the extraction and purification yield of Songguoling was 0.53%.

[0047] Example 2 (1) Crush 1.0 kg of dried Aconitum carmichaelii into coarse powder, add 10 L of 95% ethanol aqueous solution, reflux extract twice, 2.5 hours each time, combine the extracts, filter, concentrate under reduced pressure at 65℃ until there is no alcohol taste, and obtain about 2.5 L of concentrated solution.

[0048] (2) After dispersing the above concentrate with an appropriate amount of water, gradient elution was performed using D101 macroporous adsorption resin column chromatography to obtain fraction C; when identified by silica gel thin-layer chromatography, the Rf value corresponding to fraction C was 0.32. 0.45. The mobile phase for macroporous adsorption resin column chromatography was ethanol with a volume ratio of (37:63)-(43:57). Water. Preferably, ethanol is used as the mobile phase. The volume ratio of water is 40:60.

[0049] Specifically, this includes chromatography of the concentrated solution using a D101 macroporous adsorption resin column, employing ethanol at volume ratios of 0:100, 20:80, 40:60, 60:40, and 95:5, respectively. Gradient elution with aqueous solution yielded fractions 1-5, 6-12, 13-18, 19-24, and 25-30, for a total of 30 fractions. These fractions were then identified using silica gel thin-layer chromatography. Based on the brick-red spots observed by potassium bismuth iodide reagent, the Rf values ​​were 0.15-0.22 (fractions 1-5), 0.24-0.30 (fractions 6-12), 0.32-0.45 (fractions 13-18), 0.48-0.58 (fractions 19-24), and 0.60-0.68 (fractions 25-30), respectively. Similar fractions were combined to obtain fractions A, B, C, D, and E. Fraction C was selected for further separation based on the brick-red spots observed by potassium bismuth iodide in the thin-layer chromatography. Fraction 13... After merging 18, we get the flow C.

[0050] (3) Add 0.5% dilute hydrochloric acid solution to fraction C and stir to fully dissolve and disperse it, obtaining an acid-water solution. Perform cation exchange resin column chromatography on fraction C using ammonia-ethanol solution as the mobile phase to obtain fraction C3; the Rf value of fraction C3 is 0.37. 0.40; the mobile phase for cation exchange resin column chromatography is an ammonia-ethanol solution with a volume ratio of (18:82) to (22:78). Preferably, the volume ratio of the ammonia-ethanol solution in the mobile phase is 20:80.

[0051] The specific procedure included: fraction C was subjected to Diaion SK1B cation exchange resin column chromatography, eluted with gradients of ammonia-ethanol solutions at ratios of 5:95, 10:90, 20:80, and 30:70, collecting fractions 1-6, 7-13, 14-20, and 21-27 sequentially, for a total of 27 fractions. These fractions were then identified using silica gel thin-layer chromatography. Based on the brick-red spots observed by potassium bismuth iodide reagent, the Rf values ​​were 0.20-0.26 (fractions 1-6) and 0.28, respectively. 0.35 (streams 7-13), 0.37 0.40 (flow branches 14-20), 0.44-0.54 (flow branches 21-27), similar flow branches are merged to obtain four flow branches C1~C4. Among them, flow branch 14 After merging 20, we get flow segment C3.

[0052] Select fraction C3 for the next step of separation based on the brick-red spots shown by the potassium bismuth iodide reagent.

[0053] (4) Dissolve fraction C3 in an appropriate amount of ethyl acetate Dissolve the aconite root in ethanol (3:2), allow it to stand to crystallize, wash away impurities on the crystal surface with an appropriate amount of ethanol, and dry the resulting wet crystals under vacuum at 50℃ for 6 hours to obtain approximately 4.80 g of white needle-like crystals. The purity of Songguoling was determined by high-performance liquid chromatography (column: C18, 4.6 × 250 mm, 5 μm; mobile phase: methanol-0.1% trifluoroacetic acid aqueous solution, gradient elution; detection wavelength: 210 nm), ≥99.4%. Based on dried aconite root raw material, the extraction and purification yield of Songguoling was 0.48%.

[0054] Example 3 (1) 1.0 kg of dried Aconitum carmichaelii was crushed into coarse powder, and 9 L of 95% ethanol aqueous solution was added. The mixture was refluxed and extracted 3 times, each time for 2 hours. The extracts were combined, filtered, and concentrated under reduced pressure at 60°C until there was no alcohol taste, yielding a concentrate of about 2.2 L.

[0055] (2) After dispersing the above concentrate with an appropriate amount of water, gradient elution was performed using AB-8 macroporous adsorption resin column chromatography to obtain fraction C; when identified by silica gel thin-layer chromatography, the Rf value corresponding to fraction C was 0.32. 0.45. The mobile phase for macroporous adsorption resin column chromatography was ethanol with a volume ratio of (37:63)-(43:57). Water. Preferably, ethanol is used as the mobile phase. The volume ratio of water is 40:60.

[0056] Specifically, this includes the concentrate being processed by AB. 8-porosity adsorption resin column chromatography was performed using ethanol at volume ratios of 0:100, 20:80, 40:60, 60:40, and 95:5, respectively. Gradient elution with aqueous solution yielded fractions 1-6, 7-14, 15-21, 22-28, and 29-34, totaling 34 fractions. These fractions were then identified using silica gel thin-layer chromatography. Based on the brick-red spots observed by potassium bismuth iodide reagent, the Rf values ​​were 0.14-0.21 (fractions 1-6), 0.23-0.29 (fractions 7-14), 0.32-0.45 (fractions 15-21), 0.47-0.57 (fractions 22-28), and 0.59-0.67 (fractions 29-34), respectively. Similar fractions were combined to obtain fractions A, B, C, D, and E. Fraction C was selected for further separation based on the brick-red spots observed by potassium bismuth iodide in the thin-layer chromatography. Fraction 15... After merging 21, we get the flow component C.

[0057] (3) Add 0.5% dilute hydrochloric acid solution to fraction C and stir to fully dissolve and disperse it to obtain an acidic aqueous solution. Perform cation exchange resin column chromatography on fraction C using an ammonia-ethanol mobile phase to obtain fraction C3; the Rf value of fraction C3 is 0.37-0.40; the mobile phase for cation exchange resin column chromatography is an ammonia-ethanol solution with a volume ratio of (18:82)-(22:78). Preferably, the volume ratio of the ammonia-ethanol mobile phase is 20:80.

[0058] The specific procedure includes: Fraction C is subjected to D001 type cation exchange resin column chromatography, eluted with gradients of ammonia-ethanol solutions at ratios of 5:95, 10:90, 20:80, and 30:70, collecting fractions 1-7, 8-15, 16-22, and 23-30 sequentially, for a total of 30 fractions. These fractions are then identified using silica gel thin-layer chromatography. Based on the brick-red spots observed by bismuth potassium iodide reagent, the Rf values ​​are 0.21-0.27 (fractions 1-7), 0.29-0.36 (fractions 8-15), 0.37-0.40 (fractions 16-22), and 0.45-0.55 (fractions 23-30), respectively. Similar fractions are combined to obtain four fractions C1-C4. Fractions 16-22 are then combined to obtain fraction C3.

[0059] Select fraction C3 for the next step of separation based on the brick-red spots shown by the potassium bismuth iodide reagent.

[0060] (4) Dissolve fraction C3 in an appropriate amount of pure ethanol, allow it to stand to crystallize, wash away impurities on the crystal surface with an appropriate amount of ethanol, and dry the obtained wet crystals under vacuum at 50°C for 6 hours to obtain approximately 5.10 g of white needle-like crystals. The crystals were determined by high performance liquid chromatography (column: C18, 4.6 × 250 mm, 5 μm; mobile phase: methanol). The purity of Songguoling was ≥99.2% (using a 0.1% trifluoroacetic acid aqueous solution with gradient elution; detection wavelength: 210 nm). Based on dried Aconitum carmichaelii raw material, the extraction and purification yield of Songguoling was 0.51%.

[0061] Comparative Example 1: Except for omitting the macroporous adsorption resin column chromatography step (i.e., S2), the remaining operations were the same as in Example 1. The results of this comparative example show that without macroporous adsorption resin enrichment, direct subsequent processing resulted in a final purity of only 68.2% for Songguoling, and the content of impurities such as pigments and polysaccharides in the product was significantly higher. This demonstrates that macroporous adsorption resin plays a crucial role in removing water-soluble impurities and achieving preliminary enrichment of Songguoling.

[0062] Comparative Example 2: Except for omitting the cation exchange resin column chromatography step (i.e., S3), the remaining operations were the same as in Example 1. The results of this comparative example show that skipping cation exchange resin purification and relying solely on macroporous resin enrichment followed by direct crystallization resulted in a final product purity of only 72.5%. Furthermore, thin-layer chromatography revealed the presence of multiple structurally similar alkaloid spots, indicating that the cation exchange step is indispensable for removing similarly basic impurities and improving chemical purity.

[0063] Comparative Example 3: Except that the ethanol concentration of the elution fraction C in S2 was changed to 20% ethanol Except for the aqueous solution, the remaining operations were the same as in Example 1. This comparative result indicates that when eluting with a lower concentration of ethanol, sinecuronium was not fully desorbed, resulting in a final yield of only 0.13%, and a significant amount of impurities were eluted, demonstrating that a 40% ethanol concentration is crucial for the selective elution of sinecuronium.

[0064] Comparative Example 4: Except that the concentration of the ammonia-ethanol solution in the elution fraction C3 of S3 was changed to 10% ammonia Except for the ethanol solution, the remaining operations were the same as in Example 1. This comparative result shows that when using a lower concentration of ammonia-ethanol for elution, the target component was not completely eluted, resulting in a final yield of only 0.12%, and impurities were not thoroughly removed. This demonstrates that a 20% ammonia-ethanol concentration is the optimal condition for achieving efficient desorption and purification.

[0065] Comparative Example 5: Except for changing the extraction solvent in S1 to a 70% aqueous ethanol solution, the other operations were the same as in Example 1. The results of this comparative example show that when using a lower concentration of ethanol for extraction, the extraction efficiency of sine alkaloids decreased, with a final yield of 0.28%, lower than that in Example 1, proving that high concentrations of ethanol (≥85%) are more advantageous for the extraction of fat-soluble diterpenoid alkaloids.

[0066] Comparative Example 6: Except for changing the extraction solvent in step S1 to anhydrous methanol, the remaining operations were the same as in Example 1. The results of this comparative example show that although the methanol extraction efficiency was high, significant solvent compatibility issues arose during the subsequent resin treatment, leading to a decrease in resin adsorption capacity and a final product purity of only 91.8%. This demonstrates that the ethanol-water solution system is more suitable in terms of overall process compatibility.

[0067] Comparative Example 7: Except for replacing the recrystallization solvent in S4 with a single petroleum ether, the rest of the operation was the same as in Example 1. The results of this comparative example show that when using a non-polar solvent for recrystallization, the crystallization rate of songguoling is low, with a crystal yield of only 35.0% of the theoretical value, and the crystal form is not uniform, proving that the mixed solvent system has more advantages in terms of solubility regulation and crystallization induction.

[0068] Comparative Example 8: Except that the cation exchange resin used in S3 was changed to a weakly acidic cation exchange resin (such as type D151), the rest of the operation was the same as in Example 1. The results of this comparative example show that the weakly acidic resin has a weak adsorption capacity for sine phosphate, the separation degree between the target component and impurities is poor during the elution process, and the purity of the final product is only 86.7%, proving that a strongly acidic cation exchange resin (such as type 732) plays a key role in this process.

[0069] The above embodiments are merely examples of several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent.

[0070] It should be noted that those skilled in the art can make various modifications and improvements without departing from the inventive concept, and these all fall within the scope of protection of this invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

[0071] Table 1 Comparative Experiment Results

[0072] As shown in Table 1, compared with the preparation of Songguoling by patent CN112694441A, the process of this invention is simpler, with less sample loss, saving solvent and time (no need for alumina column chromatography, etc.). The yield and purity of the Songguoling active pharmaceutical ingredient are both higher, making it more suitable for industrial production. Furthermore, systematic verification through comparative examples 1-8 shows that the macroporous adsorption resin enrichment step, the strong acid cation exchange resin purification step, the specific concentration ethanol / ammoniaethanol elution system, and the optimized recrystallization process are all key technical steps to achieve high yield and high purity Songguoling. Substitution of any step or deviation from the conditions will lead to a significant decrease in product yield, purity, or process efficiency, fully demonstrating the necessity and overall synergy of the process design of this invention. The process of this invention has significant advantages for industrial production.

[0073] The physical properties and detection data of compound 1 obtained in Example 1 are as follows: White amorphous powder. High-resolution mass spectrometry (HR-ESI-MS) m / z 357.2298 [M + H] + The calculated value is 357.2298. Combined with NMR data, its molecular formula is determined to be C. 22 H 31 NO3 has an unsaturation degree of 7. The NMR data are as follows: 1 H NMR (400 MHz, DMSO- d 6) d H 5.11 (1H, s, H-17a), 5.07 (1H, s, H-17b), 4.29 (1H, t, J = 2.4 Hz, H-15), 3.84 (1H, s, H-20), 3.81 (1H, d, J = 3.5 Hz, H-1), 3.34 (1H, d, J = 6.0 Hz, H-14a), 3.16 (1H, overlapped, H-13), 3.16 (1H, overlapped, H-21a), 3.16 (1H, overlapped, H-19a), 2.96 (1H, d, J = 3.8 Hz, H-21b), 2.87 (1H, overlapped, H-6a), 2.87 (1H, overlapped, H-19b), 2.52 (1H, overlapped, H-7), 2.15 (1H, d, J = 7.3 Hz, H-14b),2.07 (1H, s, H-9), 2.04 (1H,t, J = 5.4 Hz, H-11a), 1.87 (1H, overlapped, H-2a), 1.76 (1H, tdd, J = 14.7,10.5, 4.6 Hz, H-2b), 1.54 (1H, d, J = 4.1 Hz, H-3a), 1.52 (1H, overlapped, H-6b), 1.52 (1H, overlapped, H-11b), 1.40 (1H, d, J= 7.8 Hz, H-5), 1.27 (1H, overlapped, H-3b), 1.27 (3H, overlapped, H-22), 0.81 (3H, s, H-18); 13 C NMR (100 MHz, DMSO- d 6) d C 208.1 (C-12), 150.0 (C-16), 110.8 (C-17),75.0 (C-15), 66.8 (C-1), 64.1 (C-20), 56.1 (C-19), 53.4 (C-13), 53.2 (C-21),51.9 (C-10), 47.6 (C-8), 46.4 (C-5), 43.0 (C-7), 37.5 (C-14), 36.1 (C-9), 35.5 (C-3), 35.0 (C-4), 30.7 (C-11), 30.2 (C-2), 24.8 (C-18), 21.4 (C-6), 9.8(C-22). The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A process for the preparation of songorin, characterized in that, The method includes the following steps: S1. After pulverizing the dried Aconitum carmichaelii, add it to an ethanol-water solution for reflux extraction, filter and concentrate to obtain a concentrated solution; S2. The concentrate was separated by column chromatography using a macroporous adsorption resin column with gradient elution using ethanol-water solution as the eluent. The eluent fractions with Rf values ​​of 0.32 to 0.45 as identified by silica gel thin-layer chromatography were collected and combined to obtain fraction C. S3. After acidification, fraction C is separated by cation exchange resin column chromatography with gradient elution using ammonia ethanol solution. The eluted fractions with Rf values ​​of 0.37 to 0.40 as identified by silica gel thin-layer chromatography are collected and combined to obtain fraction C3. S4. Dissolve the fraction C3 in a recrystallization solvent, allow it to stand and crystallize, then recrystallize it, wash it with the recrystallization solvent and dry it to obtain Songguoling raw material, wherein the recrystallization solvent is one or more of ethanol, ethyl acetate and petroleum ether.

2. The production method according to claim 1, characterized by, In step S1, the volume fraction of ethanol in the ethanol-water solution is 80% to 98%; preferably 93% to 97%.

3. The production method according to claim 2, characterized by, The amount of ethanol-water solution added in step S1 is 8 to 10 times the mass of Aconitum carmichaelii, and the reflux extraction is performed 2 to 4 times, with each extraction lasting 1 to 3 hours.

4. The process according to any one of claims 1 to 3, characterized in that, After the reflux extraction in step S1 is completed, the extract is concentrated under reduced pressure at 50-70°C until there is no obvious ethanol odor.

5. The preparation method according to claim 1, characterized in that, The macroporous adsorption resin mentioned in step S2 is selected from one of D101, AB-8, ADS-8, LK1300S, HPD100 or HPD300.

6. The preparation method according to claim 1 or 5, characterized in that, In step S2, when eluting fraction C, the volume ratio of ethanol to water in the ethanol-water solution is 37:63 to 43:

57.

7. The preparation method according to claim 1, characterized in that, The cation exchange resin mentioned in step S3 is selected from one of Amberlite IR-120, Amberlite FPC22, Diaion SK1B, Diaion HPK25, 732, D001 or 201×7.

8. The preparation method according to claim 1 or 7, characterized in that, The acidification treatment in step S3 uses a hydrochloric acid solution or sulfuric acid solution with a mass fraction of 0.1% to 1.0%.

9. The preparation method according to claim 1 or 7, characterized in that, In step S3, when eluting fraction C3, the volume fraction of the ammonia-ethanol solution is 18% to 22%.

10. The preparation method according to claim 1, characterized in that, In step S4, the drying conditions are vacuum drying at 40–60°C for 4–8 hours.

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