CN106632542B - Preparation method of cimicidin glycoside and 5-O-methylvisammioside reference substance - Google Patents
Preparation method of cimicidin glycoside and 5-O-methylvisammioside reference substance Download PDFInfo
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Abstract
The invention discloses a method for preparing a reference substance of linarin glycoside and 5-O-methylvisammioside. Specifically, the method of the present invention comprises the steps of: (i) providing an alcoholic extract mixture of linarin glycoside and 5-O-methylvisammioside; (ii) mixing the alcohol extraction mixture with a solvent to obtain a first mixture, and separating the first mixture by using a medium-pressure preparation column to obtain a crude product of the cimetidine glycoside and a crude product of the 5-O-methylvisammol glycoside; and (iii) recrystallizing the crude linalooside and/or the crude 5-O-methylvisammioside with a solvent to obtain a linalooside control and/or a 5-O-methylvisammioside control. The method can realize the synchronous separation of the linarin glycoside and the 5-O-methylvisammioside, has large preparation amount, high yield and high product purity, and is suitable for industrial production.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine modernization, relates to a preparation method of a traditional Chinese medicine chemical reference substance, and particularly provides a method for separating a high-purity cimicidin glycoside and 5-O-methylvisammioside glycoside reference substance from an anti-rheumatic drug.
Background
Radix Saposhnikoviae is the dried root of Saposhnikovia divaricata (Turcz.) Schischk. Nowadays, the traditional Chinese medicine composition is mainly distributed in Heilongjiang, inner Mongolia, Liaoning, Jilin, Hebei and Shandong, has wide distribution and strong adaptability, is one of the most commonly used traditional Chinese medicines in China, is pungent in taste, warm in nature, enters bladder, liver and spleen channels, and has the effects of relieving exterior syndrome, dispelling wind, eliminating dampness and relieving spasm. Can be used for treating wind cold, headache, blurred vision, general pain, wind cold paralysis, arthralgia, and spasm of limbs.
Linarin and 5-O-methylvisammioside are index components collected in 2010 edition, linarin (Prim-O-glucopyranosyl cimicifugin) has molecular formula C22H28O115-O-methylvisammiol glycoside (5-O-methylvisamminol β -D-glucopyranoside) with molecular formula C22H28O10Their structural formula is as follows:
many patents are issued for the detection methods of the linarin glycoside and the 5-O-methylvisammioside in the Chinese medicinal preparation (such as the biond magnolia rhinitis pill, the yupingfeng powder, the tongkongping and the like) composed of the divaricate saposhnikovia root, for example, the patent application with the application number of 201310504883.5 discloses a method for determining the content of the biond magnolia rhinitis pill. At present, domestic literature on separation and purification of the reference substances of the cimicidin and the 5-O-methylvisammioside is few, in addition, the cimicidin and the 5-O-methylvisammioside are listed as standards in 2010 pharmacopeia, the two reference substances are more and more in demand, particularly, the content of the 5-O-methylvisammioside in medicinal materials is very low, and the separation and purification of monomers from the medicinal materials are very difficult, so that the reference substances of the cimicidin and the 5-O-methylvisammioside from divaricate saposhnikovia root are very necessary and have wide application value.
Disclosure of Invention
The invention aims to provide a method for separating and purifying a reference substance of the cimetidine glycoside and the 5-O-methylvisammioside glycoside. The method can realize the synchronous separation of the reference substance of the cimicidin and the 5-O-methylvisammioside, has high separation efficiency, stable process and low cost, and can prepare a large amount of the reference substances of the cimicidin and the 5-O-methylvisammioside with the purity of more than 99 percent.
In a first aspect of the invention, a method for preparing a reference substance of linarin glycoside and 5-O-methylvisammioside is provided, which comprises the following steps:
(i) providing an alcoholic extract mixture of linarin glycoside and 5-O-methylvisammioside;
(ii) mixing the alcohol extraction mixture with a solvent to obtain a first mixture, and separating the first mixture by using a medium-pressure preparation column to obtain a crude product of the cimetidine glycoside and a crude product of the 5-O-methylvisammol glycoside; and
(iii) recrystallizing the crude linarin glycoside and/or the crude 5-O-methylvisammioside with a solvent to obtain a linarin glycoside reference substance and/or a 5-O-methylvisammioside reference substance.
In another preferred example, the step (ii) further comprises the steps of:
(ii-1) performing a first stage elution with 30-35% (v/v) methanol solution as a first eluent for a medium pressure preparative column, concentrating the eluate to obtain a crude linarin glycoside; and
(ii-2) performing a second-stage elution with a 45-50% (v/v) methanol solution as a second eluent for the medium-pressure preparative column, and concentrating the eluate to obtain a crude product of 5-O-methylvisammioside.
In another preferred embodiment, the medium pressure preparation column in step (ii) is provided with a column pressure of 0-2MPa, preferably 0.5-2 MPa.
In another preferred embodiment, the lower column pressure limit of the medium pressure preparation column in step (ii) is selected from the group consisting of: 0MPa, 0.1MPa, 0.2MPa, 0.3MPa, 0.4MPa, or 0.5 MPa.
In another preferred embodiment, the upper limit of the column pressure of the medium pressure preparation column in step (ii) is selected from the group consisting of: 2MPa, 2.1MPa, 2.2MPa, 2.3MPa, 2.4MPa, or 2.5 MPa.
Here, when the column pressure of the medium-pressure preparative column is 0MPa, it means that the medium-pressure preparative column is under a standard atmospheric pressure.
In another preferred example, the filler of the medium pressure preparation column in step (ii) is octadecylsilane chemically bonded silica.
In another preferred embodiment, the filler particle size of the medium pressure preparation column in step (ii) is 40-60 μm.
In another preferred embodiment, the purity of the crude product of the l-glucosylcimifugin is more than or equal to 93.0%.
In another preferred embodiment, the purity of the crude 5-O-methylvisammioside is more than or equal to 98.0%.
In another preferred embodiment, the step (iii) further includes the steps of:
(iii-1) mixing the crude linarin with pure water and acetone, and recrystallizing the crude linarin in a mixed solvent; separating and drying the obtained crystal to obtain a cimicidin reference substance; and/or
(iii-2) mixing the crude 5-O-methylvisammioside with methanol and acetone, and recrystallizing the crude 5-O-methylvisammioside in a mixed solvent; separating and drying the obtained crystal to obtain the 5-O-methylvisammioside control product.
In another preferred embodiment, said step (iii-2) is performed before said step (iii-1), or simultaneously.
In another preferred embodiment, 45-55% (v/v) methanol solution is used in said step (ii) to mix with said alcoholic extract mixture of linalooside and 5-O-methylvisammioside.
In another preferred embodiment, the pure water in the step (iii-1) has an electric conductivity of 18.2. mu.s/cm or less.
In another preferred embodiment, the purity of the methanol in the step (iii-2) is more than or equal to 99.5%.
In another preferred embodiment, the purity of the acetone in the steps (iii-1) and (iii-2) is more than or equal to 99.5%.
In another preferred example, in the step (iii-1), acetone is added in an amount of 5 to 10 times (v/v) the amount of pure water; and/or
In the step (iii-2), the amount of acetone added is 5-10 times (v/v) the amount of methanol.
In another preferred embodiment, the number of times of repetition of the recrystallization operation in the step (iii-1) is 2 or more.
In another preferred embodiment, the number of times of repetition of the recrystallization operation in the step (iii-2) is 2 or more.
In another preferred embodiment, the purity of the l-ephedrine reference substance is more than or equal to 99.0%, preferably, the purity is more than or equal to 99.3%, and more preferably, the purity is more than or equal to 99.5%.
In another preferred embodiment, the purity of the 5-O-methylvisammioside reference substance is more than or equal to 99.0%, preferably, the purity is more than or equal to 99.5%, and more preferably, the purity is more than or equal to 99.6%.
In another preferred embodiment, the alcohol extraction mixture of the linalooside and the 5-O-methylvisammioside is prepared by a method comprising the following steps:
(i-1) mixing a divaricate saposhnikovia root medicinal material and an ethanol solution, cold-soaking and extracting the mixture with ethanol, and concentrating a liquid part after solid-liquid separation to obtain a first concentrated solution;
(i-2) adsorbing the first concentrated solution by using macroporous resin, eluting impurities by using water with 4-5 times of column volume respectively, eluting a target by using 55-65% ethanol solution with 1-2 times of column volume, collecting eluent, and concentrating to obtain a second concentrated solution; and
(i-3) separating the second concentrated solution by silica gel column chromatography, eluting with dichloromethane-methanol eluent, collecting the eluent, and concentrating to obtain the alcoholic extract mixture of linalooside and 5-O-methylvisammioside in step (i).
In another preferred embodiment, the ethanol solution is mixed with the divaricate saposhnikovia root medicinal material in an amount of 5-8(L/kg) times.
In another preferred embodiment, the ethanol solution has a concentration of 70-90% (v/v), preferably 80% (v/v).
In another preferred example, the macroporous resin is polystyrene type nonpolar adsorption resin.
In another preferred embodiment, the macroporous resin has a porosity of 42-46%.
In another preferred embodiment, the volume ratio of the dichloromethane to methanol eluent is 10:1-5: 1.
In a second aspect of the invention, a reference linarin and/or 5-O-methylvisammioside is provided, which is prepared by a method according to the first aspect of the invention.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 is a HPLC detection profile of the linarin control obtained in example 1.
FIG. 2 is a HPLC chromatogram of the 5-O-methylvisammioside control obtained in example 1.
Detailed Description
The present inventors have extensively and intensively studied and developed a method for preparing a high-purity linalooside and 5-O-methylvisammioside reference substance for the first time. The preparation method adopts a medium-pressure preparation column to separate and purify the linarin glycoside and the 5-O-methylvisammioside alcohol extract, greatly improves the separation efficiency under certain pressure, and further improves the purity of a target substance by recrystallization. On the basis of this, the present invention has been completed.
In order to achieve the purpose, the invention adopts the following technical scheme:
a preferred technical scheme of the invention is as follows:
A. raw material extraction: adding a divaricate saposhnikovia root medicinal material into 5-8 times of ethanol solution according to the volume-to-mass ratio L/Kg, carrying out cold leaching extraction for 72 hours, carrying out suction filtration, combining filtrates, and concentrating until no alcohol smell exists;
the volume percentage concentration of the ethanol solution is 80 percent;
B. and (3) macroporous resin enrichment: statically adsorbing the concentrated solution obtained in the step A by using macroporous resin overnight, then respectively eluting impurities by using water with 4-5 times of column volume, eluting the target by using 65% ethanol solution with 1-2 times of column volume, collecting eluent, and concentrating to 1/10 of the original volume;
C. silica gel column chromatography: and B, mixing the concentrated solution in the step B with 100-mesh 200-mesh silica gel, drying, grinding, taking 200-mesh 300-mesh silica gel, and packing the dichloromethane into a column by a wet method. After loading, dichloromethane was used: eluting with methanol, performing high-to-low gradient elution according to a volume ratio of 10:1-5:1, tracking and detecting by TLC, collecting eluate, and concentrating to obtain a semi-finished product of linarin and a semi-finished product of 5-O-methylvisammioside;
D. c, medium-pressure preparation, namely dissolving the semi-finished product of the glucoroniside and the 5-O-methylvisammioside in the step C by 50 percent methanol, loading the semi-finished product into a medium-pressure preparation column, eluting the semi-finished product by 33 percent methanol, and concentrating to obtain a crude product of the glucoroniside with the purity of about 93 percent; eluting with 48% methanol solution, and concentrating to obtain crude 5-O-methylvisammioside with purity of about 98%;
E. and (3) product recovery: heating and dissolving the crude linarin product with the purity of about 93 percent obtained in the step D by using a small amount of pure water, adding 5 times of acetone for diluting, transferring to a refrigerator with the temperature of 4 ℃ for refrigeration and crystallization, after crystals are separated out, carrying out suction filtration on the solid, heating and dissolving the solid by using pure water again, diluting and recrystallizing by using 5 times of acetone, and carrying out freeze drying for 2-3 times to obtain a linarin reference product with the purity of more than 99 percent; and D, heating and dissolving the crude product of the 5-O-methylvisammioside with about 98 percent obtained in the step D by using a small amount of methanol, adding 5 times of acetone for diluting, transferring to a refrigerator with the temperature of 4 ℃ for refrigerating and crystallizing, after crystals are separated out, carrying out suction filtration on the solid, heating and dissolving the solid by using methanol again, diluting and recrystallizing by using 5 times of acetone, and repeating the operation for 2-3 times for freeze drying to obtain the 5-O-methylvisammioside reference product with the purity of more than 99 percent.
The main advantages of the invention
1. The product is recovered by adopting a recrystallization method, so that the product yield is high and the purity is high;
2. the solvent consumption is low, and the solvent in the process can be recycled;
3. the solvent has low toxicity, the used solvents are ethanol, methanol and pure water, and the environmental pollution is low;
4. the method has the advantages of simple operation, short production period, high separation efficiency, stable and reliable process, good reproducibility and low cost, and is suitable for industrial production;
the invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.
Example 1
The preparation method of the reference substance of the cimicidin and the 5-O-methylvisammioside is carried out according to the following process steps:
A. extracting raw materials; adding 10Kg of radix Saposhnikoviae into 50L of 80% ethanol solution, cold soaking for 72 hr, filtering, mixing filtrates, and concentrating to 5L;
B. and (3) macroporous resin enrichment: weighing 4KgAB-8 macroporous adsorption resin, statically adsorbing the concentrated solution obtained in the step A for 12 hours by using the macroporous adsorption resin, eluting impurities by using 40L of water, eluting the target object by using 6L of 65% ethanol solution, collecting the eluent, and concentrating to 100 ml;
C. silica gel column chromatography: and B, mixing 100ml of the concentrated solution in the step B with 100g of 200-mesh silica gel, drying, grinding, taking 500g of 200-mesh 300-mesh silica gel, and filling the silica gel into a column by using a dichloromethane wet method. After loading, dichloromethane was used: eluting with methanol at a volume ratio of 10:1-5:1 from high to low, tracking and detecting by TLC, collecting eluate, and concentrating to obtain 8.7g of cimicidin semi-finished product and 6.5g of 5-O-methylvisammioside semi-finished product;
D. medium pressure preparation: c, dissolving 8.7g of the semi-finished product of the linarin in the step C with 30ml of water, then eluting the solution by using 33 percent methanol on a medium-pressure preparation column, and concentrating to obtain 5.2g of a crude product of the linarin with the purity of 93 percent; c, dissolving 6.5g of the semi-finished product of the 5-O-methylvisammioside in the step C in 20ml of methanol, loading the solution into a medium-pressure preparation column, eluting the column with 48% of methanol, and concentrating the solution to obtain 4.2g of a 98% crude product of the 5-O-methylvisammioside;
E. and (3) product recovery: heating and dissolving 5.2g of the crude linarin with the purity of about 93 percent obtained in the step D by using 10ml of pure water, adding 50ml of acetone for diluting, transferring the solution to a refrigerator with the temperature of 4 ℃ for refrigerating for 12 hours, after crystals are separated out, carrying out suction filtration on the solid, heating and dissolving the solid again by using pure water, diluting and recrystallizing by using 5 times of acetone, and repeating the operation for 2-3 times for freeze drying to obtain 4.1g of a linarin reference product with the purity of more than 99 percent; and D, heating and dissolving 4.2g of the crude product of the 5-O-methylvisammioside with the purity of about 98 percent obtained in the step D by using 10ml of methanol, adding 5 times of acetone for diluting, transferring to a refrigerator with the temperature of 4 ℃ for refrigerating for 12 hours, after crystals are separated out, carrying out suction filtration on the solid, heating and dissolving again by using methanol, diluting and recrystallizing by using 5 times of acetone, and repeating the operation for 2-3 times for freeze drying to obtain 3.6g of the reference product of the 5-O-methylvisammioside with the purity of more than 99 percent.
FIG. 1 is a HPLC detection spectrum of a linarin glycoside control obtained in example 1, and FIG. 2 is a HPLC detection spectrum of a 5-O-methylvisammioside glycoside control obtained in example 1. The obtained cimicidin reference substance has purity of 99.3% by HPLC analysis, and 5-O-methylvisammioside reference substance has purity of 99.6% by HPLC analysis.
Example 2
The preparation method of the reference substance of the cimicidin and the 5-O-methylvisammioside is carried out according to the following process steps:
A. extracting raw materials; adding 5Kg of radix Saposhnikoviae into 35L of 80% ethanol solution, cold soaking for 72 hr, filtering, mixing filtrates, and concentrating to no alcohol smell, 3L in total;
B. and (3) macroporous resin enrichment: weighing 2KgAB-8 macroporous adsorption resin, statically adsorbing the concentrated solution obtained in the step A for 12 hours by using the macroporous adsorption resin, eluting impurities by using 20L of water, eluting the target object by using 3L of 65% ethanol solution, collecting the eluent, and concentrating to 50 ml;
C. silica gel column chromatography: and B, mixing 50ml of the concentrated solution in the step B with 50g of 100-mesh 200-mesh silica gel, drying, grinding, taking 300g of 200-mesh 300-mesh silica gel, and filling the mixture into a column by using a dichloromethane wet method. After loading, dichloromethane was used: eluting with methanol at a volume ratio of 10:1-5:1 from high to low, tracking and detecting by TLC, collecting eluate, and concentrating to obtain cimicidin semi-finished product 4.8g and 5-O-methylvisammioside semi-finished product 4.3 g;
D. medium pressure preparation: c, dissolving 4.8g of the semi-finished product of the linarin in the step C with 20ml of water, then eluting the solution by using 33 percent methanol on a medium-pressure preparation column, and concentrating to obtain 3.7g of a crude product of the linarin with the purity of 95 percent; dissolving 4.2g of the semi-finished product of 5-O-methylvisammioside obtained in the step C in 10ml of methanol, loading the solution into a medium-pressure preparation column, eluting the column with 48% of methanol, and concentrating the solution to obtain 3.2g of a 97% crude product of 5-O-methylvisammioside;
E. and (3) product recovery: d, heating and dissolving 3.7g of the crude linarin with the purity of about 95 percent obtained in the step D by using 6ml of pure water, adding 30ml of acetone for diluting, transferring the solution to a refrigerator with the temperature of 4 ℃ for refrigerating for 12 hours, after crystals are separated out, carrying out suction filtration on the solid, heating and dissolving the solid again by using the pure water, diluting the solid by using 5 times of acetone for recrystallization, and repeating the operation for 2-3 times for freeze drying to obtain 3.2g of a linarin reference substance with the purity of more than 99 percent; and D, heating and dissolving 3.2g of the crude product of the 5-O-methylvisammioside with the purity of about 98 percent obtained in the step D by using 100ml of methanol, adding 5 times of acetone for diluting, transferring to a refrigerator with the temperature of 4 ℃ for refrigerating for 12 hours, after crystals are separated out, carrying out suction filtration on the solid, heating and dissolving again by using methanol, diluting and recrystallizing by using 5 times of acetone, and repeating the operation for 2-3 times for freeze drying to obtain 2.5g of the reference product of the 5-O-methylvisammioside with the purity of more than 99 percent.
The obtained cimicidin reference substance has purity of 99.5% by HPLC analysis, and 5-O-methylvisammioside reference substance has purity of 99.5% by HPLC analysis.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (9)
1. A method for preparing a reference substance of linarin glycoside and 5-O-methylvisammioside, which is characterized by comprising the following steps:
(i) providing a semifinished product of linarin glycoside and a semifinished product of 5-O-methylvisammioside;
(ii) dissolving the semi-finished products of the glucoroniside and the 5-O-methylvisammioside in 50% methanol respectively, and separating by using a medium-pressure preparation column to obtain a crude product of the glucoroniside and a crude product of the 5-O-methylvisammioside respectively; wherein, the filler of the medium-pressure preparation column is octadecylsilane chemically bonded silica, and the separation comprises the following steps:
(ii-1) using a methanol solution with the volume ratio of 30-35% as a first eluent of a medium-pressure preparation column, carrying out first-stage elution, and concentrating the eluent to obtain a crude product of the l-ephedrine glycoside; and
(ii-2) using a methanol solution with a volume ratio of 45-50% as a second eluent of the medium-pressure preparation column, performing second-stage elution, and concentrating the eluent to obtain a crude product of 5-O-methylvisammioside; and
(iii) recrystallizing the crude linarin glycoside and/or the crude 5-O-methylvisammioside with a solvent to obtain a linarin glycoside reference substance and/or a 5-O-methylvisammioside reference substance;
wherein the semi-finished product of the linarin glycoside and the semi-finished product of the 5-O-methylvisammioside are prepared by a method comprising the following steps:
(i-1) mixing a divaricate saposhnikovia root medicinal material and an ethanol solution, carrying out cold leaching and ethanol extraction, carrying out solid-liquid separation, and concentrating a liquid part to obtain a first concentrated solution, wherein the volume percentage concentration of the ethanol solution is 80%;
(i-2) adsorbing the first concentrated solution by using macroporous resin, eluting impurities by using water with 4-5 times of column volume respectively, eluting a target by using 55-65% ethanol solution with 1-2 times of column volume, collecting eluent, and concentrating to obtain a second concentrated solution; and
(i-3) separating the second concentrated solution by silica gel column chromatography using dichloromethane to methanol as eluent in a volume ratio of 10:1-5:1 with gradient elution from high to low, collecting the eluent, and concentrating to obtain the semi-finished product of the cimicidin and the semi-finished product of 5-O-methylvisammioside in step (i)
And, the step (iii) further comprises the steps of:
(iii-1) mixing the crude linarin with pure water and acetone, and recrystallizing the crude linarin in a mixed solvent;
separating and drying the obtained crystal to obtain a cimicidin reference substance; and
(iii-2) mixing the crude 5-O-methylvisammioside with methanol and acetone, and recrystallizing the crude 5-O-methylvisammioside in a mixed solvent;
separating and drying the obtained crystal to obtain 5-O-methylvisammioside reference substance;
and the purity of the l-ephedrine glycoside reference substance is more than or equal to 99.0 percent, and the purity of the 5-O-methylvisammioside glycoside reference substance is more than or equal to 99.0 percent.
2. The process according to claim 1, wherein the medium pressure preparative column in step (ii) is provided with a column pressure of 0 to 2 MPa.
3. The process according to claim 1, wherein the medium pressure preparative column in step (ii) is provided with a column pressure of 0.5 to 2 MPa.
4. The method of claim 1, wherein step (iii-2) is performed before or simultaneously with step (iii-1).
5. The production method as claimed in claim 1, wherein in the step (iii-1), acetone is added in a volume 5 to 10 times that of pure water; and/or
In the step (iii-2), the volume of the acetone added is 5-10 times of that of the methanol.
6. The method of claim 1, wherein the linarin control has a purity of 99.3% or more.
7. The method of claim 1, wherein the linarin control has a purity of 99.5% or more.
8. The method of claim 1, wherein the 5-O-methylvisammioside control is at least 99.5% pure.
9. The method of claim 1, wherein the 5-O-methylvisammioside control is at least 99.6% pure.
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| CN112505232B (en) * | 2020-12-15 | 2023-06-16 | 北京中医药大学 | Radix saposhnikoviae chromone control extract, and preparation method and application thereof |
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