Method for preparing reference substance of antofine

Abstract

Provided in the present disclosure is a method for preparing a reference substance of antofine, which method comprises the steps of: soaking the Chinese herbal medicine Cynanchum komarovii Al. Iljinski in ethanol, extracting and filtering same, performing acid adjustment, extracting same with ethyl acetate, performing alkali adjustment, extracting same with chloroform, performing silica gel column chromatography, and then performing C18-column chromatography and precipitation, so as to obtain antofine with a purity of 99% or more. The extraction and separation efficiency is effectively improved by means of specific pre-treatments, and a target reference substance with high HPLC purity can be obtained with high throughput. In addition, the method for preparing a solid reference substance of antofine obtained in the present disclosure has very good industrial production applicability.

Description

Preparation method of reference substance of 7-demethoxysilane Technical Field

[0001] The present disclosure relates to a method for preparing 7-demethoxysilane (Antofine), and in particular to a method for preparing a solid reference substance of 7-demethoxysilane suitable for industrialization. Background Art

[0002] The 7-demethoxysilane (also known as antofine, CAS No. 32671-82-2) studied in this paper is a natural phenanthenic indolizidine alkaloid, also known as tylophorine B, with excellent biological activity. Wang et al. reported that in traditional Chinese medicine, antofine has analgesic, antitussive, antiasthmatic, expectorant, antidiarrheal, antipyretic, immune, and cardiovascular therapeutic effects. Tripathi reported antofine's insecticidal activity in 1990, and it also has antifungal and antibacterial activity. Antofine has potential cytotoxic and anticancer activities. In 2002, Staerk's group demonstrated in vitro inhibition of the drug-sensitive KB-3-1 and multidrug-resistant KB-V1 human cervical cancer cell lines. In 2007, Fu et al. reported its inhibitory activity against the human colorectal cancer cell lines HCT116, human colorectal adenocarcinoma HT-1080, and lung cancer A549.

[0003] 7-Demethoxysilane

[0004] Due to the significant cytotoxicity and biochemical properties of antofine, this class of compounds has attracted considerable attention as potential drug leads, resulting in a significant demand for antofine raw material compounds. However, currently, only total synthetic production methods are available, and there are no inexpensive and easily implemented solutions for producing antofine raw material compounds. Studies have shown that 7-demethoxysilanthelline has the highest content among the total alkaloids of Cynanchum serratum, suggesting a potential plant source. However, the extraction and separation of plant-derived 7-demethoxysilanthelline is difficult, with no readily available processes. Its total content is low, and it is difficult to crystallize from a mixture. Therefore, existing methods for extracting and preparing 7-demethoxysilanthelline are very limited, and there are currently no efficient, industrially available methods for extracting and preparing 7-demethoxysilanthelline. As an important characteristic component, there is an urgent need to develop a high-throughput, high-purity 7-demethoxysilanthelline preparation method suitable for large-scale production.

[0005] Non-patent document 1: Staerk, D., Lykkeberg, AK, Christensen, J., et al.. In vitro cytotoxic activity of

[0006] phenanthroindolizidine alkaloids from Cynanchum vincetoxicum and Tylophora tanakae against drug-sensitive and multidrug-resistant cancer cells. J Nat Prod, 2002, 65(9):1299-1302

[0007] Non-patent document 2: Wang, QG, Xie, L, Zhai, J. Tylophorine B benzene solvate. Acta Crystallographica Section C - Crystal Structure Communications, 2000, 56: 197-198

[0008] Non-patent document 3: Tripathi, AK, Singh, D..Persistency of Tylophorine as an Insect Antifeedant against Spilosoma-Obliqua Walker. Phytother Res, 1990, 4: 144-147

[0009] Non-patent literature 4: Fu, Y, Lee, SK, Min, HY, et al..Synthesis and structure-activity studies of antomineanalogues as potential anticancer agents. Bioorganic&Medicinal Chemistry Letters, 2007, 17(1):97-100

[0010] Non-patent literature 5: Research on chemical components and biological activities of Cynanchum rotundum. Northwest Agriculture and Forestry University, 2004.

[0011] Summary of the Invention

[0012] The present disclosure aims to provide a high-throughput method for preparing a 7-demethoxysilanine reference substance that can provide high-purity 7-demethoxysilanine and is suitable for large-scale production. In the present disclosure, 7-demethoxysilanine is sometimes also referred to as the separation target. The present disclosure significantly improves the separation and purification of 7-demethoxysilanine, introducing a unique and easy-to-use combined column adsorption and solvation method, making the entire extraction method suitable for extracting 7-demethoxysilanine from the herbal material of Cynanchum rotundum. Further exploration has found a cost-effective extraction and separation method, realizing an industrially scalable method for purifying the target compound, which has greatly improved the amount and purity of the compound, thereby completing the present disclosure.

[0013] The present disclosure uses a simple and efficient operating process to separate and obtain 7-demethoxysilanine with high purity. The preparation and separation method disclosed in the present disclosure comprises the following steps in sequence:

[0014] Normal phase silica gel chromatography purification step: The crude extract solution containing 7-demethoxysilane was loaded onto a normal phase chromatography column and eluted with ethyl acetate for 8 to 15 column volumes, then eluted with an alcohol solvent containing 0.5 to 1.5 wt% ammonia. The target fraction was collected, and the eluate was concentrated and dried to obtain a crude extract I of 7-demethoxysilane.

[0015] Solvent treatment purification step: The crude extract I of 7-demethoxysilane obtained in the normal phase silica gel chromatography purification step is added to methanol / ethanol and sonicated, the precipitate is filtered and dried to obtain a crude extract II of 7-demethoxysilane;

[0016] Reverse-phase chromatography purification process: The crude extract II of 7-demethoxysilantheline was loaded onto a reverse-phase chromatography column and eluted with a mixed solvent of acetonitrile and water or a mixed solvent of methanol and water. The target product was collected and concentrated under reduced pressure to dryness to obtain the semi-finished product of 7-demethoxysilantheline (Antofine). The eluate was collected for TLC or HPLC detection, concentrated under reduced pressure, and dried to obtain a 7-demethoxysilantheline reference substance with a purity of 99%.

[0017] In a preferred embodiment of the present disclosure, the preparation method of the total extract solution of the herbal material of Cynanchum wilfordii is as follows: the dried Cynanchum wilfordii herb is crushed, 5 to 15 times of an alcohol solvent is added, and the mixture is soaked at room temperature for 2 to 5 days. The soaking liquid is filtered and concentrated under reduced pressure to a thick extract, and an acid aqueous solution is added to dissolve and filter. The acid aqueous layer is extracted with an organic solvent, and the acid aqueous phase is adjusted to a pH of 9 to 10 with concentrated ammonia water, extracted with an organic solvent, and the organic layers are combined and concentrated under reduced pressure to obtain a crude extract solution containing 7-demethoxysilane.

[0018] In a preferred embodiment of the present disclosure, the alcohol-based aqueous solution in the method for preparing the total extract solution of Cynanchum wilfordii herbal materials is a 60% to 90% methanol aqueous solution or a 60% to 90% ethanol aqueous solution.

[0019] In a preferred embodiment of the present disclosure, in the normal phase chromatography purification process, the filler of the normal phase chromatography column is silica gel with a particle size of 100 to 300 mesh, and elution is performed using an alcohol solvent containing 0.9 to 1.1 wt % ammonia.

[0020] In a preferred embodiment of the present disclosure, in the reverse phase chromatography purification process, elution is performed at a pressure of 0.1 to 0.3 MPa, using a 48% v / v to 70% v / v acetonitrile-water mixed solvent or a 70% v / v to 95% v / v methanol-water mixed solvent, and a 40 to 60 μm octadecylsilane bonded silica gel filler.

[0021] In a preferred embodiment of the present disclosure, in the acid-base treatment step, the acid aqueous solution is 0.06 to 0.10 M hydrochloric acid.

[0022] In a preferred embodiment of the present disclosure, in the acid-base treatment step, the organic solvent used for extraction is an ester solvent or a halogenated hydrocarbon solvent.

[0023] In a preferred embodiment of the present disclosure, the ester solvent is selected from ethyl acetate, butyl acetate, and methyl acetate.

[0024] The halogenated hydrocarbon solvent is a combination of one or more selected from dichloromethane, chloroform, carbon tetrachloride, 1,1-dichloroethane, 1,2-dichloroethane, 1,1,1-trichloroethane, 1,1,2-trichloroethane, 1,1,1,2-tetrachloroethane, 1,1,2,2-tetrachloroethane, pentachloroethane, 1,1-dichloroethylene, 1,2-dichloroethylene, trichloroethylene and tetrachloroethylene.

[0025] Another aspect of the present disclosure further provides a solid reference substance of 7-demethoxysilanine with a purity of 99% or more, which is prepared by the preparation method of the present disclosure.

[0026] The present disclosure has the following features:

[0027] The disclosed method is simple and efficient to operate, capable of process scale-up to isolate and obtain large quantities of 7-demethoxyanthophylline monomer. The method for preparing and isolating high-purity 7-demethoxyanthophylline from the Chinese herbal medicine Cynanchum rotundum utilizes the unique chemical properties of 7-demethoxyanthophylline to achieve specificity and simplification, meeting the requirements of Chinese herbal chemical reference materials and enabling its use as a reference substance. The method features a simple process, convenient preparation and separation operations, low equipment costs, ease of commissioning, large single-batch processing capacity, a stable and reliable process, good reproducibility, and high yield, enabling large-scale industrial production. This method is also the first patented method for the preparation and isolation of high-purity 7-demethoxyanthophylline (Antofine), filling a gap in the industry. Through production practice, the disclosed method has isolated over 7 g of 7-demethoxyanthophylline from 50 kg of Cynanchum rotundum herbal material, with a purity exceeding 99% and a yield of approximately 0.014%. This method can be used as a reference substance for testing, marking a first in the literature. According to demand, it can also be expanded to the 100-gram level, so there are significant improvements in preparation methods and throughput. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG1 is a typical HPLC spectrum of Example 1 to Example 4;

[0029] Figure 2 is the HPLC spectrum of Comparative Example 1;

[0030] Figure 3 is the HPLC spectrum of Comparative Example 2;

[0031] Figure 4 is the HPLC spectrum of Comparative Example 3;

[0032] FIG5 is the HPLC spectrum of Comparative Example 4. DETAILED DESCRIPTION

[0033] Specific embodiments of the present disclosure are described below.

[0034] Unless otherwise specified, the purity and content percentages of the target substance in this disclosure are expressed as weight ratios. Unless otherwise specified, the percentages of liquid-to-liquid ratios in this disclosure, such as the ratio of formic acid in acetonitrile-water solution, are expressed as volume fractions.

[0035] Existing methods for isolating the target substance 7-demethoxysilantherine disclosed herein primarily utilize a combination of normal-phase and reverse-phase chromatography. Taking advantage of the different polarities of the target substance and impurities, a solvent gradient elution is used to gradually and slowly separate the target substance. Generally, depending on the polarity of the anthocyanin, for example, a concentrated extract of a crushed herbal material of Cynanchum rotundum can be loaded onto a reverse-phase chromatography column, and gradient elution is performed to obtain an eluate containing the target substance. This eluate is then collected, concentrated, and freeze-dried to obtain a solid target substance. However, the inventors of the present disclosure have discovered that other secondary metabolites in Cynanchum rotundum significantly affect the purification of 7-demethoxysilantherine. Direct reverse-phase chromatography separation is difficult to achieve because many of the compounds contained in the raw material of Cynanchum rotundum herbal material have very similar polarities to the target substance. The efficiency of reverse-phase chromatography separation is low, and the purity of the obtained 7-demethoxysilantherine remains unchanged. Although the idea of ​​continuously improving the purity by using a reversed-phase chromatographic column with higher pressure and better separation can gradually improve the purity of 7-demethoxysilane, it results in a low yield and expensive solvents in the separation process.

[0036] To achieve industrial mass production and to address yield and separation efficiency issues, the inventors of the present disclosure, through in-depth research, determined that the preparation and separation method of the present disclosure includes the following steps: a normal phase silica gel chromatography purification step: loading a crude extract solution containing 7-demethoxysilane onto a normal phase chromatography column, eluting with ethyl acetate for 8 to 15 column volumes, and then eluting with an alcohol solvent containing 0.5 to 1.5 wt% ammonia, collecting the target fraction, and concentrating and drying the eluate to obtain a crude extract I of 7-demethoxysilane;

[0037] Solvent treatment purification step: The crude extract I of 7-demethoxysilane obtained in the normal phase silica gel chromatography purification step is added to methanol / ethanol and sonicated, the precipitate is filtered and dried to obtain a crude extract II of 7-demethoxysilane;

[0038] Reverse phase chromatography purification step: The crude extract II of 7-demethoxysilantheline was loaded onto a reverse phase chromatography column and eluted with a mixed solvent of acetonitrile and water or a mixed solvent of methanol and water. The target product was collected and concentrated under reduced pressure to dryness to obtain the semi-finished product of 7-demethoxysilantheline (Antofine). The eluate was collected according to TLC or HPLC detection, concentrated under reduced pressure, and dried to obtain a 7-demethoxysilantheline reference product with a purity of 99%.

[0039] The purpose of the normal phase chromatography purification process is generally to distinguish impurities with large polarity differences from the target. The filler of the normal phase chromatography column used in the normal phase chromatography purification process can be a well-known normal phase filler. The so-called normal phase filler is a filler in which the polarity of the stationary phase is greater than that of the mobile phase. If the mobile phase is an organic solvent, commonly used fillers are silica gel (specifically SiO2, silicon dioxide), Al2O3, polar bonded phase fillers, etc. However, the present disclosure requires the use of a silica gel chromatography column. The normal phase chromatography purification process of the present disclosure is not quite the same as this. It does not use a gradient elution method for fine separation, but still has a good target enrichment effect. The key to the present disclosure is that it was found that the normal phase silica gel chromatography column is particularly beneficial for removing non-anthophan components in Herba Cynanchifoliae, and the reversibility of the adsorption and desorption of the anthophone components on the normal phase silica gel chromatography column is good. Based on these characteristics, ethyl acetate can be used for elution after sample loading. This elution primarily removes non-antofine components. Generally, 8 to 15 column volumes are sufficient to remove most components other than 7-demethoxysilane (Antofine). These components often have polarities very similar to 7-demethoxysilane and are difficult to separate in reversed-phase chromatography. Therefore, while the normal-phase silica gel chromatography purification process disclosed herein is simple to operate, it plays a crucial role in efficiently separating the target component. It should be noted that the methanol used for the final elution needs to be more alkaline. Theoretically, adding both organic and inorganic bases can help increase the desorption rate. However, research has found that adding aqueous ammonia can simply and effectively desorb the target compound from the normal-phase silica gel column, while also reducing impurities. The silica gel particle size is not particularly limited. From the perspectives of efficiency and readily available fillers, in the preferred preparation method disclosed herein, the filler for the normal-phase column in the first normal-phase chromatography column purification step is silica gel with a particle size of 100 to 300 mesh. In order to better obtain separation efficiency, and from the perspective of separation degree and adsorption loss, silica gel is preferably used, and 100-300 mesh silica gel is further preferably used. In a preferred embodiment of the present disclosure, in the normal phase chromatography purification process, the filler of the normal phase chromatography column is silica gel with a particle size of 100-300 mesh, and elution is performed with an alcohol solvent containing 0.9-1.1wt% ammonia. For the chromatographic purification process, there is no particular restriction on the method of loading the crude raw material in the present disclosure. It can be dissolved in a good solvent and added dropwise, or it can be loaded by mixing the sample with the filler. In order to compress the sample volume to a lower level, it is preferred to load the sample by mixing the filler.

[0040] The starting raw material of the separation method disclosed in the present invention can be any form of crude extract of Cynanchum wilfordii herbal medicine, which can be purchased raw materials or self-extracted raw materials. Since the target product of the present invention is an alkaloid, if the crude extract is subjected to acid-base treatment, the target product will be further enriched, which is beneficial to increase the extraction efficiency. If the acid-base treatment process in the present invention is added, the separation effect can be greatly improved, but it is not necessary. Therefore, the preferred method for preparing the starting raw material in the present invention is to crush the dried Cynanchum wilfordii herbal medicine, add 5 to 15 times of alcohol solvent, soak at room temperature for 2 to 5 days, filter the soaking liquid and concentrate it under reduced pressure to a thick extract, dissolve it with acid water solution, filter it, extract the acid water layer with an organic solvent, adjust the pH of the acid water phase to 9 to 10 with concentrated ammonia water, extract it with an organic solvent, combine the organic layers, and concentrate under reduced pressure. In a preferred embodiment of the present invention, in the acid-base treatment process, the organic solvent used for extraction is an ester solvent or a halogenated hydrocarbon solvent. In a preferred embodiment of the present disclosure, the alcoholic aqueous solution in the method for preparing the total extract solution of the herbal material of Cynanchum wilfordii is a 60% to 90% methanol aqueous solution or a 60% to 90% ethanol aqueous solution. In a preferred embodiment of the present disclosure, the alcoholic aqueous solution is a 60% to 90% methanol aqueous solution or a 60% to 90% ethanol aqueous solution. From the perspective of process safety, a 60% to 90% ethanol aqueous solution is preferably used. In a preferred embodiment of the present disclosure, the ester solvent is selected from ethyl acetate, butyl acetate, and methyl acetate, and the halogenated hydrocarbon solvent is selected from one or more of dichloromethane, chloroform, carbon tetrachloride, 1,1-dichloroethane, 1,2-dichloroethane, 1,1,1-trichloroethane, 1,1,2-trichloroethane, 1,1,1,2-tetrachloroethane, 1,1,2,2-tetrachloroethane, pentachloroethane, 1,1-dichloroethylene, 1,2-dichloroethylene, trichloroethylene, and tetrachloroethylene. In a preferred embodiment of the present disclosure, in the acid-base treatment step, the acid aqueous solution is 0.06-0.10 M hydrochloric acid; the base can be an organic base or an inorganic base, and sodium bicarbonate is preferably used.

[0041] The solvent treatment purification step in this disclosure is also crucial. The inventors discovered that the solubility of substances eluted from a normal-phase column in methanol and / or ethanol varies significantly. Furthermore, the target compound is not soluble in methanol and / or ethanol, while a large number of impurities are soluble in methanol and / or ethanol. This solvent treatment purification step can significantly improve the purity of 7-demethoxysilane. More importantly, this step can remove most of the impurities that are difficult to separate from 7-demethoxysilane during reverse-phase chromatography purification, reducing the difficulty of reverse-phase chromatography purification.

[0042] The order of the steps in this disclosure also significantly impacts the efficiency of extraction and separation. While solvent purification, such as recrystallization, is typically performed last, the present inventors have discovered that performing solvent purification at specific steps not only significantly improves purity but also increases recovery. The sequential arrangement of the steps is also a key feature of this disclosure.

[0043] The crude extract II of 7-demethoxysilanine is loaded onto a reverse-phase chromatography column and eluted with a mixed solvent of acetonitrile and water or a mixed solvent of methanol and water to obtain a very high-purity 7-demethoxysilanine reference substance. In the present disclosure, the reverse-phase chromatography purification step is used to separate other components with different polarity from the target compound.

[0044] In a preferred embodiment of the present disclosure, the reverse phase chromatography purification process can be carried out as a medium-low pressure reverse phase chromatography purification process, and there is no need to carry out high-pressure reverse phase chromatography purification. Therefore, common preparative liquid phases, flash columns, etc. can be used. The so-called medium-low pressure and high pressure are not strictly conceptual distinctions. In the present disclosure, the medium-low pressure reverse phase chromatography purification process can be considered to be used to separate other components that have different polarities from the target. The use of an elution pressure of 0.05 to 1 MPa can achieve a balance between resolution and speed. In the medium-low pressure column separation process, this pressure is particularly preferred because it can balance the separation effect and separation speed. The reverse phase filler of the reverse phase chromatography column used in the chromatographic column preparation process of the present disclosure can be a well-known non-polar silica gel with a bonded functional group being an alkane (for example: C18 (ODS), C8, C4, etc.). A C18 (ODS) silica gel column, i.e., octadecylsilane bonded silica gel, is preferred. For so-called medium- and low-pressure reversed-phase chromatography purification processes, a packing material of 40 to 60 μm is preferably used. A reasonable particle size is beneficial for maintaining appropriate column pressure and resolution, and octadecylsilane bonded silica gel is readily available on the market. From the perspectives of efficiency and readily available packing materials, in the preferred preparation method disclosed herein, the packing material for the reversed-phase chromatography column is octadecylsilane bonded silica gel with a particle size of 40 to 60 μm. In the reversed-phase chromatography purification process, preferably, elution is performed at a pressure of 0.1 to 0.3 MPa, using a 48% v / v to 70% v / v acetonitrile-water mixed solvent or a 70% v / v to 95% v / v methanol-water mixed solvent. More preferably, a 48% v / v to 70% v / v acetonitrile-water mixed solvent is used, and octadecylsilane bonded silica gel with a particle size of 40 to 60 μm is selected. When an eluent with excessively high polarity is used for elution, the resulting product purity is low.

[0045] Example

[0046] The following examples further illustrate typical extraction methods disclosed herein. The following experimental schemes are merely examples and are not intended to limit the present disclosure. Those skilled in the art may make any changes based on their understanding of the principles of the embodiments and the spirit of the invention.

[0047] Example 1

[0048] 50 kg of Cynanchum wilfordii herbal material was added with 10 times the amount of 90% ethanol and soaked at room temperature for 3 days. The soaking liquid was filtered and concentrated under reduced pressure to a thick extract, which was then dissolved in 5% aqueous acid and filtered. The acidic aqueous layer was extracted with ethyl acetate, the acidic aqueous phase was adjusted to pH 9-10 with aqueous ammonia, extracted with chloroform, the chloroform layers were combined, concentrated under reduced pressure, and mixed with silica gel. Silica gel column chromatography was performed, eluting with ethyl acetate for 10 column volumes and then with methanol (containing 1% aqueous ammonia) to collect the target fraction. The solvent was removed from the target fraction, sonicated with methanol, filtered, precipitated, and dried. The sample was dissolved in a small amount of a mixed solvent of dichloromethane and methanol and loaded onto a C18 column for high-efficiency preparation. The eluent was eluted with acetonitrile (48% v / v to 70% v / v). The target was collected, concentrated, and dried under reduced pressure to obtain 7.084 g of pure 7-demethoxysilane (Antofine) with an HPLC purity greater than 99% and a yield of 35.42%.

[0049] Comparative Example 1

[0050] 50 kg of Cynanchum wilfordii herbal material was added with 10 times the amount of 90% ethanol and soaked at room temperature for 3 days. The soaking liquid was filtered and concentrated under reduced pressure to a thick extract, which was then dissolved in 5% aqueous acid and filtered. The acidic aqueous layer was extracted with ethyl acetate, and the acidic aqueous phase was adjusted to a pH of 9-10 with aqueous ammonia and extracted with chloroform. The chloroform layers were combined, concentrated under reduced pressure, and mixed with silica gel. Silica gel column chromatography was performed, eluting with ethyl acetate for 10 column volumes and then with methanol (containing 1% aqueous ammonia) to collect the target fraction. The solvent was removed from the target fraction, and methanol was added for ultrasonic treatment, then filtered, precipitated, and dried. The sample was dissolved in a small amount of a mixed solvent of dichloromethane and methanol and loaded onto a C18 column for high-efficiency preparation. The elution was performed with acetonitrile (45% v / v). The target was collected, concentrated, and dried under reduced pressure to obtain pure 7-demethoxysilane (Antofine) with an HPLC purity greater than 94.0%.

[0051] Example 2

[0052] 50 kg of Cynanchum wilfordii herbal material was added with 10 times the amount of 90% ethanol and soaked at room temperature for 3 days. The soaking liquid was filtered and concentrated under reduced pressure to a thick extract, which was dissolved in 5% aqueous acid and filtered. The acid-water layer was extracted with ethyl acetate, the acid-water phase was adjusted to pH 9-10 with aqueous ammonia, extracted with chloroform, the chloroform layers were combined, concentrated under reduced pressure, and mixed with silica gel. Silica gel column chromatography was performed, eluting with ethyl acetate for 10 column volumes and then with methanol (containing 1% aqueous ammonia) to collect the target fraction; the target fraction was desolvated, ultrasonically treated with methanol, filtered, precipitated, and dried; the sample was dissolved in a small amount of a mixed solvent of dichloromethane and methanol and loaded onto a C18 column for efficient preparation. The eluent was eluted with methanol (70% v / v to 95% v / v), the target was collected, concentrated, and dried under reduced pressure to obtain 7.026 g of pure 7-demethoxysilane (Antofine) with an HPLC purity greater than 99% and a yield of 35.13%.

[0053] Comparative Example 2

[0054] 50 kg of Cynanchum wilfordii herbal material was added with 10 times the amount of 90% ethanol and soaked at room temperature for 3 days. The soaking liquid was filtered and concentrated under reduced pressure to a thick extract, which was dissolved in 5% aqueous acid and filtered. The acid-water layer was extracted with ethyl acetate, and the acid-water phase was adjusted to pH 9-10 with aqueous ammonia and extracted with chloroform. The chloroform layers were combined, concentrated under reduced pressure, and mixed with silica gel. Silica gel column chromatography was performed, eluting with ethyl acetate for 10 column volumes and then with methanol (containing 1% aqueous ammonia) to collect the target fraction. The solvent was removed from the target fraction, sonicated with methanol, filtered, precipitated, and dried. The sample was dissolved in a small amount of a mixed solvent of dichloromethane and methanol and loaded onto a C18 column for high-efficiency preparation. The elution was performed with methanol (65% v / v). The target was collected, concentrated, and dried under reduced pressure to obtain 7.339 g of pure 7-demethoxysilane (Antofine) with an HPLC purity of 91.5%.

[0055] Example 3

[0056] 50 kg of Cynanchum wilfordii herbal material was added with 10 times the amount of 90% ethanol and soaked at room temperature for 3 days. The soaking liquid was filtered and concentrated under reduced pressure to a thick extract, which was then dissolved in 5% aqueous acid and filtered. The acidic aqueous layer was extracted with ethyl acetate, the acidic aqueous phase was adjusted to pH 9-10 with aqueous ammonia, extracted with chloroform, the chloroform layers were combined, concentrated under reduced pressure, and mixed with silica gel. Silica gel column chromatography was performed, eluting with ethyl acetate for 10 column volumes and then with methanol (containing 1% aqueous ammonia) to collect the target fraction. The solvent was removed from the target fraction, sonicated with ethanol, filtered, precipitated, and dried. The sample was dissolved in a small amount of a mixed solvent of dichloromethane and methanol and loaded onto a C18 column for high-efficiency preparation. The eluent was eluted with acetonitrile (48% v / v to 70% v / v). The target was collected, concentrated, and dried under reduced pressure to obtain 7.580 g of pure 7-demethoxysilane (Antofine) with an HPLC purity greater than 99% and a yield of 37.90%.

[0057] Comparative Example 3

[0058] 50 kg of Cynanchum wilfordii herbal material was added with 10 times the amount of 90% ethanol and soaked at room temperature for 3 days. The soaking liquid was filtered and concentrated under reduced pressure to a thick extract, which was then dissolved in 5% aqueous acid and filtered. The acidic aqueous layer was extracted with ethyl acetate, the acidic aqueous phase was adjusted to a pH of 9-10 with aqueous ammonia, extracted with chloroform, the chloroform layers were combined, concentrated under reduced pressure, and mixed with silica gel. Silica gel column chromatography was performed, eluting with ethyl acetate for 10 column volumes and then with methanol (containing 1% aqueous ammonia) to collect the target fraction. The solvent was removed from the target fraction, sonicated with ethanol, filtered, precipitated, and dried. The sample was dissolved in a small amount of a mixed solvent of dichloromethane and methanol and loaded onto a C18 column for high-efficiency preparation. The elution was performed with acetonitrile (75% v / v). The target was collected, concentrated, and dried under reduced pressure to obtain 7-demethoxysilane (Antofine) with a maximum HPLC purity of 98.4%, a weight of 4.55 g, and a yield of 22.75%.

[0059] Example 4

[0060] 50 kg of Cynanchum wilfordii herbal material was added with 10 times the amount of 90% ethanol and soaked at room temperature for 3 days. The soaking liquid was filtered and concentrated under reduced pressure to a thick extract, which was then dissolved in 5% aqueous acid and filtered. The acidic aqueous layer was extracted with ethyl acetate, the acidic aqueous phase was adjusted to a pH of 9-10 with aqueous ammonia, extracted with chloroform, the chloroform layers were combined, concentrated under reduced pressure, and mixed with silica gel. Silica gel column chromatography was performed, eluting with ethyl acetate for 10 column volumes and then with methanol (containing 1% aqueous ammonia) to collect the target fraction. The solvent was removed from the target fraction, sonicated with ethanol, filtered, precipitated, and dried. The sample was dissolved in a small amount of a mixed solvent of dichloromethane and methanol and loaded onto a C18 column for high-efficiency preparation. The elution was performed with methanol (70% v / v to 95% v / v). The target was collected, concentrated, and dried under reduced pressure to obtain 7.030 g of pure 7-demethoxysilane (Antofine) with an HPLC purity greater than 99% and a yield of 35.15%.

[0061] Comparative Example 4

[0062] 50 kg of Cynanchum wilfordii herbal material was added with 10 times the amount of 90% ethanol and soaked at room temperature for 3 days. The soaking liquid was filtered and concentrated under reduced pressure to a thick extract, which was then dissolved in 5% aqueous acid and filtered. The acidic aqueous layer was extracted with ethyl acetate, and the acidic aqueous phase was adjusted to a pH of 9-10 with aqueous ammonia and extracted with chloroform. The chloroform layers were combined, concentrated under reduced pressure, and mixed with silica gel. Silica gel column chromatography was performed, eluting with ethyl acetate for 10 column volumes and then with methanol (containing 1% aqueous ammonia) to collect the target fraction. The solvent was removed from the target fraction, ultrasonically treated with ethanol, filtered, precipitated, and dried. The sample was dissolved in a small amount of a mixed solvent of dichloromethane and methanol and loaded onto a C18 column for high-efficiency preparation. The elution was performed with methanol (100% v / v). The target was collected, concentrated, and dried under reduced pressure to obtain 7-demethoxysilane (Antofine) with a maximum HPLC purity of 96.4%.

[0063] Comparative Example 5 (without introducing acid-base treatment process)

[0064] 50 kg of Cynanchum wilfordii herbal material was added to a 10-fold volume of 90% ethanol and soaked at room temperature for 3 days. The soaking solution was filtered and concentrated under reduced pressure to a thick extract, which was then mixed with silica gel. Silica gel column chromatography was performed, eluting the extract with ethyl acetate for 10 column volumes, followed by methanol (containing 1% ammonia) to collect the target fraction. The solvent was removed from the target fraction, sonicated with ethanol, filtered, precipitated, and dried. The sample was dissolved in a small amount of a mixed solvent of dichloromethane and methanol and loaded onto a C18 column for high-efficiency preparation. The extract was eluted with acetonitrile (48% v / v to 70% v / v). The target extract was collected, concentrated, and dried under reduced pressure to obtain 6.89 g of pure 7-demethoxysilane (Antofine) with an HPLC purity of 89%.

[0065] Comparative Example 6 (without solvent treatment)

[0066] 50 kg of Cynanchum wilfordii herbal material was added with 10 times the amount of 90% ethanol and soaked at room temperature for 3 days. The soaking liquid was filtered and concentrated under reduced pressure to a thick extract, which was dissolved in 5% aqueous acid and filtered. The acid-water layer was extracted with ethyl acetate, and the acid-water phase was adjusted to pH 9-10 with aqueous ammonia and extracted with chloroform. The chloroform layers were combined, concentrated under reduced pressure, and mixed with silica gel. Silica gel column chromatography was performed, eluting with ethyl acetate for 10 column volumes and then with methanol (containing 1% aqueous ammonia) to collect the target fraction. The target fraction was desolvated and dried. The sample was dissolved in a small amount of a mixed solvent of dichloromethane and methanol and loaded onto a C18 column for high-efficiency preparation. The eluent was eluted with methanol (70% v / v to 95% v / v). The target was collected, concentrated, and dried under reduced pressure to obtain 7.12 g of pure 7-demethoxysilane (Antofine) with an HPLC purity of 95.0%.

[0067] Comparative Example 7 (Solvent Treatment Order Adjustment)

[0068] 50 kg of Cynanchum wilfordii herbal material was added with 10 times the amount of 90% ethanol and soaked at room temperature for 3 days. The soaking liquid was filtered and concentrated under reduced pressure to a thick extract, which was dissolved in 5% aqueous acid and filtered. The acid-water layer was extracted with ethyl acetate, and the acid-water phase was adjusted to pH 9-10 with aqueous ammonia and extracted with chloroform. The chloroform layers were combined, concentrated under reduced pressure, and then mixed with silica gel. Silica gel column chromatography was performed, eluting with ethyl acetate for 10 column volumes and then with methanol (containing 1% aqueous ammonia) to collect the target fraction. After the target fraction was concentrated, C18 high pressure preparation was performed, eluting with methanol (70% v / v to 95% v / v). The target fraction was collected, concentrated, dried under reduced pressure, sonicated with ethanol, filtered, and the precipitate was dried to obtain 4.030 g of pure 7-demethoxysilane (Antofine). The HPLC purity was greater than 99%, the yield (20.15%) was much lower than that of Examples 1 to 4, and the process time was 5 times that before the order was changed.

[0069] Analysis and testing conditions

[0070] Chromatographic conditions:

[0071] Test solution: Take 1.0 mg of sample, dissolve in 1 mL of methanol, make up to 1.0 mg / mL, and filter through a 0.22 μm filter membrane.

[0072] Instrument: Waters 2695 HPLC chromatograph;

[0073] Chromatographic column: Agilent Plus C18, 4.6×150 mm, 5 μm;

[0074] Column temperature: 30°C;

[0075] Injection volume: 10 μL;

[0076] Detection wavelength: 280nm;

[0077] Mobile phase gradient setting (A: acetonitrile, B: water)

[0078] The HPLC spectrum of the final target product can be referred to FIG1 , which shows that the present disclosure can stably provide a high-purity target compound.

[0079] The various technical features disclosed above are not limited to the combinations disclosed with other features. Those skilled in the art may also make other combinations between the various technical features according to the purpose of the invention to achieve the purpose of this disclosure. Without departing from the design spirit of this disclosure, various improvements made by ordinary technicians in this field to the technical solutions of this disclosure should fall within the scope of protection determined by the claims of this disclosure. Industrial Applicability

[0080] The present disclosure can provide a method for preparing a high-purity 7-demethoxysilanine reference substance that is suitable for large-scale preparation, and has broad prospects for industrial application.

Claims

1. A method for preparing a reference substance of 7-demethoxysilane, characterized in that: The following steps are included in sequence: Normal phase silica gel chromatography purification process: the crude extract solution containing 7-demethoxysilane is loaded onto a normal phase chromatography column, eluted with ethyl acetate solvent for 8 to 15 column volumes, eluted with an alcohol solvent containing 0.5 to 1.5 wt% ammonia, the target portion is collected, and the eluate is concentrated and dried to obtain a crude extract I of 7-demethoxysilane; Solvent treatment purification step: adding methanol / ethanol to the crude extract I of 7-demethoxysilane obtained in the normal phase silica gel chromatography purification step for ultrasonic treatment, filtering to obtain a precipitate, and drying to obtain a crude extract II of 7-demethoxysilane; Reverse phase chromatography purification process: The crude extract II of 7-demethoxysilanine is loaded onto a reverse phase chromatography column, eluted with a mixed solvent of acetonitrile and water or a mixed solvent of methanol and water, the target is collected, and concentrated under reduced pressure to obtain the semi-finished product of 7-demethoxysilanine (Antofine), and the eluate is collected according to TLC or HPLC detection, and the eluate is concentrated under reduced pressure and dried to obtain a 7-demethoxysilanine reference substance with a purity of 99%.

2. The method for preparing a reference substance of 7-demethoxysilanine according to claim 1, characterized in that: The preparation method of the total extract solution of Cynanchum wilfordii herb is as follows: the dried Cynanchum wilfordii herb is crushed, 5 to 15 times of an alcohol solvent is added, and the mixture is soaked at room temperature for 2 to 5 days. The soaking liquid is filtered and concentrated under reduced pressure to a thick extract, and an acid aqueous solution is added to dissolve and filter the mixture. The acid aqueous layer is extracted with an organic solvent, the acid aqueous phase is adjusted to a pH of 9 to 10 with concentrated ammonia water, and extracted with an organic solvent. The organic layers are combined and concentrated under reduced pressure to obtain a crude extract solution containing 7-demethoxysilane.

3. The method for preparing a reference substance of 7-demethoxysilanine according to claim 2, characterized in that: The alcohol-based aqueous solution is a 60% to 90% methanol aqueous solution or a 60% to 90% ethanol aqueous solution.

4. The method for preparing a reference substance of 7-demethoxysilanine according to claim 1, characterized in that: In the normal phase chromatography purification process, the filler of the normal phase chromatography column is silica gel with a particle size of 100 to 300 meshes, and elution is performed with methanol containing 0.9 to 1.1 wt % ammonia.

5. The method for preparing a reference substance of 7-demethoxysilanine according to claim 1, characterized in that: In the reverse phase chromatography purification process, elution is performed at a pressure of 0.1 to 0.3 MPa, using a 48% v / v to 70% v / v acetonitrile-water mixed solvent or a 70% v / v to 95% v / v methanol-water mixed solvent, and a 40 to 60 μm octadecylsilane bonded silica gel filler.

6. The method for preparing a reference substance of 7-demethoxysilanine according to claim 1, characterized in that: In the acid-base treatment step, the aqueous acid solution is 0.06 to 0.10 M hydrochloric acid.

7. The method for preparing a reference substance of 7-demethoxysilanine according to claim 2, characterized in that: In the acid-base treatment process, the organic solvent used for extraction is an ester solvent or a halogenated hydrocarbon solvent.

8. The method for preparing a reference substance of 7-demethoxysilanine according to claim 7, characterized in that: The ester solvent is selected from ethyl acetate, butyl acetate, methyl acetate, The halogenated hydrocarbon solvent is a combination of one or more selected from the group consisting of dichloromethane, chloroform, carbon tetrachloride, 1,1-dichloroethane, 1,2-dichloroethane, 1,1,1-trichloroethane, 1,1,2-trichloroethane, 1,1,1,2-tetrachloroethane, 1,1,2,2-tetrachloroethane, pentachloroethane, 1,1-dichloroethylene, 1,2-dichloroethylene, trichloroethylene and tetrachloroethylene.

9. A solid reference substance of 7-demethoxysilane with a purity of more than 99%, which is prepared by the preparation method of claim 1.

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