Preparation method of neorin

Abstract

This invention relates to the field of pharmaceutical technology, and in particular to a method for preparing neoryl. The method includes the following steps: S1, extracting Aconitum carmichaelii with an ethanol-water solution to obtain an extract and concentrating it to obtain a concentrate; S2, dispersing the concentrate in water and performing column chromatography with macroporous adsorption resin, using an ethanol-water solution as the eluent for gradient elution to obtain fraction B; S3, acidifying fraction B and then separating it with cation exchange resin column chromatography, using an ammonia-ethanol solution for isocratic elution, and combining and collecting the eluted fractions to obtain fraction B2; S4, further separating fraction B2 with silica gel column chromatography, using a petroleum ether-ethyl acetate solution as the eluent for gradient elution to obtain fraction E8; S5, dissolving fraction E8 in a recrystallization solvent, allowing it to stand and crystallize, recrystallizing it, washing it with the recrystallization solvent, and drying it to obtain neoryl raw material.

Description

Technical Field

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

[0002] Neoline is C 19 Neoline, a type of diterpenoid alkaloid, is one of the main active components of the traditional Chinese medicine Aconitum carmichaelii, possessing pharmacological activities such as improving cognitive impairment, analgesia, and anti-inflammation. Existing methods for preparing Neoline include methanol extraction, chloroform extraction, alumina column chromatography, and silica gel column chromatography, which suffer from drawbacks such as complex processes, high production costs, low yields and purity, and unsuitability for industrial-scale production. To evaluate the drug potential of Neoline and develop new drugs, a simple, practical, stable, and controllable method for enriching Neoline active pharmaceutical ingredients is urgently needed. Summary of the Invention

[0003] This invention covers the following technical solutions: This invention relates to a method for preparing nystatin, characterized in that the method comprises the following steps: S1. Extract Aconitum carmichaelii with ethanol-water solution to obtain the extract and concentrate it to obtain the concentrate. S2. After dispersing the concentrate with water, the concentrate is subjected to column chromatography using macroporous adsorption resin column chromatography with gradient elution using ethanol-water solution as eluent and silica gel thin-layer chromatography detection as the collection criterion. The eluent fractions with Rf values ​​of 0.24-0.38 identified by silica gel thin-layer chromatography are combined and collected to obtain fraction B. S3. After acidification treatment of the fraction B, separation is performed by cation exchange resin column chromatography, isocratic elution is performed with ammonia ethanol solution, and silica gel thin-layer chromatography is used as the collection criterion. The eluted fractions with Rf values ​​of 0.26-0.35 identified by silica gel thin-layer chromatography are combined and collected to obtain fraction B2. S4. Fiber B2 was further separated by silica gel column chromatography with gradient elution using petroleum ether-ethyl acetate solution as the eluent. Fiber thin-layer chromatography (TLC) was used as the collection criterion. The eluted fractions with Rf values ​​of 0.28-0.30 identified by TLC were combined to obtain fraction E8. S5. Dissolve fraction E8 in an organic solvent, allow it to stand to crystallize, recrystallize it, wash it with the recrystallization solvent, and dry it to obtain nylon raw material, wherein the recrystallization solvent is one or more of ethanol, ethyl acetate, and petroleum ether.

[0004] This invention constructs a stable and controllable process for preparing Neoline active pharmaceutical ingredient (API) by combining ethanol-water extraction, macroporous adsorption resin enrichment, cation exchange resin selective purification, silica gel column fine separation, and recrystallization. This method uses the Rf value of silica gel thin-layer chromatography as the separation criterion, effectively improving the accuracy and repeatability of target component separation and significantly reducing impurity interference. Compared with existing technologies, this invention has a simplified process flow, high solvent safety, avoids the use of highly toxic organic solvents, and is suitable for continuous and large-scale production. Through optimized control of key process parameters, this invention can stably obtain high-purity, high-yield Neoline API, with a purity of over 98%, and a yield significantly superior to existing methods, demonstrating good industrial application value. Attached Figure Description

[0005] 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.

[0006] Figure 1 The images show the high-performance liquid chromatograms of Neoline active pharmaceutical ingredient obtained in Examples 1-3 of this invention.

[0007] 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: differential refractive index detector; column temperature: 30℃. Detailed Implementation

[0008] 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.

[0009] 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 traditional Chinese medicine chemistry and natural product chemistry, medicinal chemistry, separation and analytical chemistry, chromatography, pharmaceutical engineering and pharmaceutical formulation processes, and drug quality control and analytical testing techniques, 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.

[0010] 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.

[0011] 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.

[0012] 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.

[0013] 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.

[0014] As used in this invention, unless otherwise stated, the singular forms of the articles “a,” “an,” and “the” include plural referents.

[0015] In this invention, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity of 2 or more.

[0016] 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.

[0017] 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.

[0018] All references to this invention are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the inventive purpose and / or technical solution of this invention, the referenced documents are incorporated herein by reference in their entirety and for all purposes. When references are made in this invention, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the referenced technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this invention. It should be understood that when the cited content conflicts with the description in this invention, this invention shall prevail or modifications shall be made adaptively according to the description in this invention.

[0019] In this invention, "Neoline" refers to Aconitum carmichaelii, a plant derived from the Ranunculaceae family and the Aconitum genus. Aconite of Carichalcus A C19 type diterpenoid alkaloid compound contained in Aconitum carmichaelii (Debx.), a processed product of the root of Aconitum carmichaelii., has the CAS number 466-26-2 and the molecular formula C. 24 H 39 NO6, molecular weight 437.57; white powder, soluble in methanol, ethanol, DMSO, and acetone, slightly soluble in water.

[0020] 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.

[0021] In this invention, "macroporous adsorption resin column chromatography" refers to a separation and purification method that uses macroporous adsorption resin to selectively adsorb and elute target components, and achieves fractional separation of different components by adjusting the composition of the eluent.

[0022] In this invention, "cation exchange resin column chromatography" refers to a separation method that uses cation exchange resin to selectively adsorb and desorb compounds containing basic functional groups, thereby enriching and purifying the target alkaloid components through acidification and alkaline elution.

[0023] In this invention, "silica gel column chromatography" refers to a chromatographic method that uses silica gel as the stationary phase and an organic solvent system as the mobile phase to separate and purify compounds. By adjusting the polarity of the eluent, fine separation of structurally similar components can be achieved.

[0024] In this invention, "silica gel thin-layer chromatography identification" refers to a method for detecting and analyzing eluted fractions using silica gel thin-layer plates, and for identifying and determining target components by comparing the Rf values ​​of the spots.

[0025] In this invention, "fraction B", "fraction B2" and "fraction E8" refer to the Neoline-containing eluents identified by silica gel thin-layer chromatography and screened 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 Neoline, the method comprising the following steps: S1. Extract Aconitum carmichaelii with ethanol-water solution to obtain the extract and concentrate it to obtain the concentrate. S2. After dispersing the concentrate with water, the concentrate is subjected to column chromatography using macroporous adsorption resin column chromatography with gradient elution using ethanol-water solution as eluent and silica gel thin-layer chromatography detection as the collection criterion. The eluent fractions with Rf values ​​of 0.24-0.38 identified by silica gel thin-layer chromatography are combined and collected to obtain fraction B. S3. After acidification treatment of the fraction B, separation is performed by cation exchange resin column chromatography, isocratic elution is performed with ammonia ethanol solution, and silica gel thin-layer chromatography is used as the collection criterion. The eluted fractions with Rf values ​​of 0.26-0.35 identified by silica gel thin-layer chromatography are combined and collected to obtain fraction B2. S4. Fiber B2 was further separated by silica gel column chromatography with gradient elution using petroleum ether-ethyl acetate solution as the eluent. Fiber thin-layer chromatography (TLC) was used as the collection criterion. The eluted fractions with Rf values ​​of 0.28-0.30 identified by TLC were combined to obtain fraction E8. S5. Dissolve fraction E8 in an organic solvent, allow it to stand to crystallize, recrystallize it, wash it with the recrystallization solvent, and dry it to obtain Neoline raw material, wherein the recrystallization solvent is one or more of ethanol, ethyl acetate, and petroleum ether.

[0027] Through the coordinated efforts of the above steps, Neoline is purified step by step during extraction, enrichment, separation and purification, which not only improves separation efficiency and process stability, but also ensures the purity and yield of the final product, making it suitable for industrial-scale production.

[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%; preferably 93%–97%. By limiting the volume fraction of ethanol within the above range, it is possible to effectively avoid the introduction of excessive water-soluble impurities due to excessively low ethanol content, and also to avoid the problem of insufficient dissolution of some polar alkaloids due to excessively high ethanol content. This ensures the stability and consistency of the extract composition, provides a good raw material basis for subsequent macroporous adsorption resin separation and ion exchange purification steps, and helps to improve the repeatability, yield, and purity of the final product of the overall process.

[0029] In some embodiments, in step S1, the amount of ethanol-water solution added is 8 to 10 times, for example, 9 times, the mass of Aconitum carmichaelii.

[0030] In some implementations, the reflux extraction is performed 2 to 4 times, with each reflux extraction lasting 1 to 3 hours.

[0031] By synergistically controlling the amount of solvent, the number of reflux cycles, and the extraction time per extraction, the problem of insufficient dissolution of the target component due to insufficient solvent or too short extraction time can be avoided. At the same time, it can also prevent excessive solvent or over-extraction from introducing a large number of impurities. Thus, while ensuring the extraction efficiency of Neoline, the purity and stability of the extract are also taken into account, laying a good material foundation for subsequent separation and purification steps, and helping to improve the overall yield and reproducibility of the process.

[0032] In some embodiments, in step S1, the concentration is carried out under reduced pressure until the relative density of the resulting concentrate at 50°C is 1.10–1.15. By controlling the concentration endpoint, it is possible to avoid insufficient concentration leading to low content of active ingredients and excessive load on subsequent resin processing. It is also possible to prevent excessive concentration from causing adverse effects such as component elution, excessively high viscosity, or degradation of heat-sensitive components. This ensures that the material entering the macroporous adsorption resin column chromatography step is in a stable and consistent state, which is beneficial to improving the stability and repeatability of the separation process, and further enhancing the subsequent purification effect and the quality of the final product.

[0033] In some embodiments, in step S2, the macroporous adsorption resin is one of D101, AB-8, ADS-8, LK1300S, HPD100 or HPD300.

[0034] In some embodiments, in step S2, the volume ratio of ethanol to water in the ethanol-water solution used to elute fraction B is (27:73) to (33:67); for example, 30:70. By limiting this volume ratio range, the adsorption and desorption processes of Neoline on the macroporous adsorption resin can be kept in good equilibrium, thereby achieving effective separation of the target component from pigments, polysaccharides, and other impurities. This avoids insufficient elution of the target component due to an excessively low ethanol ratio, and also avoids the introduction of too many non-target impurities into fraction B due to an excessively high ethanol ratio. Reasonable control of the above elution ratio is beneficial for stably obtaining target fractions with Rf values ​​in the range of 0.24 to 0.38, improving the separation efficiency and repeatability of the initial enrichment stage, and providing a high-purity, compositionally stable raw material basis for subsequent ion exchange and fine separation steps.

[0035] In some embodiments, in step S3, the acid is either a hydrochloric acid solution or a 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%). This avoids the problem of incomplete salination of the target component and reduced adsorption efficiency due to insufficient acidity, while also preventing excessively high acidity from adversely affecting the structural stability of the compound and subsequent separation processes. This ensures the selectivity and controllability of the ion exchange separation process, which is beneficial for the effective separation of Neoline from coexisting impurities and further improves the purification effect and process stability.

[0036] In some embodiments, in step S3, the cation exchange resin is one of Amberlite IR-120, Amberlite FPC22, Diaion SK1B, Diaion HPK25, 732, D001, or 201×7.

[0037] In some embodiments, in step S4, the volume ratio of petroleum ether to ethyl acetate in the eluted fraction E8 is (45:55) to (55:45); for example, 50:50. By reasonably limiting this volume ratio range, Neoline can form a stable distribution equilibrium between the silica gel stationary phase and the mobile phase, thereby achieving effective separation between it and structurally similar diterpenoid alkaloids and other accompanying impurities. This avoids the problem of excessively high petroleum ether ratio leading to excessively long retention time and difficult elution of the target component, and also avoids the problem of excessively high ethyl acetate ratio causing co-elution of impurities and the target component.

[0038] In some embodiments, the recrystallization operation in step S5 is repeated 2 to 3 times. By reasonably controlling the number of recrystallizations, trace structurally similar impurities and residual solvent components that are difficult to completely separate by column chromatography can be effectively removed, further improving the purity and stability of Neoline active pharmaceutical ingredient.

[0039] 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.

[0040] Example 1 S1. Crush 500 g of dried Aconitum carmichaelii into coarse powder, add 5 L of 95% ethanol and reflux extract three times for 3 h each time. Combine the extracts, filter, concentrate under reduced pressure, and concentrate to a concentrate with a relative density of about 1.10-1.15 (measured at 50℃) (about 1.2 L). S2. 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 B. When identified by silica gel thin-layer chromatography, the Rf value corresponding to fraction B was 0.24-0.38. The mobile phase for macroporous adsorption resin column chromatography was ethanol-water with a volume ratio of (27:73)-(33:67). Preferably, the volume ratio of ethanol-water in the mobile phase was 30:70.

[0041] It should be noted that during gradient elution, ethanol-water ratios of (27:73) to (33:67) can elute fraction B. Detection using silica gel thin-layer chromatography reveals fractions with an Rf value of 0.24-0.38 as fraction B. The elution efficiency is highest for an ethanol-water ratio of 30:70. 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, using gradient elution with ethanol-water solutions at volume ratios of 0:100, 30:70, 60:40, and 95:5, yielding fractions 1-5, 6-10, 11-14, and 15-18, for a total of 18 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 0.16-0.20 (fractions 1-5), 0.24-0.38 (fractions 6-10), 0.42-0.50 (fractions 11-14), and 0.55-0.64 (fractions 15-18), respectively. Similar fractions were combined to obtain fractions A, B, C, and D. Based on the brick-red spots observed by potassium bismuth iodide in the thin-layer chromatography, fraction B was selected for further separation. Fractions 21-30 were combined to obtain fraction B.

[0043] S3. Add 0.5% dilute hydrochloric acid solution to fraction B and stir to fully dissolve and disperse it, obtaining an acid-water solution. Perform cation exchange resin column chromatography on fraction B using an ammonia-ethanol mobile phase to obtain fraction B2; the Rf value of fraction B2 is 0.26-0.35; the mobile phase for cation exchange resin column chromatography is 10% ammonia-ethanol solution (ammonia:ethanol = 10:90, v / v).

[0044] The specific procedure included: fraction B2 was subjected to chromatography using a 732 type cation exchange resin column, eluted isocratically with 10% ammonia-ethanol solution (ammonia:ethanol = 10:90, v / v), and fractions 1-6, 7-14, 15-20, and 21-29 were collected sequentially, for a total of 29 fractions. These fractions were then identified using silica gel thin-layer chromatography. Based on the brick-red spots observed by bismuth potassium iodide reagent, the Rf values ​​were 0.18-0.23 (fractions 1-6), 0.26-0.35 (fractions 7-14), 0.40-0.47 (fractions 15-20), and 0.50-0.55 (fractions 21-29), respectively. Similar fractions were combined to obtain four fractions, B1 to B4. Fractions 7-14 were then combined to obtain fraction B2.

[0045] Select fraction B2 for the next separation step based on the brick-red spots shown by the potassium bismuth iodide reagent.

[0046] S4. B2 was subjected to gradient elution using petroleum ether-ethyl acetate silica gel column chromatography to obtain fraction E; when identified by silica gel thin-layer chromatography, the Rf value corresponding to fraction E was 0.28-0.30. The mobile phase for silica gel column chromatography was petroleum ether-ethyl acetate with a volume ratio of (45:55)-(55:45). Preferably, the volume ratio of petroleum ether-ethyl acetate was 50:50.

[0047] Specifically, fraction B2 was subjected to silica gel column chromatography, eluted with a petroleum ether-ethyl acetate gradient at volume ratios of 100:0, 95:5, 92:8, 90:10, 85:15, 75:25, 65:35, 50:50, and 0:100, yielding fractions 1-10, 11-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, and 81-90, for a total of 90 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 observed to be 0.80-0.82 (fractions 1-10). Fractions 11-20 (0.76-0.78), 21-30 (0.71-0.73), 31-40 (0.64-0.68), 41-50 (0.58-0.62), 61-70 (0.50-0.55), 81-90 (0.42-0.48), 71-80 (0.28-0.30), and 81-90 (0.25-0.27) were combined to obtain fractions E1-E9. Fraction E8 was selected for further separation based on the brick-red spot appearance of potassium bismuth iodide in thin-layer chromatography. Fractions 71-80 were combined to obtain fraction E8.

[0048] S5. Dissolve fraction E8 in an appropriate amount of ethyl acetate-ethanol (2:8), let it stand to allow crystals to precipitate, wash away impurities on the crystal surface with an appropriate amount of ethanol, recrystallize 3 times, and the purity of Neoline reaches 99.2%. After vacuum drying, approximately 3.25g of Neoline raw material is obtained, with a yield of 0.65%.

[0049] Example 2 S1. Crush 500 g of dried Aconitum carmichaelii into coarse powder, add 4.5 L of 95% ethanol and reflux extract twice, 2 h each time. Combine the extracts, filter, concentrate under reduced pressure, and concentrate to a concentrate with a relative density of about 1.10–1.15 (measured at 50℃) (about 1 L). S2. 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 B. When identified by silica gel thin-layer chromatography, the Rf value corresponding to fraction B was 0.24-0.38. The mobile phase for macroporous adsorption resin column chromatography was ethanol-water with a volume ratio of (27:73)-(33:67). Preferably, the mobile phase was ethanol-water with a volume ratio of 30:70.

[0050] Specifically, the concentrated solution was subjected to D101 macroporous adsorption resin column chromatography, with gradient elution using ethanol-water solutions at volume ratios of 0:100, 30:70, 60:40, and 95:5, yielding fractions 1-6, 7-14, 15-20, and 21-26, for a total of 26 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 0.16-0.22 (fractions 1-6), 0.24-0.38 (fractions 7-14), 0.40-0.48 (fractions 15-20), and 0.55-0.62 (fractions 21-26), respectively. Similar fractions were combined to obtain fractions A, B, C, and D. Based on the brick-red spots observed by potassium bismuth iodide in the thin-layer chromatography, fraction B was selected for further separation. Fractions 7-14 were combined to obtain fraction B.

[0051] S3. Add 0.4% dilute hydrochloric acid solution to fraction B and stir to fully dissolve and disperse it, obtaining an acid-water solution. Perform cation exchange resin column chromatography on fraction B using an ammonia-ethanol mobile phase to obtain fraction B2; the Rf value of fraction B2 is 0.27-0.33; the mobile phase for cation exchange resin column chromatography is 20% ammonia-ethanol solution (ammonia:ethanol = 20:80, v / v).

[0052] The specific procedure included: passing the acidic aqueous solution through a 732-type cation exchange resin column, eluting isocratically with 20% ammonia-ethanol solution (ammonia:ethanol = 20:80, v / v), and collecting fractions 1-7, 8-16, 17-24, and 25-32 sequentially, for a total of 32 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 0.18-0.24 (fractions 1-7), 0.27-0.33 (fractions 8-16), 0.38-0.45 (fractions 17-24), and 0.48-0.54 (fractions 25-32), respectively. Similar fractions were combined to obtain four fractions, B1 to B4. Fractions 8-16 were combined to obtain fraction B2.

[0053] Select fraction B2 for the next separation step based on the brick-red spots shown by the potassium bismuth iodide reagent.

[0054] S4. B2 was subjected to gradient elution using petroleum ether-ethyl acetate silica gel column chromatography to obtain fraction E; when identified by silica gel thin-layer chromatography, the Rf value corresponding to fraction E was 0.28-0.30. The mobile phase for silica gel column chromatography was petroleum ether-ethyl acetate with a volume ratio of (45:55)-(55:45). Preferably, the volume ratio of petroleum ether-ethyl acetate was 50:50.

[0055] Specifically, fraction B2 was subjected to silica gel column chromatography, eluted with a gradient of petroleum ether-ethyl acetate at volume ratios of 100:0, 95:5, 90:10, 85:15, 75:25, 65:35, 48:52, and 0:100, yielding fractions 1-10, 11-20, 21-30, 31-40, 41-50, 51-60, 61-70, and 71-80, for a total of 80 fractions. These fractions were then identified using silica gel thin-layer chromatography, and the Rf values ​​(fractions) were observed based on the brick-red spots revealed by potassium bismuth iodide reagent, ranging from 0.80 to 0.82. Fractions 1-10, 0.75-0.78 (fractions 11-20), 0.70-0.73 (fractions 21-30), 0.64-0.68 (fractions 31-40), 0.58-0.62 (fractions 41-50), 0.50-0.55 (fractions 51-60), 0.42-0.48 (fractions 61-70), and 0.28-0.30 (fractions 71-80) were combined to obtain fractions E1-E8. Fraction E8 was selected for further separation based on the brick-red spot appearance of potassium bismuth iodide in thin-layer chromatography. Fractions 71-80 were combined to obtain fraction E8. S5. Dissolve fraction E8 in an appropriate amount of ethyl acetate-ethanol (3:7), let it stand to allow crystals to precipitate, wash away impurities on the crystal surface with an appropriate amount of ethanol, recrystallize twice, and the purity of Neoline reaches 98.2%. After vacuum drying, approximately 2.60 g of Neoline raw material is obtained, with a yield of 0.52%.

[0056] Example 3 S1. Crush 500 g of dried Aconitum carmichaelii into coarse powder, add 5 L of 95% ethanol and reflux extract three times for 2.5 h each time. Combine the extracts, filter, concentrate under reduced pressure, and concentrate to a concentrate with a relative density of about 1.10-1.15 (measured at 50℃) (about 1.2 L). S2. After dispersing the above concentrate with an appropriate amount of water, gradient elution is performed using AB-8 macroporous adsorption resin column chromatography to obtain fraction B. When identified by silica gel thin-layer chromatography, the Rf value corresponding to fraction B is 0.26-0.36. The mobile phase for macroporous adsorption resin column chromatography is ethanol-water with a volume ratio of (27:73)-(33:67). Preferably, the mobile phase for macroporous adsorption resin column chromatography is ethanol-water with a volume ratio of 30:70.

[0057] Specifically, the concentrated solution was subjected to AB-8 macroporous adsorption resin column chromatography, using gradient elution with ethanol-water solutions at volume ratios of 0:100, 30:70, 60:40, and 95:5 to obtain fractions 1-5, 6-12, 13-18, and 19-24, 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 0.16-0.20 (fractions 1-5), 0.26-0.36 (fractions 6-12), 0.40-0.48 (fractions 13-18), and 0.55-0.63 (fractions 19-24), respectively. Similar fractions were combined to obtain fractions A, B, C, and D. Based on the brick-red spots observed by potassium bismuth iodide in the thin-layer chromatography, fraction B was selected for further separation. Fractions 6-12 were combined to obtain fraction B.

[0058] S3. Add 0.5% dilute hydrochloric acid solution to fraction B and stir to fully dissolve and disperse it, obtaining an acid-water solution. Perform cation exchange resin column chromatography on fraction B using an ammonia-ethanol mobile phase to obtain fraction B2; the Rf value of fraction B2 is 0.28-0.32; the mobile phase for cation exchange resin column chromatography is 5% ammonia-ethanol solution (ammonia:ethanol = 5:95, v / v).

[0059] The specific procedure included: passing the acidic aqueous solution through a 732-type cation exchange resin column, eluting isocratically with 5% ammonia-ethanol solution (ammonia:ethanol = 5:95, v / v), and collecting fractions 1-6, 7-15, 16-22, and 23-30 sequentially, 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 0.18-0.23 (fractions 1-6), 0.28-0.32 (fractions 7-15), 0.38-0.45 (fractions 16-22), and 0.48-0.54 (fractions 23-30), respectively. Similar fractions were combined to obtain four fractions, B1 to B4. Fractions 7-15 were combined to obtain fraction B2.

[0060] Select fraction B2 for the next separation step based on the brick-red spots shown by the potassium bismuth iodide reagent.

[0061] S4. B2 was subjected to gradient elution using petroleum ether-ethyl acetate silica gel column chromatography to obtain fraction E; when identified by silica gel thin-layer chromatography, the Rf value corresponding to fraction E was 0.28-0.30. The mobile phase for silica gel column chromatography was petroleum ether-ethyl acetate with a volume ratio of (45:55)-(55:45). Preferably, the volume ratio of petroleum ether-ethyl acetate was 50:50.

[0062] Specifically, fraction B2 was subjected to silica gel column chromatography, eluted with a petroleum ether-ethyl acetate gradient at volume ratios of 100:0, 95:5, 92:8, 90:10, 85:15, 75:25, 65:35, 50:50, and 0:100, yielding fractions 1-10, 11-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, and 81-90, for a total of 90 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 observed to be 0.80-0.82 (fractions 1-10). Fractions 11-20 (0.76-0.78), 21-30 (0.71-0.73), 31-40 (0.64-0.68), 41-50 (0.58-0.62), 61-70 (0.50-0.55), 81-90 (0.42-0.48), 71-80 (0.28-0.30), and 81-90 (0.25-0.27) were combined to obtain fractions E1-E9. Fraction E8 was selected for further separation based on the brick-red spot appearance of potassium bismuth iodide in thin-layer chromatography. Fractions 71-80 were combined to obtain fraction E8.

[0063] S5. Dissolve fraction E8 in an appropriate amount of ethanol, let it stand to allow crystals to precipitate, wash away impurities on the crystal surface with an appropriate amount of ethanol, recrystallize 3 times, and the purity of Neoline reaches 99.0%. After vacuum drying, approximately 3.00g of Neoline raw material is obtained, with a yield of 0.60%.

[0064] To further compare the effects of the preparation method of this invention with the prior art, we compared Neoline obtained from the existing patent document "An alcohol amine type diterpenoid alkaloid with analgesic effect, preparation method and application" (CN 114507183 A) with the method in the patent application of this invention using the method disclosed in that patent (extraction-alumina column chromatography-silica gel column chromatography). The specific results are shown in Table 1 below.

[0065] In addition, in order to further compare the effect of the specific preparation method selected in this invention, since the Neoline yield of Example 1 is as high as 0.65% and the purity is as high as 99.2%, the following comparative example is set up to compare with Example 1 of this application. The specific results are shown in Table 1 below.

[0066] Comparative Example 1: Except for not adding dilute hydrochloric acid solution to fraction B for acidification in S3, but directly dissolving in water and loading the sample onto a 732 cation exchange resin column, the other steps and parameters were the same as in Example 1; the Neoline purity obtained in this comparative example was only 85.3%, indicating that the lack of an acidification step will seriously affect the selective adsorption and impurity separation effect of the target component on the ion exchange resin, and the purity is significantly lower than that of the process of the present invention.

[0067] Comparative Example 2: Except for not using silica gel column chromatography, the fraction B2 obtained in S3 was directly recrystallized in S5, and the other steps and parameters were the same as in Example 1; the Neoline purity obtained in this comparative example was only 85.7%, indicating that the lack of silica gel column chromatography could not effectively separate structurally similar diterpenoid alkaloids, and high-purity products could not be obtained, and the effect was worse than that of this application.

[0068] Comparative Example 3: Except for not using macroporous adsorption resin for purification, the concentrated solution obtained in S1 was directly treated with cation exchange resin in S3. The other steps and parameters were the same as in Example 1. The Neoline purity obtained in this comparative example was only 83.5%, indicating that the lack of macroporous adsorption resin enrichment and impurity removal resulted in a large number of pigments, polysaccharides and other impurities entering the subsequent steps, which seriously affected the final purity and was worse than the effect of this application.

[0069] Comparative Example 4: Except for not performing the recrystallization operation in S5, i.e., directly evaporating the fraction E8 obtained in S4 under reduced pressure and then drying, the other steps and parameters are the same as in Example 1; the Neoline purity obtained in this comparative example is only 92.7%, indicating that the lack of a recrystallization step cannot remove trace co-crystallized impurities, the product purity cannot meet the standard of active pharmaceutical ingredient, and the effect is worse than that of this application.

[0070] Comparative Example 5: Except for replacing 95% ethanol with acetone in S1, the other steps and parameters were the same as in Example 1; the final Neoline yield obtained in this comparative example was only 0.38%, indicating that the non-preferred extraction solvent was not conducive to the dissolution of the target component, and the yield was significantly lower than that of the process of the present invention.

[0071] Comparative Example 6: Except for the extraction solvent of S1 being 70% ethanol, the rest is the same as in Example 1; this comparative example shows that the yield of Neoline obtained by using a solvent with a ratio lower than the preferred extraction solvent is only 0.40%, which is worse than the effect of this application.

[0072] Comparative Example 7: Except for adjusting the ethanol-water ratio of eluent B in the S2 macroporous adsorption resin column chromatography from 30:70 to 20:80, the other steps and parameters were the same as in Example 1; the final Neoline yield obtained in this comparative example was only 0.43%, which was worse than the effect of this application.

[0073] Comparative Example 8: Except for adjusting the ratio of petroleum ether to ethyl acetate in the eluent E8 from 50:50 to 40:60 in the S4 silica gel column elution, the other steps and parameters were the same as in Example 1. The final Neoline yield obtained in this comparative example was only 0.45%, which was worse than the effect of this application.

[0074] 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.

[0075] 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.

[0076] Table 1 Comparative Experiment Results

[0077] As can be seen from Table 1, compared with Neoline prepared by patent CN114507183A, the process of this invention is simpler, with less sample loss, saving solvent and time (no need for alumina column chromatography, etc.), and the yield and purity of Neoline active pharmaceutical ingredient are higher, making it more suitable for industrial production. In addition, compared with comparative examples 1-8, the specific selection of solvent extraction, macroporous adsorption resin enrichment, cation exchange resin specific purification and silica gel column fine separation and recrystallization steps, as well as the specific selection of process conditions in the preparation process, have an important impact on the yield and purity of the final product.

[0078] The physical properties and detection data of compound 1 obtained in Example 1 are as follows: White amorphous powder, readily soluble in methanol. High-resolution mass spectrometry (HR-ESI-MS) m / z 438.2846 ([M+H] + The calculated value is 438.2839. Combined with NMR data, its molecular formula is determined to be C. 24 H 39 NO6 has an unsaturation degree of 6, and its NMR data are as follows: 1 H NMR (400MHz, CDCl3) δ : H 4.21 (1H, t, J = 5.1 Hz, H-14), 4.16 (1H, d, J = 6.6 Hz, H-6), 3.66 (1H, s, H-1), 3.65 (1H, d,J = 8.1 Hz, H-18a), 3.37 (1H,m, H-16), 3.33 (3H, s, 18-OCH3), 3.33 (3H, s, 6-OCH3), 3.32 (3H, s, 16-OCH3),3.26 (1H, d, J = 8.1 Hz, H-18b), 2.71 (1H, d, J = 11.0 Hz, H-19a), 2.69 (1H,overlapped, H-17), 2.53 (2H, overlapped, H-20), 2.39 (1H, overlapped, H-15a),2.33 (1H, overlapped, H-19b), 2.28 (1H, overlapped, H-13), 2.18 (1H,overlapped, H-9), 2.16 (1H, overlapped, H-5), 2.06 (1H, overlapped, H-15b),2.02 (1H, overlapped, H-2a), 1.99 (1H, s, H-7), 1.90 (1H, overlapped, H-12a),1.83 (1H, overlapped, H-10), 1.71 (1H, dd, J = 5.1, 14.5 Hz, H-3a), 1.63 (1H,dd, J = 4.9, 14.3 Hz, H-12b), 1.56 (1H, overlapped, H-2b), 1.49 (1H,overlapped, H-3b), 1.12 (3H, t, J = 7.1 Hz, H-21); 13 C NMR (100 MHz, CDCl3) δ:C 83.2 (C-6), 81.9 (C-16), 80.4 (C-18), 76.2(C-14), 74.3 (C-8), 72.3 (C-1), 64.0 (C-17), 59.3 (18-OCH3), 58.0 (6-OCH3),57.1 (C-19), 56.5 (16-OCH3), 52.3 (C-7), 49.6 (C-11), 48.5 (C-9), 48.4 (C-20), 45.0 (C-5), 44.3 (C-10), 43.0 (C-15), 40.4 (C-13), 38.3 (C-4), 30.0 (C-3), 29.5 (C-12), 29.3 (C-2), 13.2 (C-21).

[0079] 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 method for preparing neoryl, characterized in that, The method includes the following steps: S1. Extract Aconitum carmichaelii with ethanol-water solution to obtain the extract and concentrate it to obtain the concentrate. S2. After dispersing the concentrate with water, the concentrate is subjected to column chromatography using macroporous adsorption resin column chromatography with gradient elution using ethanol-water solution as eluent and silica gel thin-layer chromatography detection as the collection criterion. The eluent fractions with Rf values ​​of 0.24-0.38 identified by silica gel thin-layer chromatography are combined and collected to obtain fraction B. S3. After acidification treatment of the fraction B, separation is performed by cation exchange resin column chromatography, isocratic elution is performed with ammonia ethanol solution, and silica gel thin-layer chromatography is used as the collection criterion. The eluted fractions with Rf values ​​of 0.26-0.35 identified by silica gel thin-layer chromatography are combined and collected to obtain fraction B2. S4. Fiber B2 was further separated by silica gel column chromatography with gradient elution using petroleum ether-ethyl acetate solution as the eluent. Fiber thin-layer chromatography (TLC) was used as the collection criterion. The eluted fractions with Rf values ​​of 0.28-0.30 identified by TLC were combined to obtain fraction E8. S5. Dissolve fraction E8 in an organic solvent, allow it to stand to crystallize, recrystallize it, wash it with the recrystallization solvent, and dry it to obtain nylon raw material, wherein the recrystallization solvent is one or more of ethanol, ethyl acetate, and petroleum ether.

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

3. The preparation method according to claim 2, characterized in that, In step S1, the amount of ethanol-water solution added is 8 to 10 times the mass of Aconitum carmichaelii; preferably, the reflux extraction is performed 2 to 4 times, and the reflux extraction time is 1 to 3 hours each time.

4. The preparation method according to any one of claims 1 to 3, characterized in that, In step S1, the solution is concentrated under reduced pressure until the relative density of the resulting concentrate is 1.10 to 1.15 at 50°C.

5. The preparation method according to claim 1, characterized in that, In step S2, the macroporous adsorption resin is 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, the volume ratio of ethanol to water in the ethanol-water solution during the washing out of fraction B is (27:73) to (33:67).

7. The preparation method according to claim 1, characterized in that, In step S3, the acid is either a hydrochloric acid solution or a sulfuric acid solution with a mass fraction of 0.1% to 1.0%.

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

9. The preparation method according to claim 1, characterized in that, In step S4, the volume ratio of petroleum ether to ethyl acetate in the eluting fraction E8 is (45:55) to (55:45).

10. The preparation method according to claim 1, characterized in that, In step S5, the recrystallization operation is repeated 2 to 3 times.

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