Preparation method of aristolochic acid D reference substance

Abstract

The present invention provides a method for preparing a reference substance of aristolochic acid D, comprising an acid-base pretreatment process, a specific polyamide column chromatography process, and a normal phase and reverse phase chromatography column purification process, etc. Through the specific pretreatment, the extraction and separation efficiency is effectively improved, and a target substance reference substance with high HPLC purity can be obtained in high throughput. In addition, the preparation method of the solid reference substance of aristolochic acid D obtained by the present invention has very good applicability for industrial production.

Description

Technical Field

[0001] The present invention relates to a method for preparing aristolochic acid D, and in particular to a method for preparing a solid reference substance of aristolochic acid D suitable for industrialization. Background Art

[0002] Aristolochic acid components are a class of nitrophenanthrane carboxylic acids that are nephrotoxic and carcinogenic. Among the large number of reported cases of aristolochic acid nephropathy, some patients with aristolochic acid nephropathy subsequently developed urothelial carcinoma and bladder cancer. The discovery of urinary system carcinogenicity has led to aristolochic acid being classified as a Class I carcinogen. Aristolochic acid components are a class of plant secondary metabolites that are widely found in Aristolochaceae plants. There are more than 500 species of Aristolochaceae plants, which are used as traditional medicines in Asia to treat a variety of diseases. There are mainly 4 genera and more than 70 species of Aristolochaceae plants in China, most of which are medicinal plants and are often used as Chinese herbal medicines. They have the effects of relieving cough and asthma, dispelling wind and relieving pain, and clearing heat and activating blood circulation. At present, there is only one Aristolochaceae plant in the 2020 edition of the Pharmacopoeia, Asarum, which is widely used in clinical practice, mainly for the treatment of allergic asthma, rhinitis, cough, and new coronavirus infection. In addition to being used in decoctions, a large number of Chinese patent medicine preparations also contain Asarum. The Chinese Pharmacopoeia contains 50 varieties containing Asarum. Many varieties are very commonly used, such as Xiaoqinglong Granules, Children's Qingfei Oral Liquid, Children's Kechuan Granules, Shaqi Pills, Biyuanshu Oral Liquid, Children's Feibining Tablets, Xinqin Granules, Jiuwei Qianghuo Tablets, Chuanxiong Tea Tablets, Biyan Tablets, Weiyanning Granules, Ginseng Zaizao Pills, Zhengtian Pills, Eucommia Bone Strengthening Pills, etc., many of which are commonly used for children. Aristolochic acid components have been detected in Chinese patent medicines containing medicinal materials such as Aristolochic acid, Inula, Akebia, Stephania Tetrandra, and Xungufeng. In order to avoid the toxic damage caused by aristolochic acid components in drugs in actual applications, early diagnosis of aristolochic acid toxicity is extremely important for its safety monitoring.

[0003] Common aristolochic acid ingredients include aristolochic acid A, aristolochic acid B, aristolochic acid C, and aristolochic acid D. At present, there are many studies on aristolochic acid A, aristolochic acid B, and aristolochic acid C at home and abroad, but few studies on aristolochic acid D, and there is little ready-made aristolochic acid D supply on the market. However, aristolochic acid D has been listed as an indicator ingredient of toxic medicinal materials stipulated in the Pharmacopoeia of the People's Republic of China, and its importance cannot be ignored.

[0004]

[0005] Aristolochic acid D, molecular formula: C17H11NO8

[0006] Since aristolochic acid D has been listed as a toxicity indicator component in the pharmacopoeia, isolating aristolochic acid D standard can greatly improve the quality control level of related medicinal materials, which is very necessary and has extremely high application value.

[0007] There are many patent documents on the detection of aristolochic acid D, but there are few documents on a large number of extraction processes, most of which are about the separation of aristolochic acid D analogs. There are literature reports that aristolochic acid I and II were isolated from Aristolochic acid Root by alcohol extraction-alkali dissolution and acid precipitation-macroporous resin purification-high pressure preparation, with purities of 96% and 94.45% respectively, but no yield data. There are also literature reports that Aristolochic acid was extracted with alcohol, the alcohol extract was mixed with silica gel, and then dried and extracted with petroleum ether, chloroform, and ethanol Soxhlet in turn. The chloroform extract was chromatographed on a silica gel column and recrystallized to obtain aristolochic acid, and another 2 aristolochic acid analogs were isolated from other solvent extracts. No structural identification was done, and there was no purity and yield data. There are also literature reports that aristolochic acid was isolated from Cinnabarinia by alcohol extraction-concentration precipitation-precipitation washed with alcohol multiple times-silica gel column chromatography-recrystallization, with a yield of 0.45%, but no purity data.

[0008] Patent Literature

[0009] [1] A method for determining the content of six aristolochic acid components in Asarum CN113252810 A

[0010] Non-patent literature

[0011] [1] Qu Biqiong, Liu Jie, Yang Jiao, Yang Xiaoqin, Pan Qixue, Liu Mingyan, Liu Lulu, Xiao Hongbin. Study on the preparation technology of aristolochic acid I and II[J]. West China Journal of Pharmaceutical Sciences, 2022, 37(3): 245-248.

[0012] [2] Li Shuyang, Yao Qi. Isolation and identification of aristolochic acid from Aristolochum strychnifolium[J]. Chinese Pharmaceutical Impurities, 1981, (02): 15-18.

[0013] [3] Qiu Yunping, Chen Xiuzhen, Quan Dejian, Wen Lingfang, Wu Yanmei. Study on the chemical components of Aristolochic acid in Aristolochic acid I. Isolation of aristolochic acid [J]. Guangxi Botany, 1980, (01): 24-27.

[0014] The current situation is that the plant-derived aristolochic acid D is difficult to extract and separate, there is no ready-made process, its total content is low, and it is difficult to crystallize from a mixture. Therefore, the existing extraction and preparation methods of aristolochic acid D that have been disclosed are very limited, and there is currently no efficient industrial extraction and preparation method for aristolochic acid D. As an important characteristic component, it is urgent to develop a high-throughput, high-purity aristolochic acid D preparation method suitable for mass preparation. Summary of the invention

[0015] The object of the present invention is to provide a method for preparing aristolochic acid D reference substance with high throughput, which can provide high-purity aristolochic acid D and is suitable for mass preparation. In the present invention, aristolochic acid D is sometimes also referred to as a separation target. The present invention has made great improvements in the separation and purification of aristolochic acid D, introduced a unique pH adjustment method, and made the entire extraction method suitable for the extraction of chemical components of Tianxianteng medicinal materials. After in-depth exploration, a cost-controllable extraction and separation method was found, and a method for purifying target compounds that can be industrialized was realized, which greatly improved the amount and purity of the compound, thereby completing the present invention.

[0016] The invention can separate aristolochic acid D with high purity through a simple and efficient operation process.

[0017] The preparation and separation method of the present invention comprises the following steps in sequence:

[0018] In the acid-base treatment step, the powder or extract of the total extract of the Tianxianteng medicinal material is suspended with 3 to 7 times the weight of an alkaline aqueous solution with a pH value of 8 to 9, and after standing for 0.5 to 1.5 hours, it is extracted with 0.8 to 4 times the volume of an organic solvent, the aqueous phase is recovered, an acidic aqueous solution is added, the pH is adjusted to 3 to 4, and the organic phase is extracted with 0.8 to 4 times the volume of the organic solvent, and the organic phase is recovered and concentrated to obtain the Tianxianteng total extract II.

[0019] In a polyamide purification step, the total extract II of the scutellaria baicalensis is loaded onto a 100-200 mesh polyamide column for chromatography, and eluted with an eluent of ethyl acetate:methanol=20:1-10:1 with gradually increasing polarity, and the eluent is collected according to TLC or HPLC detection, and the eluent is concentrated and dried to obtain a crude extract I of the scutellaria baicalensis, wherein the methanol contains 8 volume % to 12 volume % of formic acid and 8 volume % to 12 volume % of water;

[0020] Reverse phase chromatography purification process: the crude extract I of Aristolochum sibiricum is loaded onto a reverse phase chromatography column, and a mixed eluent of acetonitrile aqueous solution or methanol aqueous solution is used as an eluent, wherein the ratio of acetonitrile or methanol to water is 0.4 to 0.7:1 by volume, and the eluent is collected according to TLC or HPLC detection, and the eluent is concentrated under reduced pressure at a temperature below 50° C., and dried to obtain a crude product II solution of aristolochic acid D, and the mixed eluent of acetonitrile aqueous solution or methanol aqueous solution optionally contains 0.1% to 0.3% acetic acid;

[0021] In the solvent treatment step, the crude product II solution of aristolochic acid D obtained in the reverse phase chromatography purification step is concentrated at a temperature below 50°C to obtain a precipitate, which is then washed and dried to obtain a reference substance of aristolochic acid D.

[0022] The reference substance of aristolochic acid D obtained in the solvent treatment step can be purified by a recrystallization method, and the recrystallization is preferably repeated twice or more. For example, in a preferred embodiment of the present invention, the crude product II of aristolochic acid D obtained in the reverse phase chromatography purification step is dissolved in an alcohol solvent, crystallized at -30°C to 0°C, filtered to obtain crystals, and then recrystallized twice or more.

[0023] In a preferred embodiment of the present invention, the preparation method of the total extract solution of the Tianxianteng medicinal material is as follows: crush the Tianxianteng medicinal material, add 5 to 20 times the weight of an alcohol aqueous solution, soak at room temperature or perform hot reflux extraction for 10 to 60 hours, filter, and concentrate to obtain the total extract solution of the Tianxianteng medicinal material.

[0024] In a preferred embodiment of the present invention, the reverse phase chromatography purification process can first be carried out as a medium-low pressure reverse phase chromatography purification process, and then as a high pressure reverse phase chromatography purification process, wherein, for example, the medium-low pressure reverse phase chromatography purification process: the crude extract I of the Herba Cibotii is loaded onto a reverse phase chromatography column, and eluted with a mixed eluent of acetonitrile aqueous solution or methanol aqueous solution at a pressure of 0.5 to 1 MPa, wherein the ratio of acetonitrile or methanol to water is 0.4 to 0.6:1 by volume, and the eluate is collected according to TLC or HPLC detection, and the eluate is heated at a temperature below 50°C Concentrate under reduced pressure to remove acetonitrile or methanol to obtain a crude product II-1 containing aristolochic acid D; high-pressure reverse phase chromatography purification step: load the crude product II-1 of aristolochic acid D onto a reverse phase chromatography column, and elute with a mixed eluent of an acetonitrile aqueous solution or a methanol aqueous solution optionally containing 0.1% to 0.3% acetic acid at a pressure of 1 to 15 MPa, wherein the ratio of acetonitrile or methanol to water is 0.4 to 0.67:1 by volume, and collect the eluate according to TLC or HPLC detection to obtain a crude product II solution of aristolochic acid D.

[0025] It should be noted that medium-low pressure and high pressure are not based on strict definitions in the art. Unless otherwise specified, the medium-low pressure chromatography in the present invention is described above, representing chromatography with a pressure of 0.5 to 1 MPa. Chromatography above 1 MPa is called high-pressure chromatography in the present invention.

[0026] In a preferred embodiment of the present invention, the alcohol solvent is preferably methanol, ethanol, n-butanol, etc., and the alcohol aqueous solution is 60% to 90% methanol aqueous solution or 60% to 90% ethanol aqueous solution.

[0027] In a preferred embodiment of the present invention, in the reverse phase chromatography purification process, elution is performed at a pressure of 0.1 to 0.3 MPa, a mixture of acetonitrile and water is used for elution, and an octadecylsilane bonded silica gel filler of 40 to 60 μm is selected; in the high-pressure reverse phase chromatography purification process, the filler of the reverse phase chromatography column is an octadecylsilane bonded silica gel with a particle size of 5-10 μm, and an acetonitrile: water eluent is used, and the eluent is eluted in a ratio of 0.5 to 0.7:1 by volume.

[0028] In a preferred embodiment of the present invention, in the acid-base treatment step, the acidified aqueous solution is 8 wt % to 10 wt % hydrochloric acid, and the alkaline solution is a sodium bicarbonate solution.

[0029] In a preferred embodiment of the present invention, in the acid-base treatment step, the organic solvent used for extraction is one selected from a halogenated hydrocarbon solvent and an ester solvent, the halogenated hydrocarbon solvent is selected from a combination of 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, and the ester solvent is selected from a combination of one or more of methyl acetate, ethyl acetate, phenyl acetate, methyl benzoate, butyl acetate and octyl acrylate.

[0030] The present invention also provides a solid reference substance of aristolochic acid D with a purity of more than 99.5%, which is prepared by the preparation method of the present invention. The reference substance prepared by the method of the present invention has high HPLC purity and obvious cost advantage.

[0031] The present invention has the following characteristics:

[0032] 1. The present invention is simple and efficient to operate, and can be scaled up to obtain a large amount of aristolochic acid D monomer;

[0033] 2. The present invention has separated 60g of aristolochic acid D from 500Kg of Tianxianteng medicinal materials through production practice, with a purity of 99.8% and a yield of 0.012%. The purity has been greatly improved relative to the literature, and can be used as a reference substance for detection, which is the first time in the literature patent report. The present invention separates 60g of aristolochic acid D, and according to demand, it can also be scaled up to 100 grams, so there are great improvements in the preparation method and flux.

[0034] 3. The combination of acid and base treatment combined with polyamide achieved rapid enrichment of a large number of target substances and increased the final recovery rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1HPLC spectrum of aristolochic acid D obtained by the separation method of Example 1;

[0036] Figure 2 Aristolochic acid D pure 1 H-NMR spectrum;

[0037] Figure 3 Aristolochic acid D pure 13 C-NMR spectrum;

[0038] Figure 4 This is the mass spectrum of pure aristolochic acid D;

[0039] Figure 5 Schematic diagram of the separation and purification process of aristolochic acid D in Example 1 of the present invention. DETAILED DESCRIPTION

[0040] Specific embodiments of the present invention are described below.

[0041] If not otherwise specified, the purity and content percentage of the target substance in the present invention are the content measured by mass ratio. If not otherwise specified, the percentage of the liquid to liquid content ratio in the present invention, such as the content ratio of formic acid in acetonitrile aqueous solution, is measured by volume fraction.

[0042] The existing method for separating the target aristolochic acid D of the present invention mainly utilizes a combination of normal phase and reverse phase chromatography, and utilizes solvent gradient elution through the different polarities of the target and impurities to gradually and slowly separate the target. Generally speaking, for example, the extract concentrate of the crushed Tianxianteng medicinal material can be loaded onto a normal phase chromatography column, and gradient eluted with petroleum ether and ethyl acetate, and the target eluted at the rear is collected, and concentrated to obtain a crude concentrate containing the target, and then a second normal phase chromatography column purification process is performed: the semi-finished solid powder of the target is loaded onto a normal phase chromatography column, and eluted with a mixed solvent comprising dichloromethane, ethyl acetate, and triethylamine, and first eluted with an eluent of dichloromethane: ethyl acetate to obtain an eluent containing the target, collect the eluent, concentrate, and freeze-dry to obtain a solid target. However, the inventors of the present invention have found that, first of all, due to the presence of carboxyl groups, aristolochic acid D is irreversibly adsorbed by normal phase silica gel, and is not suitable for large-scale extraction and separation. Reverse phase chromatography can also be used to separate aristolochic acid D with higher purity. If only reverse phase chromatography is used for separation, the separation degree is poor and the recovery rate is low. In addition, the inventors found that other secondary metabolites in Tianxian Caulis will greatly affect the purification of aristolochic acid D. Directly using reverse phase chromatography to separate the compounds contained in Tianxian Caulis medicinal materials, many of which are very close to the target polarity, the final purity is difficult to improve; the idea of ​​using a chromatographic column with higher pressure and better separation degree to continuously improve the purity can gradually improve the purity of aristolochic acid D, but it leads to low yield, and the solvent filler is very expensive during the separation process, and the most important thing is that the flux is extremely low.

[0043] In order to be suitable for industrial mass production and to solve the problems of yield and separation efficiency, the inventors of the present invention have conducted in-depth research and determined that the preparation and separation method of the present invention comprises the following steps in sequence: A method for preparing a reference substance of aristolochic acid D, characterized in that it comprises the following steps in sequence:

[0044] In the acid-base treatment step, the powder or extract of the total extract of the Tianxianteng medicinal material is suspended with 3 to 7 times the weight of an alkaline aqueous solution with a pH value of 8 to 9, and after standing for 0.5 to 1.5 hours, it is extracted with 0.8 to 4 times the volume of an organic solvent, the aqueous phase is recovered, an acidic aqueous solution is added, the pH is adjusted to 3 to 4, and the organic phase is extracted with 0.8 to 4 times the volume of the organic solvent, and the organic phase is recovered and concentrated to obtain the Tianxianteng total extract II.

[0045] In a polyamide purification step, the total extract II of the scutellaria baicalensis is loaded onto a 100-200 mesh polyamide column for chromatography, and eluted with an eluent of ethyl acetate:methanol=20:1-10:1 with gradually increasing polarity, and the eluent is collected according to TLC or HPLC detection, and the eluent is concentrated and dried to obtain a crude extract I of the scutellaria baicalensis, wherein the methanol contains 8 volume % to 12 volume % of formic acid and 8 volume % to 12 volume % of water;

[0046] Reverse phase chromatography purification process: the crude extract I of Aristolochum sibiricum is loaded onto a reverse phase chromatography column, and a mixed eluent of acetonitrile aqueous solution or methanol aqueous solution is used as an eluent, wherein the ratio of acetonitrile or methanol to water is 0.4 to 0.7:1 by volume, and the eluent is collected according to TLC or HPLC detection, and the eluent is concentrated under reduced pressure at a temperature below 50° C., and dried to obtain a crude product II solution of aristolochic acid D, and the mixed eluent of acetonitrile aqueous solution or methanol aqueous solution optionally contains 0.1% to 0.3% acetic acid;

[0047] In the solvent treatment step, the crude product II solution of aristolochic acid D obtained in the reverse phase chromatography purification step is concentrated at a temperature below 50°C to obtain a precipitate, which is then washed and dried to obtain a reference substance of aristolochic acid D.

[0048] In a preferred embodiment of the present invention, the preparation method of the total extract solution of the Tianxianteng medicinal material is as follows: crush the Tianxianteng medicinal material, add 5 to 20 times the weight of an alcohol aqueous solution, soak at room temperature or perform hot reflux extraction for 10 to 60 hours, filter, and concentrate to obtain the total extract solution of the Tianxianteng medicinal material.

[0049] The key to the present invention is the combination of the acid-base treatment process and polyamide, as well as the connection order between the various process steps. The conventional normal phase separation method and reverse phase separation method are used to obtain the target compound, but the results are not ideal. It is always difficult to obtain a large amount of aristolochic acid D. However, if the acid-base treatment and polyamide treatment processes in the present invention are added, the separation effect can be greatly improved. The reason is not very clear. It is speculated that there may be the following reasons: the glycosides and esters of aristolochic acid D may exist in the secondary metabolites of Aristolochic acid D. In the acid-base treatment process, they can be hydrolyzed to produce more aristolochic acid D, directly increasing the source of aristolochic acid D. It should be noted here that in the present invention, Long or short standing time during acid-base treatment affects the final recovery rate. The final yield is best after standing for 0.5 to 1.5 hours. The secondary metabolites of Aristolochic acid scutellariae contain many N-type natural products and carboxyl-containing natural products. These components have serious solvation phenomena during chromatographic elution, resulting in asymmetric chromatographic peaks of these impurities. Under the elution conditions with better separation of aristolochic acid D, there is serious tailing, which makes it difficult for these components to be completely separated from aristolochic acid D. Acid-base treatment can efficiently separate and enrich these components.

[0050] The acid-base treatment is placed before the polyamide purification process, and the overall separation efficiency is the highest, and a good separation effect can be obtained. The order between the various separation steps of the present invention is also an important technical feature of the present invention. In a preferred embodiment of the present invention, in the acid-base treatment process, the acidified aqueous solution is 8wt% to 10wt% hydrochloric acid, and the alkaline solution is a sodium bicarbonate solution.

[0051] The starting raw material in the method of the present invention can be commercially available Tianxianteng medicinal material extract powder, which can be obtained by those skilled in the art from the market, or can prepare the Tianxianteng medicinal material total extract solution by themselves. The inventor also recommends the following extraction method. For example, the preparation method of the Tianxianteng medicinal material total extract solution is as follows: add 5 to 20 times the weight of an alcohol aqueous solution, soak at room temperature or perform hot reflux extraction for 10 to 60 hours, filter, and concentrate to obtain a Tianxianteng medicinal material total extract solution.

[0052] In a preferred embodiment of the present invention, the alcohol-based 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.

[0053] Polyamide purification is a crucial process. The inventors found that after macroporous resin enrichment, if combined with polyamide purification, the target compound can be accurately enriched, and the impurity components mixed with the target compound are particularly easy to remove by other methods. It can be said that the introduction of polyamide separation in a suitable process is the key to the present invention. In the present invention, polyamide column chromatography with nylon 6 or nylon 66 structure can be used. For the chromatographic purification process, the method of loading the raw material crude product in the present invention is not particularly limited. It can be dissolved in a good solvent and added dropwise, or it can be mixed with a filler such as polyamide for loading. In order to compress the loading volume to a lower level, it is preferred to mix the sample with a filler for loading.

[0054] In the polyamide purification process, in order to increase the separation degree in the polyamide purification process, the methanol contains 8 volume % to 12 volume % of formic acid and 8 volume % to 12 volume % of water, preferably 9 volume % to 10 volume % of formic acid.

[0055] In the present invention, the reverse phase chromatography purification process is used to separate other components with different polarity from the target. In order to obtain better separation efficiency, the medium-low pressure reverse phase chromatography purification process and the high-pressure reverse phase chromatography purification process are carried out successively in the present invention. It should be noted that medium-low pressure and high pressure are not based on strict definitions in the art. The medium-low pressure chromatography in the present invention is described above as long as there is no special limitation. The chromatography representing the pressure of 0.5 to 1Mpa and the chromatography above 1Mpa are called high-pressure chromatography in the present invention.

[0056] In a preferred embodiment of the present invention, the reverse phase chromatography purification process can first be carried out as a medium-low pressure reverse phase chromatography purification process, and then as a high-pressure reverse phase chromatography purification process. Medium-low pressure reverse phase chromatography purification process: The crude extract I of Caulis Cibotii is loaded onto a reverse phase chromatography column, and eluted with a mixed eluent of acetonitrile aqueous solution or methanol aqueous solution at a pressure of 0.5 to 1 MPa. This pressure is particularly preferred because it can balance the separation effect and separation speed, wherein the ratio of acetonitrile or methanol: water is 0.4 to 0.6:1 by volume, and the eluate is collected according to TLC or HPLC detection, and the eluate is concentrated under reduced pressure at a temperature below 50°C to remove the acetonitrile or methanol therein to obtain To obtain a crude product II-1 containing aristolochic acid D; high-pressure reverse phase chromatography purification step: load the crude product II-1 of aristolochic acid D onto a reverse phase chromatography column, and elute with a mixed eluent of an acetonitrile aqueous solution or a methanol aqueous solution optionally containing 0.1% to 0.3% acetic acid, at a pressure of 1 to 15 MPa, wherein the ratio of acetonitrile or methanol to water is 0.4 to 0.67:1 by volume, and collect the eluate according to TLC or HPLC detection to obtain a crude product II solution of aristolochic acid D.

[0057] The reverse phase filler of the reverse phase chromatographic column used in the chromatographic column preparation process of the present invention can be a well-known non-polar silica gel with a bonded functional group being an alkane (for example: C18 (ODS), C8, C4, etc.). Preferably, a C18 (ODS) silica gel column, i.e., octadecylsilane bonded silica gel, is used. In the so-called medium and low pressure reverse phase chromatography purification process, a filler of 40 to 60 μm is preferably used. A reasonable particle size is conducive to maintaining an appropriate column pressure and resolution. Octadecylsilane bonded silica gel is easy to purchase on the market. From the perspective of efficiency and the availability of fillers, in the preferred preparation method of the present invention, the filler of the reverse phase chromatographic column is an octadecylsilane bonded silica gel with a particle size of 40 to 60 μm.

[0058] The so-called high-pressure reverse-phase chromatography column purification refers to a chromatography column separation technology with an elution pressure greater than 1Mpa. In the present invention, elution is performed at a pressure of 1 to 15.0Mpa to achieve a balance between resolution and speed. In the high-pressure reverse-phase chromatography purification process of the present invention, it is further preferred to elute with an acetonitrile aqueous solution at a pressure of 8 to 10Mpa. The high-pressure reverse-phase chromatography column can be any high-pressure reverse-phase chromatography column as long as the filler loading capacity can meet the capacity of the target. The use of an elution pressure of 5 to 15Mpa in this process can achieve a balance between resolution and speed, and the more preferred column pressure is 5 to 10Mpa. In the separation process, the pressure of the reverse-phase chromatography column is further preferably 5 to 8Mpa, which is particularly preferred because it can balance the separation effect and separation speed. As the reverse-phase filler in the high-pressure reverse-phase chromatography separation process of the present invention, a known non-polar silica gel with an alkane as the bonded functional group (for example: C18 (ODS), C8, C4, etc.) can be used. Preferably, a C18 (ODS) silica gel column, i.e., octadecylsilane bonded silica gel, is used, preferably with a filler of 5-60 μm, and a reasonable particle size is conducive to maintaining appropriate column pressure and resolution, and octadecylsilane bonded silica gel is easily available on the market. From the perspective of efficiency and easy availability of fillers, in the preferred preparation method of the present invention, the filler of the reverse phase chromatographic column is octadecylsilane bonded silica gel with a particle size of 5 to 10 μm.

[0059] In the further solvent treatment purification step, a sparingly soluble solvent treatment method or a recrystallization method may be used to further improve the purity.

[0060] In a preferred embodiment of the present invention, in the acid-base treatment step, the organic solvent used for extraction is one selected from a halogenated hydrocarbon solvent and an ester solvent, the halogenated hydrocarbon solvent is selected from a combination of 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, and the ester solvent is selected from a combination of one or more of methyl acetate, ethyl acetate, phenyl acetate, methyl benzoate, butyl acetate and octyl acrylate.

[0061] Example

[0062] Below, typical extraction methods of the present invention are further described in detail by examples. The following experimental schemes are merely examples and are not limitations of the present invention. Those skilled in the art can make any changes under the premise of understanding the principles of the embodiments and the spirit of the invention.

[0063] Example 1

[0064] The preparation method of aristolochic acid D reference substance is carried out according to the following process steps:

[0065] Step (A): Extraction of raw materials: 500 kg of Tianxianteng medicinal materials were taken, and 10 times 80% ethanol was added for hot reflux extraction twice, filtered, and the filtrate was concentrated to 500 L to obtain Tianxianteng total extract, which quantitatively contained 178 g of aristolochic acid D;

[0066] Step (B): Acid-base extraction: Add 10% sodium hydroxide water to the concentrate, adjust the pH to 8-9, and then extract with an equal volume of ethyl acetate for 3 times to remove a large amount of extract. After extraction, add 10% hydrochloric acid water to the water layer, adjust the pH to 3-4, and extract with an equal volume of ethyl acetate for 2 times to obtain aristolochic acid D crude product 1, with a quantitative amount of 142g;

[0067] Step (C): separation by polyamide column chromatography: the crude product 1 of aristolochic acid D obtained in step B is mixed with 100-200 mesh polyamide, dried and ground, and separated by polyamide column chromatography, ethyl acetate: methanol: formic acid: water = 20: 1: 0.1: 0.1-10: 1: 0.1: 0.1 gradient elution, small polar impurities are eluted first, and the target is eluted later. This step removes a large amount of impurity extracts to obtain aristolochic acid D crude product 2, with a quantitative amount of 121 g;

[0068] Step (D): Medium-pressure purification: The crude product 2 of aristolochic acid D obtained in step C was dissolved in 80% tetrahydrofuran water, and purified by medium-pressure C18 column chromatography, with a gradient elution of 30% to 38% acetonitrile water to obtain 84.7 g of crude product 3 of aristolochic acid D, with HPLC purity of 85%;

[0069] Step (E): High-pressure preparation and purification: The crude product 3 of aristolochic acid D obtained in step D is dissolved in 80% tetrahydrofuran, subjected to high-pressure preparation, and eluted with 40% acetonitrile-0.2% acetic acid water to obtain the target fraction of HPLC 99.8%.

[0070] Step (F): Product recovery: The target fraction obtained in step E was concentrated to a small volume at 50°C in the dark, cooled, and a large amount of target was precipitated. It was filtered and rinsed with pure water to remove acetonitrile and acetic acid residues. The filter cake was dispersed, placed in a blast drying oven, and dried at 55°C for 20h. After taking it out, it was ground evenly, and placed in a blast drying oven again, and dried at 55°C for 24h to obtain 60g of aristolochic acid D powder with a purity of 99.8%.

[0071] Analysis and testing conditions

[0072] Chromatographic conditions:

[0073] 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.

[0074]

[0075]

[0076] Mobile phase composition A-0.2% acetic acid, B-acetonitrile

[0077]

[0078] Its structure can be confirmed by mass spectrometry and nuclear magnetic resonance spectra. For mass spectrometry and nuclear magnetic resonance spectra, please refer to Figure 3-Figure 5 The HPLC spectrum of the final target can be found at Figure 2 (The upper part is the test result, and the lower part is the baseline control of the blank solution), which can stably provide high-purity target compounds.

[0079] Comparative Example 1 (without introducing acid-base treatment process)

[0080] Step (A) Extraction of medicinal materials: 500 kg of Tianxianteng medicinal materials were taken, and 10 times 80% ethanol was added for hot reflux extraction twice, filtered, and the filtrate was concentrated to 500 L to obtain Tianxianteng total extract, which quantitatively contained 175 g of aristolochic acid D;

[0081] Step (B) Polyamide column chromatography separation: The crude product 1 of aristolochic acid D obtained in step B is mixed with 100-200 mesh polyamide, dried and ground, and separated by polyamide column chromatography, ethyl acetate: methanol: formic acid: water

[0082] =20:1:0.1:0.1-10:1:0.1:0.1 gradient elution, small polar impurities are eluted first, and the target is eluted later. This step removes a large amount of impurity extracts and obtains aristolochic acid D crude product 2, with a quantitative amount of 87.5g;

[0083] Step (C) medium pressure purification: The crude product 2 of aristolochic acid D obtained in step C was dissolved in 80% tetrahydrofuran water, and purified by medium pressure C18 column chromatography, with a gradient elution of 30% to 38% acetonitrile water, to obtain 49 g of crude product 3 of aristolochic acid D, with HPLC purity of 70%;

[0084] Step (D) high pressure preparation and purification: The crude product 3 of aristolochic acid D obtained in step D is dissolved in 80% tetrahydrofuran, subjected to high pressure preparation, and eluted with 40% acetonitrile-0.2% acetic acid water to obtain the target fraction of HPLC 98.2%.

[0085] Process (E) Product Recovery: The target fraction obtained in step E was concentrated to a small volume at 50°C in the dark, cooled, and a large amount of target was precipitated. It was filtered and rinsed with pure water to remove acetonitrile and acetic acid residues. The filter cake was dispersed, placed in a forced air drying oven, and dried at 55°C for 20h. After taking it out, it was ground evenly, placed in a forced air drying oven again, and dried at 55°C for 24h to obtain 25g of aristolochic acid D powder with a purity of 98.2%.

[0086] Comparative Example 2 (Switching the order of processes)

[0087] Step (A) Extraction of medicinal materials: 500 kg of Tianxianteng medicinal materials were taken, and 10 times 80% ethanol was added for hot reflux extraction twice, filtered, and the filtrate was concentrated to 500 L to obtain Tianxianteng total extract, which quantitatively contained 172 g of aristolochic acid D;

[0088] Step (B) Polyamide column chromatography separation: The crude product 1 of aristolochic acid D obtained in step B is mixed with 100-200 mesh polyamide, dried and ground, and separated by polyamide column chromatography, ethyl acetate: methanol: formic acid: water

[0089] =20:1:0.1:0.1-10:1:0.1:0.1 gradient elution, small polar impurities are eluted first, and the target is eluted later. This step removes a large amount of impurity extracts and obtains aristolochic acid D crude product 2, with a quantitative amount of 85g;

[0090] Step (C) Acid-base extraction: Add 10% sodium hydroxide water to the concentrate, adjust the pH to 8-9, and then extract with an equal volume of ethyl acetate for 3 times to remove a large amount of extract. After extraction, add 10% hydrochloric acid water to the water layer, adjust the pH to 3-4, and extract with an equal volume of ethyl acetate for 2 times to obtain aristolochic acid D crude product 1, with a quantitative amount of 74.5g;

[0091] Step (D) medium pressure purification: The crude product 2 of aristolochic acid D obtained in step C was dissolved in 80% tetrahydrofuran water, and purified by medium pressure C18 column chromatography, with a gradient elution of 30% to 38% acetonitrile water, to obtain 52.1 g of crude product 3 of aristolochic acid D, with HPLC purity of 75%;

[0092] Step (E) high pressure preparation and purification: The crude product 3 of aristolochic acid D obtained in step D is dissolved in 80% tetrahydrofuran, subjected to high pressure preparation, and eluted with 40% acetonitrile-0.2% acetic acid water to obtain the target fraction of HPLC 98.6%.

[0093] Process (F) Product Recovery: The target fraction obtained in step E was concentrated to a small volume at 50°C in the dark, cooled, and a large amount of target was precipitated. It was filtered and rinsed with pure water to remove acetonitrile and acetic acid residues. The filter cake was dispersed, placed in a forced air drying oven, and dried at 55°C for 20 hours. After taking it out, it was ground evenly, placed in a forced air drying oven again, and dried at 55°C for 24 hours to obtain 36.5g of aristolochic acid D powder with a purity of 98.6%.

[0094] Comparative Example 3 (without using polyamide)

[0095] Step (A) Extraction of medicinal materials: 500 kg of Tianxianteng medicinal materials were taken, and 10 times 80% ethanol was added for hot reflux extraction twice, filtered, and the filtrate was concentrated to 500 L to obtain Tianxianteng total extract, which quantitatively contained 177 g of aristolochic acid D;

[0096] Step (B) normal phase silica gel column separation: dissolve the dichloromethane extract and mix with 2 times the weight of 100-200 mesh silica gel, dry and grind evenly; then wet-load the column with 10 times the weight of 100-200 mesh silica gel using petroleum as solvent; load the mixed sample onto the silica gel column and perform gradient elution with dichloromethane-methanol = 15:1→10:1; detect and collect the target fractions by TLC spot plate, concentrate under reduced pressure at 45°C, and obtain a crude product of aristolochic acid D 2, which quantitatively contains 53.4 g of aristolochic acid D;

[0097] Step (C) medium pressure purification: The crude product 2 of aristolochic acid D obtained in step C was dissolved in 80% tetrahydrofuran water, and purified by medium pressure C18 column chromatography, with a gradient elution of 30% to 38% acetonitrile water, to obtain 32.0 g of crude product 3 of aristolochic acid D, with HPLC purity of 68%;

[0098] Step (D) high-pressure preparation and purification: The crude product 3 of aristolochic acid D obtained in step D is dissolved in 80% tetrahydrofuran, subjected to high-pressure preparation, and eluted with 40% acetonitrile-0.2% acetic acid water to obtain the target fraction of HPLC 95.8%.

[0099] Process (E) Product Recovery: The target fraction obtained in step E was concentrated to a small volume at 50°C in the dark, cooled, and a large amount of target was precipitated. It was filtered and rinsed with pure water to remove acetonitrile and acetic acid residues. The filter cake was dispersed, placed in a blast drying oven, and dried at 55°C for 20h. After taking it out, it was ground evenly, placed in a blast drying oven again, and dried at 55°C for 24h to obtain 14.6g of aristolochic acid D powder with a purity of 98.0%.

[0100] The mass spectrum information of aristolochic acid D obtained in Example 1 is as follows: Figure 4 .

[0101] Instrument Sciex TripleTOF 4600LC / MS

[0102] Detection mode Negative ion mode

[0103] ESI source parameters:

[0104]

[0105] The technical features disclosed above are not limited to the combinations disclosed with other features. Those skilled in the art may also make other combinations of the technical features according to the purpose of the invention to achieve the purpose of the invention. Without departing from the design spirit of the invention, various improvements made to the technical solutions of the invention by those of ordinary skill in the art shall fall within the protection scope determined by the claims of the invention.

Claims

1. A method for preparing a reference substance of aristolochic acid D, characterized in that: The following steps are included in sequence: In the acid-base treatment step, the total extract of Tianxianteng medicinal material is suspended with 3 to 7 times the weight of an alkaline aqueous solution with a pH value of 8 to 9, and after standing for 0.5 to 1.5 hours, it is extracted with 0.8 to 4 times the volume of an organic solvent, the aqueous phase is recovered, an acidic aqueous solution is added, the pH is adjusted to 3 to 4, and 0.8 to 4 times the volume of an organic solvent is extracted, the organic phase is recovered, and concentrated to obtain the total extract II of Tianxianteng. In this step, the acidified aqueous solution is 8wt% to 10wt% hydrochloric acid, the alkaline solution is a sodium bicarbonate solution, and the organic solvent used for extraction is a halogenated hydrocarbon solvent selected from , one of the ester solvents, the halogenated hydrocarbon solvent is selected from one or more combinations 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, and the ester solvent is selected from one or more combinations of methyl acetate, ethyl acetate, phenyl acetate, methyl benzoate, butyl acetate and octyl acrylate; Polyamide purification step, loading the total extract II of Cnidium monnieri onto a 100-200 mesh polyamide column for chromatography, eluting with an eluent of ethyl acetate:methanol=20:1-10:1 with gradually increasing polarity, wherein the methanol contains 9 volume %-10 volume % of formic acid and 9 volume %-10 volume % of water, collecting the eluent according to TLC or HPLC detection, concentrating and drying the eluent, and obtaining the crude extract I of Cnidium monnieri; Reverse phase chromatography purification process: The reverse phase chromatography purification process first performs a medium-low pressure reverse phase chromatography purification process, and then performs a high pressure reverse phase chromatography purification process, wherein in the medium-low pressure reverse phase chromatography purification process, the Caulis Cibotii crude extract I is loaded onto a reverse phase chromatography column, and eluted with a mixed eluent of acetonitrile aqueous solution or methanol aqueous solution at a pressure of 0.5 to 1 MPa, wherein the ratio of acetonitrile or methanol to water is 0.4 to 0.6:1 by volume, and the eluate is collected according to TLC or HPLC detection, and the eluate is decompressed at a temperature below 50°C Concentrate to remove acetonitrile or methanol to obtain a crude product II-1 containing aristolochic acid D; in the high-pressure reverse phase chromatography purification step, load the crude product II-1 of aristolochic acid D onto a reverse phase chromatography column, and elute with a mixed eluent of acetonitrile aqueous solution or methanol aqueous solution containing 0.1% to 0.3% acetic acid at a pressure of 1 to 15 MPa, wherein the ratio of acetonitrile or methanol to water is 0.4 to 0.67:1 by volume, and collect the eluate according to TLC or HPLC detection to obtain a crude product II solution of aristolochic acid D; In the solvent treatment step, the crude product II solution of aristolochic acid D obtained in the reverse phase chromatography purification step is concentrated at a temperature below 50°C to obtain a precipitate, which is then washed and dried to obtain a reference substance of aristolochic acid D.

2. The method for preparing a reference substance of aristolochic acid D according to claim 1, characterized in that: The preparation method of the total extract of Tianxianteng medicinal material is as follows: crush the Tianxianteng medicinal material, add 5 to 20 times the weight of an alcohol aqueous solution, soak at room temperature or extract under hot reflux for 10 to 60 hours, filter, and concentrate to obtain the total extract of Tianxianteng medicinal material, wherein the alcohol aqueous solution is 60% to 90% methanol aqueous solution or 60% to 90% ethanol aqueous solution.

3. The method for preparing a reference substance of aristolochic acid D according to claim 1, characterized in that: The method further comprises a purification step, in which the reference substance of aristolochic acid D obtained in the solvent treatment purification step is purified by a recrystallization method.

4. The method for preparing a reference substance of aristolochic acid D according to claim 3, characterized in that: In the refining process, the reference substance of aristolochic acid D obtained in the solvent treatment process is dissolved in an alcohol solvent, crystallized at -30°C to 0°C, filtered to obtain crystals, and then recrystallized twice or more.

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