CN114195627A - Preparation method of sheep maleic acid ABA reference substance - Google Patents
Preparation method of sheep maleic acid ABA reference substance Download PDFInfo
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- CN114195627A CN114195627A CN202111592623.9A CN202111592623A CN114195627A CN 114195627 A CN114195627 A CN 114195627A CN 202111592623 A CN202111592623 A CN 202111592623A CN 114195627 A CN114195627 A CN 114195627A
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- Prior art keywords
- aba
- solvent
- acid
- aspic
- aqueous solution
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- 238000002360 preparation method Methods 0.000 title claims abstract description 23
- 239000013558 reference substance Substances 0.000 title claims abstract description 23
- 239000011976 maleic acid Substances 0.000 title description 4
- 241001494479 Pecora Species 0.000 title description 3
- OFOBLEOULBTSOW-UHFFFAOYSA-N Propanedioic acid Natural products OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 title description 3
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 title description 3
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 title description 3
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- 238000000034 method Methods 0.000 claims abstract description 58
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- 238000007711 solidification Methods 0.000 claims abstract description 12
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- 238000000746 purification Methods 0.000 claims abstract description 9
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 69
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- 239000002994 raw material Substances 0.000 claims description 19
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- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
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- QCDYQQDYXPDABM-UHFFFAOYSA-N phloroglucinol Chemical compound OC1=CC(O)=CC(O)=C1 QCDYQQDYXPDABM-UHFFFAOYSA-N 0.000 description 1
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- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
- C07C45/78—Separation; Purification; Stabilisation; Use of additives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
- C07C45/78—Separation; Purification; Stabilisation; Use of additives
- C07C45/79—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
- C07C49/76—Ketones containing a keto group bound to a six-membered aromatic ring
- C07C49/86—Ketones containing a keto group bound to a six-membered aromatic ring containing —CHO groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/07—Optical isomers
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2601/00—Systems containing only non-condensed rings
- C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
- C07C2601/16—Systems containing only non-condensed rings with a six-membered ring the ring being unsaturated
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention provides a preparation method of an aspic acid ABA reference substance, which sequentially comprises the following steps: normal phase chromatography purification, crude product solvent treatment, high pressure reverse phase chromatography purification and solidification. The preparation method disclosed by the invention is a preparation method of the aspic acid ABA reference substance, which has high flux, can provide high-purity aspic acid ABA and is suitable for large-scale preparation, and the extraction rate of the aspic acid ABA prepared by the method is higher. The invention can rapidly obtain the aspidic acid ABA with the purity of more than 99.5 percent.
Description
Technical Field
The invention relates to a preparation method of aspic acid ABA, in particular to a preparation method of a solid reference substance capable of preparing high-purity aspic acid ABA.
Background
The rhizoma Dryopteris Crassirhizoma Nakai is dry rhizome and petiole residue of Dryopteris crassirhizophila of Dryopteridaceae, and is mainly distributed in 3 provinces of northeast China. Clinically, the rhizoma dryopteris crassirhizomae is commonly used for treating diseases such as cancer, tumor, influenza virus and the like by using single or compound medicines. The rhizoma dryopteris crassirhizomae is a hot spot of pharmaceutical research in antimalarial, antitumor and antibacterial activities, and is a promising medicinal resource plant. The processed product of male fern rhizome, male fern rhizome charcoal, is recorded in the 'Chinese pharmacopoeia' of 2015 edition, has the efficacy of astringing and stopping bleeding, is clinically used for metrorrhagia, metrostaxis and bleeding, and the identification and the establishment of quality standards are very important for the modernization of traditional Chinese medicines.
Aspic acid ABA (filixic acid ABA, also known as trisalbasaridin ABA) is a phloroglucinol compound, is a characteristic component of traditional Chinese medicinal materials of aspidium elatum and traditional Chinese medicinal aspidium elatum charcoal, and has the following specific chemical structure:
although the traditional Chinese medicine rhizoma dryopteris crassirhizomae and traditional Chinese medicine rhizoma dryopteris crassirhizomae charcoal are characteristic components, the channels for purchasing the obtained standard products in the market are not rich, and the standard product providers with high purity of more than 99% are difficult to find. The extraction or the preparation efficiency of the ABA-pamoic acid is not high, Wushoujin and the like are reported in the research on chemical components of the male fern rhizome (Chinese herbal medicine, 1996,27(8):458-459), the male fern rhizome is extracted by ether hot reflux, the total extract is subjected to repeated silica gel column chromatography and crystallization to obtain the ABA-pamoic acid, and the yield and the purity are not reported; laura Domi i Ninguez et al extracted Dryopteris crassirhizoma 138g with ether and then prepared 27.5mg of aspic acid ABA by silica gel, LH-20 and medium and high pressure, in 0.02% yield (Phytochemistry,2012, 80: 115-. The two methods adopt ether for extraction, but the ether has lower boiling point, and the hot reflux extraction is not suitable for mass extraction and preparation. In Qiyan in the research on the separation and bacteriostatic activity of phloroglucinol in aspidistra elata (Chinese herbal medicine, 2017,48(23):4860-4864.), aspidistric acid ABA is obtained by extracting through 50% ethanol and then purifying through repeated silica gel columns and prepared liquid phases, but the yield is lower, and 40kg of aspidistra elata only obtains 11mg of aspidistric acid ABA (the yield is less than three parts per million). However, the existing methods are extraction methods which are difficult to rapidly obtain reference substances with the purity of more than 99 percent.
In summary, there is still a need to develop a method for extracting aspic acid ABA that is simple and rapid enough, has a high extraction rate, and has a purity of 99% or more.
Disclosure of Invention
The invention aims to provide a preparation method of a reference substance of aspic acid ABA, which has high flux, can provide high-purity aspic acid ABA and is suitable for large-scale preparation, and the extraction rate of aspic acid ABA of the method is higher.
Specifically, the invention provides a preparation method of a reference substance of aspic acid ABA, which can obtain aspic acid ABA with high purity at high efficiency and yield, and sequentially comprises the following steps:
normal phase chromatography purification procedure: loading the crude raw material extract containing the ABA pamoate to a normal-phase chromatographic column, and eluting by using mixed eluent containing petroleum ether, halogenated alkane solvent and alcohol solvent, wherein the weight ratio of petroleum ether: the ratio of the halogenated alkane solvent is 100-50: 1, the halogenated alkane solvent is: the ratio of the alcohol solvent is 20-5: 1, collecting eluent according to TLC or HPLC detection, concentrating and drying the eluent to obtain a first crude product powder;
a crude product solvent treatment process: heating the first crude powder at a temperature of below 60 ℃ by using a ketone solvent with 2-6 times of the mass ratio and 3-6 carbon atoms in the molecule to dissolve the first crude powder, standing the first crude powder at a temperature of below 10 ℃ for 1-24 hours, filtering the precipitate, and drying the precipitate to obtain second crude powder;
a reversed phase chromatography purification procedure: loading the second crude powder to reverse phase chromatography, and eluting with a mixed eluent of acetonitrile or methanol and an acidic aqueous solution with the pH value of 3-6 at the pressure of 1-15.0 Mpa, wherein the ratio of acetonitrile or methanol: the ratio of the acidic aqueous solution is 5.5-3.5: 1, eluent is collected according to TLC or HPLC detection, the eluent is concentrated under reduced pressure at the temperature of below 50 ℃, acetonitrile or methanol in the eluent is removed, and an aqueous solution containing the ABA of the aspic acid is obtained;
and a solidification process, namely extracting the aqueous solution containing the ABA aspic acid by using an equal-volume organic solvent, taking an organic solvent layer, and removing the organic solvent at the temperature of 20-80 ℃ to obtain an ABA aspic acid reference substance.
In a preferred embodiment of the present invention, the aforementioned aspic acid ABA control is preferably obtained by forming a dispersion using a solvent selected from acetonitrile, methanol or ethanol, filtering the precipitate, and drying the precipitate.
In a preferred embodiment of the present invention, the crude extract of raw material containing aspic acid ABA is prepared as follows: crushing raw material rhizoma dryopteris crassirhizomae, soaking the raw material rhizoma dryopteris crassirhizomae in an alcohol aqueous solution, filtering, concentrating filtrate at the temperature of 30-80 ℃ in the dark until no alcohol smell exists, adding an organic solvent into the filtrate, and calculating the volume ratio of the organic solvent: the concentrated solution is 0.5-3: 1, extracting, repeating the extraction for 2-5 times, combining organic solvents, and removing the organic solvents to obtain a crude raw material extract of the ABA aspidisate.
In a preferred embodiment of the present invention, in the normal phase chromatography purification step, the ratio of petroleum ether: the proportion of the halogenated alkane solvent is 80-60: 1, and an acidic pH regulator accounting for 0.05-0.2 wt% of the total solvent is added into the halogenated alkane solvent.
In a preferred embodiment of the present invention, in the normal phase chromatography purification step, the acidic pH adjuster added in the normal phase chromatography purification step is an organic acid, preferably acetic acid or formic acid; the halogenated alkane solvent is dichloromethane, and the alcohol solvent is methanol.
In a preferred embodiment of the present invention, the alcohol-based aqueous solution is 80% to 95% methanol aqueous solution or 80% to 95% ethanol aqueous solution, and the organic solvent is ethyl acetate.
In a preferred embodiment of the present invention, in the reverse phase chromatographic purification step, the reaction mixture is purified by a reverse phase chromatographic purification process using acetonitrile: eluting with an acidic aqueous solution at a ratio of 4.5-4: 1, wherein the acidic aqueous solution is 0.004-0.01 mol/L of Na2HPO4-citric acid buffered saline solution.
In a preferred embodiment of the present invention, in the normal phase chromatography purification step, the filler of the normal phase chromatography column is silica gel having a particle size of 100 to 300 mesh, and the filler of the reverse phase chromatography column is octadecylsilane chemically bonded silica having a particle size of 5 to 10 μm.
In a preferred embodiment of the present invention, in the crude solvent treatment step, acetone is added to the first crude powder in an amount of 3 to 5 times by mass ratio, and the mixture is heated and dissolved at a temperature of 45 ℃.
The invention also provides a solid reference substance of the aspic acid ABA with the purity of more than 99 percent, which is prepared by the preparation method.
Compared with the literature and the previous invention reports, the purity of the aspic acid ABA prepared by the method is improved a lot, the aspic acid ABA with the purity of more than 99% can be quickly obtained, and the extraction rate and the flux are greatly improved. Specifically, the invention has the following characteristics:
1. the invention provides a high-flux method for preparing aspic acid for the first time, can prepare a large amount of pure aspic acid ABA products from aspic japonica, provides a solid material foundation for the identification of traditional Chinese medicines of aspic japonica and aspic cyrtomium fortunei charcoal and the quality standard of medicinal materials, and has higher economic value.
2. Compared with the ether hot extraction of the existing extraction method, the method has the advantages that the extraction by adopting the alcohol solvent is more economic and safer; meanwhile, the yield of the aspic acid ABA prepared by the method can reach as high as 0.066%, the purity can be more than 99% and even more than 99.5%, the efficiency is high, and the cost is low.
3. The invention discovers the reason that the purity is difficult to improve in the aspic acid ABA extraction method, overcomes the technical problem that aspic acid ABA is unstable in an aqueous solution, and solves the problem that the high-pressure column chromatographic separation system is difficult to improve the aspic acid ABA.
Drawings
FIG. 1 is a schematic flow chart of the extraction of aspic acid ABA in example 1 of the present invention;
FIG. 2 is an MS spectrum of pure aspic acid ABA obtained in example 1 of the present invention;
FIG. 3 shows pure sheep maleic acid ABA1H-NMR spectrum (CD)3OD-d4，500MHz)；
FIG. 4 shows the ABA purity of aspic acidArticle of manufacture13C-NMR spectra (CD)3OD-d4，150MHz)；
FIG. 5 is an HPLC profile of aspic acid ABA product of example 1 of the present invention;
FIG. 6 is an HPLC chromatogram of aspic acid ABA product of comparative example 1 of the present invention.
Detailed Description
The following describes specific embodiments of the present invention.
In the present invention, aspic acid ABA is also sometimes referred to as a target or an isolated target. The percentage of the purity and content of the objective substance in the present invention is the content by mass ratio, if not specifically stated. The percentage ratio of the liquid to the liquid content, for example the ratio of the formic acid content in the aqueous solution, is measured in volume fraction, if not specified otherwise.
The inventor of the invention finds that a large amount of aspic acid ABA with high purity cannot be obtained by the conventional separation method of gradually increasing the gradient of chromatographic separation. The reason may be that the conventional method for gradually increasing the degree of chromatographic separation, such as slow elution by reducing the elution efficiency of the solvent, reducing the packing strength of column chromatography, etc., may result in slow extraction and separation speed, low stability of ABA of pamoic acid, and low-efficiency and low-speed separation process, the self-decomposition may result in purity reduction, so that it has been difficult to obtain a standard product with high purity in large quantity. The inventor of the invention discovers, through research, that in a conventional separation method, a special crude product solvent treatment process and a solidification process are matched, so that the separation efficiency is improved, a large amount of aspic acid ABA can be obtained, and the purity of a final product is also improved.
Specifically, the invention can separate and obtain sheep-maleic acid ABA with high purity through a simple and efficient operation process. The preparation and separation method sequentially comprises the following steps:
normal phase chromatography purification procedure: loading the crude raw material extract containing the ABA pamoate to a normal-phase chromatographic column, and eluting by using mixed eluent containing petroleum ether, halogenated alkane solvent and alcohol solvent, wherein the weight ratio of petroleum ether: the ratio of the halogenated alkane solvent is 100-50: 1, the halogenated alkane solvent is: the ratio of the alcohol solvent is 20-5: 1, collecting eluent according to TLC or HPLC detection, concentrating and drying the eluent to obtain a first crude product powder;
a crude product solvent treatment process: heating the first crude powder at a temperature of below 60 ℃ by using a ketone solvent with 2-6 times of the mass ratio and 3-6 carbon atoms in the molecule to dissolve the first crude powder, standing the first crude powder at a temperature of below 10 ℃ for 1-24 hours, filtering the precipitate, and drying the precipitate to obtain second crude powder;
a reversed phase chromatography purification procedure: loading the second crude powder to reverse phase chromatography, and eluting with a mixed eluent of acetonitrile or methanol and an acidic aqueous solution with the pH value of 3-6 at the pressure of 1-15.0 Mpa, wherein the ratio of acetonitrile or methanol: the ratio of the acidic aqueous solution is 5.5-3.5: 1, eluent is collected according to TLC or HPLC detection, the eluent is concentrated under reduced pressure at the temperature of below 50 ℃, acetonitrile or methanol in the eluent is removed, and an aqueous solution containing the ABA of the aspic acid is obtained;
and a solidification process, namely extracting the aqueous solution containing the ABA aspic acid by using an equal-volume organic solvent, taking an organic solvent layer, and removing the organic solvent at the temperature of 20-80 ℃ to obtain an ABA aspic acid reference substance.
The sequence of the above-described processes is important and cannot be easily replaced.
The normal phase chromatography purification step is intended to distinguish impurities having a large difference in polarity from the target, and in the normal phase chromatography purification step, the manner of loading the crude raw material is not particularly limited, and the crude raw material may be loaded by dissolving it in a good solvent, or may be loaded by stirring it with a filler, and in order to reduce the compression of the loading volume, it is preferable to load the raw material with a stirring filler.
The filler of the normal phase chromatographic column used in the normal phase chromatographic purification step may be a known normal phase filler, i.e., a filler in which the stationary phase has a polarity higher than that of the mobile phase, and if the mobile phase is an organic solvent, a commonly used filler is silica gel (specifically, SiO) which is an organic solvent2Silicon dioxide), Al2O3Polar bonded phase fillers, etc., silica gel is preferably used in the present invention. The particle size of the silica gel is not particularly limited, and is readily available from efficiency and fillersIn a preferred method of the present invention, in the first normal phase column purification step, the filler of the normal phase column is silica gel having a particle size of 100 to 300 mesh. In order to obtain a better separation efficiency, and from the viewpoint of the degree of separation and the adsorption loss, silica gel is preferably used, and silica gel having a particle size of 100 to 300 mesh is more preferably used.
In the normal phase chromatography purification step, petroleum ether is a light petroleum product, which is a mixture of hydrocarbons (mainly pentane and hexane) having low relative molecular mass, and although it is referred to as ether, it has no ether bond (C — O — C) in its structure, and is not a colorless transparent liquid and has a kerosene odor. Commercially available petroleum ether is generally classified into three boiling range specifications of 30 to 60 ℃, 60 to 90 ℃ and 90 to 120 ℃, and any of the above can be used in the present invention, and preferably commercially available petroleum ether having a boiling range of 60 to 90 ℃ can be used.
In the normal phase chromatography purification step, the mixed eluent of the haloalkane solvent and the alcohol solvent is preferably used to distinguish the target substance from impurities, and the halohydrocarbon solvent is a saturated or unsaturated chlorinated hydrocarbon having 1 or 2 carbon atoms, and is usually selected from the group consisting of dichloromethane, trichloromethane, 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. More preferred are dichloromethane, trichloromethane, 1, 1, 1-trichloroethane, trichloroethylene and tetrachloroethylene, and still more preferred are dichloromethane and trichloromethane. As the alcohol solvent, methanol, ethanol or isopropanol may be used. As a preferred combination in the present invention, a combination of dichloromethane and methanol can be used, resulting in a better separation effect.
In the normal phase chromatographic purification procedure, petroleum ether: the preferable ratio of the halogenated alkane solvent is 80-60: 1. And wherein the haloalkane hydrocarbon solvent: the proportion of the alcohol solvent is preferably 10-8: 1. in order to increase the degree of separation in the normal phase chromatography purification step, it is preferable to add an acidic pH adjuster in an amount of 0.05 to 0.2 wt% of the total amount of the eluent to the mixed eluent of petroleum ether, a haloalkane solvent, and an alcohol solvent. Examples of the acidic pH adjuster include organic acids such as formic acid, acetic acid, propionic acid, butyric acid, and glycolic acid, and inorganic acids such as hydrochloric acid and dilute sulfuric acid, with organic acids being preferred, and formic acid being more preferred. By using a combination of petroleum ether, methylene chloride and methanol, and adding formic acid as a pH adjuster, the separation efficiency in the normal phase chromatography purification step in the present invention can be further improved. The amount of the pH adjuster to be added is more preferably 0.08 to 0.11 wt%.
The crude solvent treatment step is the most important step in the present invention, and the order of the step and the preceding and following steps is also very important. The inventor finds that the solvent dissolution behavior of the aspic acid ABA has self characteristics in the substance with polarity similar to the aspic acid ABA obtained in the normal-phase chromatographic purification process, and the solvent dissolution behavior can be rapidly distinguished from other impurities by utilizing the characteristics. The process can obviously compress the time of the whole extraction and separation method, thereby reducing the self decomposition of the target substance, and improving the purity of the final product.
Specifically, if a ketone solvent with the carbon number of 3-6 in a molecule is used for heating and dissolving the part obtained in the normal phase chromatography purification process, and then the part is cooled to separate out a solute, most of ABA of pamoic acid is separated out, most of impurities are left in the solution, and a target crude product with the purity of more than 80% is rapidly obtained. And the step is simple and convenient to operate, the separation difficulty is integrally reduced, and the yield and the purity of a final product are improved. And the flux of the treatment in the step is very large, so that the method is very suitable for preparing a large amount of reference substances.
Examples of the ketone solvent having 3 to 6 carbon atoms in the molecule include acetone, methyl ethyl ketone, methyl isobutyl ketone and the like. Acetone is preferably used in the present invention. The temperature for heating and dissolving should not be too high in order to reduce the decomposition of the target substance itself. Preferably 60 ℃ or lower, more preferably 55 ℃ or lower, and still more preferably 50 ℃ or lower. The ketone solvent used is not particularly limited, and a ketone solvent having 3 to 5 times (by mass) the number of carbon atoms in the molecule is preferably 3 to 6.
In the invention, the aspic acid ABA can be obtained conventionally even without a crude solvent treatment process, however, in order to obtain high-purity aspic acid ABA, more solvents and time are required, and the recovery rate is greatly reduced. In contrast, the highest separation efficiency can be obtained by performing the crude solvent treatment step between the normal phase chromatography purification step and the reverse phase chromatography purification step, and if the treatment steps are exchanged, the separation efficiency and the recovery rate are significantly reduced.
The filtration method in the present invention is not particularly limited, and filtration can be carried out using a conventional filter, suction filtration apparatus, or microfiltration membrane apparatus. The drying in the present invention is not particularly limited, and an oven, an infrared lamp, a hot plate, or the like may be used, and the drying temperature may be appropriately selected by those skilled in the art.
In the present invention, the concentration method and the method for removing the organic solvent are not particularly limited, and heating evaporation concentration, rotary evaporator concentration, and the like can be used. The water used in the present invention is not particularly limited, and any water commonly used such as tap water, distilled water, and deionized water can be used.
In the present invention, the reverse phase chromatographic separation is used to separate other components having a polarity different from that of the target. In this step, the mode of loading is not particularly limited, and the loading may be dissolved in a good solvent and dropped, or may be stirred with a filler, and for reducing the compression of the loading volume, stirred filler loading is preferred. However, in the present invention, the crude target product obtained from the crude solvent treatment step is not well soluble in the eluent of the reverse phase chromatography, and it is preferable to perform the sample application after dissolving the crude target product in a mixed solvent of a halogenated hydrocarbon and acetonitrile.
The process can realize the balance between the resolution and the speed by adopting the elution pressure of 5-15 Mpa, and the further preferable column pressure is 5-10 Mpa. In the separation step, the pressure of the reverse phase column is more preferably 5 to 8MPa, and this pressure is particularly preferable because the separation effect and the separation rate can be balanced.
As the reversed-phase packing of the reversed-phase column used in the column preparation step of the present invention, known nonpolar silica gel having an alkane as a bonded functional group (e.g., C18(ODS), C8, C4, etc.) can be used. Preferably a C18(ODS) silica gel column, i.e. octadecylsilane bonded silica, preferably using 5-60 μm filler, reasonable particle size is advantageous to maintain proper column pressure and resolution, octadecylsilane bonded silica is readily available on the market. From the viewpoint of efficiency and easy availability of the filler, in the preferred preparation method of the invention, the filler of the reversed phase chromatographic column is octadecylsilane chemically bonded silica with the particle size of 5-10 μm.
According to the principle of the present invention, the reverse phase column to be used in the solidification step may be any reverse phase column, and may be a medium-pressure or high-pressure one as long as the loading of the packing satisfies the capacity of the target product, and for better separation effect, a high-pressure reverse phase column, that is, a reverse phase column having a pressure of 5Mpa or more is preferably used.
In the reverse phase chromatographic purification step of the present invention, acetonitrile or methanol may be used as the organic phase eluent, and acetonitrile is preferably used as the organic phase eluent because a better degree of separation is obtained. In the reverse phase chromatography purification process, a buffer salt is required to be added into the water phase of the eluent, so that the pH of the water phase is adjusted to 3-6, preferably 4-5. As such a buffer salt system, Na may be mentioned2HPO4+ citric acid, K2HPO4+ citric acid, NaH2PO4+ citric acid KH2PO4+ citric acid. The content of the buffer salt system is not particularly limited, and is preferably 0.004 to 0.01 mol/L.
A preferred combination of eluents for the purification step by reverse phase chromatography may include acetonitrile and 0.004 to 0.06mol/L of Na2HPO4-a combination of citric acid buffered saline solutions.
The solidification step of the present invention is intended to rapidly extract a target substance from an elution solution. The present inventors have found that the target substance having a purity of 95% or more cannot be obtained by directly concentrating the eluate containing the target substance obtained from the reverse phase chromatography separation step. For unknown reasons, it is likely that the buffer salt system in the eluent will lead to substantial decomposition of the target during concentration, resulting in a reduction in purity. Even if a technique such as freeze-drying is used, high purity cannot be easily achieved.
The reason is not clear, and the method can obtain the aspic acid ABA solid with very high purity by utilizing the processes of extracting an aqueous solution containing aspic acid ABA by using an organic solvent with the same volume and then evaporating the organic solvent to dryness. The organic solvent in the solidification step is not particularly limited, and a halogenated hydrocarbon solvent, an ester solvent, toluene, benzene, or the like can be used. It is preferable to use a halogenated hydrocarbon solvent, and methylene chloride is more preferable. In the process of organic solvent extraction, sodium bicarbonate, anhydrous sodium sulfate, etc. may be added to the organic solvent to remove acid and water in the organic solvent, thereby further improving the purity of the target product. In the solidification process, the organic solvent is removed at 20-80 ℃, preferably at 20-50 ℃, and the solid is the aspic acid ABA reference substance after drying. Drying the solid, preferably using a solvent selected from acetonitrile, methanol or ethanol to form a dispersion, then filtering to obtain a precipitate, and drying the precipitate to obtain a solid powder with a more loose form.
In the present invention, the crude extract of raw material containing aspidistric acid ABA can be any extract containing aspidistric acid ABA, such as commercially available aspidistra elatior extract. In order to obtain more excellent separation effect and recovery rate, the raw material extract containing ABA aspic is preferably obtained by the following method:
crushing raw material rhizoma dryopteris crassirhizomae, soaking the raw material rhizoma dryopteris crassirhizomae in an alcohol aqueous solution, filtering, concentrating filtrate at the temperature of 30-80 ℃ in the dark until no alcohol smell exists, adding an organic solvent into the filtrate, and calculating the volume ratio of the organic solvent: the concentrated solution is 0.5-3: 1, extracting, repeating the extraction for 2-5 times, combining organic solvents, and removing the organic solvents to obtain a crude raw material extract of the ABA aspidisate. In the invention, the organic solvent is preferably extracted and combined for 3 times, namely complete extraction of the aspidic acid ABA is realized.
In a preferred embodiment of the present invention, the alcohol-based aqueous solution is an 80% to 95% methanol aqueous solution or an 80% to 95% ethanol aqueous solution. In the present invention, the alcohol solvent in the substantially mixed solution is substantially evaporated off (content: less than 5%) by concentrating to free of alcohol smell, and only water which is difficult to evaporate is left. Therefore, the extraction process described above, at a later stage, corresponds to a two-phase extraction of water and organic solvent. The organic solvent is not particularly limited, and may be benzene, toluene, chloroform, carbon tetrachloride, methylene chloride, n-butanol, ethyl acetate, n-hexane, cyclohexane, etc., which are commonly used, and here, ethyl acetate is preferred.
The invention also provides a solid reference substance of the aspic acid ABA with the purity of more than 99%, which is prepared by the preparation method.
Based on the above description, the present invention has the following features: the method is simple and efficient to operate, can amplify the process, and can separate a large amount of aspic acid ABA monomers. According to the method, through the deep research of the solubility difference between the ABA of the aspidic acid and impurities, the separation process is greatly simplified and the separation difficulty is reduced by combining a solvent treatment process at a specific stage of the separation process. According to the invention, the stability of the aspic acid ABA is further studied, the final solidification process is improved, and the method enables the aspic acid ABA with the purity higher than 99% to be obtained quickly, and is particularly suitable for being used as a high-quality reference substance. Meanwhile, the whole process is suitable for amplification, and the preparation flux of the reference substance can be greatly improved.
Examples
The present invention is described in detail below by way of examples, but is not meant to be limited to any of the foregoing. Having described the invention in detail and having disclosed specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.
The reagents used in the examples were purchased from Aldrich, national reagents, and the like. The instrument conditions were as follows:
MS analysis conditions
The instrument comprises the following steps: sciex Triple TOF 4600LC/MS
Detection mode: negative ion mode
ESI source parameters
HPLC chromatographic conditions
The sample solution is prepared by dissolving 1.0mg sample in 1mL methanol to obtain 1.0mg/mL sample, and filtering with 0.22 μm filter membrane.
The instrument comprises the following steps: agilent 1260;
a chromatographic column: agilent Plus C18, 4.6X 150mm,5 μm;
column temperature: 30 ℃;
sample introduction amount: 10 mu L of the solution;
detection wavelength: 302 nm;
gradient setting of mobile phase (A: acetonitrile, B:0.005mol/L Na)2HPO4-citric acid buffer salt pH ═ 5)
Example 1 preparation of aspic acid ABA control in aspic elata
Referring to fig. 1, the method comprises the following process steps:
A) extracting medicinal materials: 5kg of rhizoma dryopteris crassirhizomae medicinal materials, Jilin of the producing area, and a crusher for crushing, wherein the temperature is prevented from being too high in the crushing process; after being crushed, the mixture is extracted by 50 liters of 95 percent ethanol cold soaking for 72 hours, and each 12-hour test is stirred once to ensure that the extraction is more sufficient; repeatedly extracting for 2 times, mixing extractive solutions, and filtering; concentrating under reduced pressure at 40 deg.C to obtain 3L concentrated solution, and ensuring no ethanol residue in the concentrated solution to avoid affecting subsequent extraction;
B) and (3) extraction: extracting the concentrated solution with 3L ethyl acetate, repeating for 2-3 times, evaluating by TLC or HPLC whether extraction is complete, and mixing ethyl acetate layers; concentrating under reduced pressure at 40 deg.C to obtain 165.3g ethyl acetate layer extract;
C) silica gel column chromatographic separation: dissolving ethyl acetate layer extract with 300 ml ethyl acetate, mixing with 200-mesh 300-mesh silica gel (Qingdao sea) with equal mass, drying, and grinding; packing the column by a wet method by using 10 times of 200-mesh and 300-mesh silica gel by mass and using petroleum ether as a solvent; mixing the sample with silica gel, performing gradient elution with petroleum ether, dichloromethane, methanol, acetic acid, 80:10:1:0.1 → 65:10:1:0.1, collecting every 0.2 column volumes, and detecting the target compound by TLC or HPLC; merging target fractions, concentrating at 40 ℃ under reduced pressure to enrich the ABA of the pamoic acid, and detecting the purity of the ABA by using HPLC (high performance liquid chromatography) to be 50-70 percent, wherein the weight of the ABA is 14.49 g;
D) solvent treatment: adding the ABA of the pamoic acid enriched in the step C) into 3-5 times of acetone, heating at 45 ℃ and carrying out ultrasonic treatment to dissolve the ABA of the pamoic acid; after dissolution, the solution is kept stand for 24 hours at 4 ℃; after precipitation (crystal shape), filtering to obtain 4.17g of half-finished product ABA of aspic acid, and improving the purity to 80% -95%; concentrating the filtrate under reduced pressure to a certain volume, and continuing trying to separate out a precipitate;
E) high-pressure liquid phase preparation: dissolving the precipitate obtained in the step D) with a solvent of 3-5 times the volume of dichloromethane and acetonitrile (5: 1), and then carrying out high-pressure column chromatography separation, wherein a small amount of solvent is used for dissolving the precipitate as much as possible in the step, so that the damage of excessive dichloromethane in the solvent to the high-pressure filler is avoided. The filler used for the high-pressure column chromatography is ODS reversed-phase filler with the particle size of 5 mu m; the mobile phase is A: acetonitrile, B:0.005mol/L Na2HPO4-citric acid buffer salt (pH 5), a: B80: 20; setting the detection wavelength to be 302 nm; collecting the target flow.
F) Recovery (solidification): concentrating the target fraction obtained in the step E) at 40 ℃ under reduced pressure until no acetonitrile exists, extracting for 2-3 times by using dichloromethane with the same volume, combining dichloromethane layers, and detecting whether the extraction is complete by HPLC; adding anhydrous sodium sulfate into the dichloromethane layer to remove water, filtering, and concentrating the filtrate under reduced pressure to dryness; adding acetonitrile, performing ultrasonic treatment, performing suction filtration to obtain light yellow powder, and performing vacuum drying at 25 ℃ to obtain 3.3g of aspic acid ABA with the purity of more than 99.5%. The HPLC chromatogram for purity detection is shown in FIG. 5.
Obtained aspic acid ABA1H and13the C NMR spectrum is shown in FIGS. 3 and 4, and the mass spectrum is shown in FIG. 2.
Comparative example 1
5Kg of rhizoma Dryopteris Crassirhizomatis was purchased and the pretreatment was performed in the same manner as in A, B, C, D, E of example 1. Removing acetonitrile from the aqueous solution containing the aspic acid ABA obtained in the step E) by using a rotary evaporator, freeze-drying the obtained aqueous solution to obtain solid aspic acid ABA, adding acetonitrile into the solid aspic acid ABA, performing ultrasonic treatment, and performing suction filtration to obtain light yellow powder. Referring to fig. 6, the HPLC profile of the purity test shows a significant reduction in purity compared to example 1.
The technical features disclosed above are not limited to the combinations with other features disclosed, and other combinations between the technical features can be performed by those skilled in the art according to the purpose of the invention to achieve the aim of the invention, and various modifications made to the technical scheme of the invention by those skilled in the art without departing from the design spirit of the invention shall fall within the protection scope defined by the claims of the invention.
Claims (10)
1. A preparation method of an ABA pamoate reference substance is characterized by sequentially comprising the following steps of:
normal phase chromatography purification procedure: loading the crude raw material extract containing the ABA pamoate to a normal-phase chromatographic column, and eluting by using mixed eluent containing petroleum ether, halogenated alkane solvent and alcohol solvent, wherein the weight ratio of petroleum ether: the ratio of the halogenated alkane solvent is 100-50: 1, the halogenated alkane solvent is: the ratio of the alcohol solvent is 20-5: 1, collecting eluent according to TLC or HPLC detection, concentrating and drying the eluent to obtain first crude product powder;
a crude product solvent treatment process: heating and dissolving the first crude powder at a temperature of below 60 ℃ by using a ketone solvent with 2-6 times of carbon number in a molecule of 3-6 in terms of mass ratio, standing for 1-24 hours at a temperature of below 10 ℃, filtering and precipitating, and drying the precipitate to obtain second crude powder;
a reversed phase chromatography purification procedure: loading the second crude powder to reverse phase chromatography, and eluting with a mixed eluent of acetonitrile or methanol and an acidic aqueous solution with the pH value of 3-6 at the pressure of 1-15.0 Mpa, wherein the ratio of acetonitrile or methanol: the ratio of the acidic aqueous solution is 5.5-3.5: 1, eluent is collected according to TLC or HPLC detection, the eluent is concentrated under reduced pressure at the temperature of below 50 ℃, acetonitrile or methanol in the eluent is removed, and an aqueous solution containing the ABA of the aspic acid is obtained;
and a solidification process, namely extracting the aqueous solution containing the ABA aspic acid by using an equal-volume organic solvent, taking an organic solvent layer, and removing the organic solvent at the temperature of 20-80 ℃ to obtain an ABA aspic acid reference substance.
2. The method for preparing the reference of aspic acid ABA as claimed in claim 1, wherein the crude raw material extract containing aspic acid ABA is prepared as follows:
crushing raw material rhizoma dryopteris crassirhizomae, soaking the raw material rhizoma dryopteris crassirhizomae in an alcohol aqueous solution, filtering, concentrating filtrate at the temperature of 30-80 ℃ in the dark until no alcohol smell exists, adding an organic solvent into the filtrate, and calculating the volume ratio of the organic solvent: the concentrated solution is 0.5-3: 1, extracting, repeating the extraction for 2-5 times, combining organic solvents, and removing the organic solvents to obtain a crude raw material extract of the ABA aspidisate.
3. The method for preparing a reference substance of ABA aspirates according to claim 1, wherein in the normal phase chromatography purification step, the ratio of petroleum ether: the proportion of the halogenated alkane solvent is 80-60: 1, and an acidic pH regulator accounting for 0.05-0.2 wt% of the total solvent is added into the eluent.
4. The method for preparing the reference substance of ABA aspinate according to claim 3, wherein the acidic pH adjusting agent added in the normal phase chromatography purification process is an organic acid, the haloalkane solvent is dichloromethane, and the alcohol solvent is methanol.
5. The method for preparing the reference of ABA aspinate according to claim 2, wherein the alcohol-based aqueous solution is 80-95% methanol aqueous solution or 80-95% ethanol aqueous solution, and the organic solvent is ethyl acetate.
6. The method for preparing a reference substance of ABA aspicate according to claim 1, wherein in the step of reverse phase chromatography purification, the ratio of acetonitrile: eluting with an acidic aqueous solution at a ratio of 4.5-4: 1, wherein the acidic aqueous solution is 0.004-0.01 mol/L of Na2HPO4-citric acid buffered saline solution.
7. The method for preparing ABA aspinate reference substance according to claim 1, wherein in the normal-phase chromatography purification step, the filler of the normal-phase chromatography column is silica gel with a particle size of 100-300 meshes, the filler of the reverse-phase chromatography column is octadecylsilane chemically bonded silica with a particle size of 5-10 μm, and the acidic pH adjusting agent is acetic acid or formic acid.
8. The method for preparing reference substance of ABA aspinate according to claim 1, wherein in the crude solvent treatment step, acetone is added to the first crude powder in an amount of 3 to 5 times by mass ratio, and the mixture is heated and dissolved at a temperature of 45 ℃.
9. The method for preparing a control of aspic ABA according to claim 1, wherein in the solidification step, the solid obtained by removing the organic solvent is further dispersed in a solvent selected from acetonitrile, methanol or ethanol to form a dispersion, the precipitate is filtered, and the precipitate is dried to obtain the control of aspic ABA.
10. A solid control of aspic acid ABA with a purity of 99% or more, which is prepared by the preparation method of claim 1.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102813713A (en) * | 2012-08-01 | 2012-12-12 | 中国农业大学 | Rhizoma dryopteris crassirhizomae and fructus crataegi composition, preparation method and application of composition |
| CN105699510A (en) * | 2016-01-22 | 2016-06-22 | 山东省食品药品检验研究院 | Content determination method of kaempferitrin in rhizoma dryopteris crassirhizomatis crude medicine |
| CN107661322A (en) * | 2017-01-13 | 2018-02-06 | 北京中医药大学 | The new application of phloroglucinol derivatives compound in thick wood-fern rhizome |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102813713A (en) * | 2012-08-01 | 2012-12-12 | 中国农业大学 | Rhizoma dryopteris crassirhizomae and fructus crataegi composition, preparation method and application of composition |
| CN105699510A (en) * | 2016-01-22 | 2016-06-22 | 山东省食品药品检验研究院 | Content determination method of kaempferitrin in rhizoma dryopteris crassirhizomatis crude medicine |
| CN107661322A (en) * | 2017-01-13 | 2018-02-06 | 北京中医药大学 | The new application of phloroglucinol derivatives compound in thick wood-fern rhizome |
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| 马国祥等: "薄层色谱扫描法测定7种贯众中东北贯众素及绵马酸的含量", 中国药科大学学报, vol. 25, no. 6, pages 376 * |
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