CN104926659B - The method preparing rosmarinic acid chemical reference substance from three kinds of high mountain claries - Google Patents

Abstract

The present invention relates to a kind of method of preparing rosmarinic acid chemical reference substance from three kinds of alpine sage plants, the method comprises the following steps: (1) microporous resin impurity removal: sticky sage, ganxi sage or An alcohol extract from Kangding sage is dissolved in ethanol solution, filtered, and separated on a microporous resin column. First, it is eluted with aqueous solution to remove impurities, and then eluted with step gradient with methanol solution to collect 30%~50% methanol Water eluate, the methanol water eluate was dried under reduced pressure to obtain crude phenolic acid components; (2) silica gel column enrichment: the crude phenolic acid components were separated by silica gel column chromatography and eluted, and the collected Rf value was 0.18 ~0.22 fraction; the fraction is dried under reduced pressure to obtain the crude product of rosmarinic acid in the form of light yellow powder; (3) preparation of chromatographic purification by hydrophilic liquid phase: the crude product of rosmarinic acid is dissolved in methanol solution, filtered and prepared by hydrophilic liquid phase After chromatographic separation, the chromatographic peak fraction was collected, and the chromatographic peak fraction was dried under reduced pressure to obtain a white powdery reference substance. The process of the invention is simple and easy to scale.

Description

Method for preparing rosmarinic acid chemical reference substances from three kinds of alpine sage plants

technical field

The invention relates to a method for preparing a chemical reference substance of rosmarinic acid, in particular to a method for preparing a chemical reference substance of rosmarinic acid from three kinds of alpine sage plants.

Background technique

Salvia L. is the largest genus of Lamiaceae. There are three main species of sage on the Qinghai-Tibet Plateau, Salvia Roborowskii, Salvia przewalskii Maxim. and Salvia Kangding. (Salvia prattii). Modern studies have shown that the chemical constituents in sage plants mainly include phenolic acids and terpenes. At present, there are no quality control index ingredients for the three Alpine Salvia plants, Ganxi Sage, Clary Sage and Kangding Sage. Rosmarinic acid is the content of these three Salvia plants. Higher phenolic acid compounds, due to the high polarity of the compound and severe tailing on silica gel thin-layer chromatography, it is difficult to obtain high-purity rosmarinic acid reference substances from these three sage medicinal materials. In order to speed up the quality evaluation, production and sales of Alpine Salvia plants and the research and development of related new drugs, it is particularly important to develop efficient preparation methods for high-purity rosmarinic acid, especially large-scale preparation technology.

At present, there is no report on the preparation of phenolic acid compounds from Alpine Salvia plants. Chinese patent (CN1995007) reports that extraction, addition of antioxidants or adjustment of pH to acidity and then filtration, the filtrate is passed through polyamide resin, ion exchange Resin or macroporous resin column chromatography is used to separate, then adjust the pH to acidic, stand still, crystallize, and filter to produce rosmarinic acid with a purity of about 90%. The process is cumbersome and the loss is large. , but there is no literature report on the preparation of rapid acquisition of large-scale rosmarinic acid from Ganxi sage, sticky sage and Kangding sage.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method for preparing rosmarinic acid chemical reference substances from three kinds of alpine sage plants with simple process and easy scale.

In order to solve the above problems, the method for preparing rosmarinic acid chemical reference substance from three kinds of alpine sage plants of the present invention comprises the following steps:

⑴Microporous resin impurity removal:

A kind of ethanol extract in the brown-red extract-like clary sage, ganxi sage or Kangding sage prepared by conventional methods is used in a volume fraction of 10 to 30% of 5 to 10 times its mass. Dissolved in ethanol solution, filtered to obtain filtrate A, the filtrate A was separated on a microporous resin column, first eluted with 2 to 5 times the column volume of aqueous solution to remove impurities, and then 2 to 5 times the column volume, with a volume fraction of 30 to 80% Carry out step gradient elution with methanol solution, collect 30%~50% methanol water eluate, the methanol water eluate is dried under reduced pressure to obtain the crude product of phenolic acid components; The ratio of a kind of alcohol extract in grass or sage sage to the microporous resin is 1g: 10mL;

⑵ Silica gel column enrichment:

After the crude product of the phenolic acid components was separated by silica gel column chromatography, it was eluted with 3 to 5 times the column volume of chloroform-methanol-water mixed solvent, and the fractions were collected in 500 mL portions and detected by thin-layer chromatography. The reagent is a mixed solvent of chloroform-methanol-water, the chromogenic agent is a concentrated sulfuric acid ethanol solution with a volume concentration of 10%, and the fractions with an Rf value of 0.18 to 0.22 are combined; the fractions with a Rf value of 0.18 to 0.22 are dried under reduced pressure to obtain Crude rosmarinic acid in the form of pale yellow powder;

⑶ Hydrophilic liquid phase preparative chromatographic purification:

The crude product of rosmarinic acid is dissolved in methanol solution with a volume fraction of 2 to 5 times its mass and is 80% to 100%, and then the concentration of the prepared sample is 100.0 to 150.0 mg/mL, and filtered through a 0.45 μm microporous membrane to obtain Filtrate B, the filtrate B is separated by hydrophilic liquid phase preparative chromatography, detected by an ultraviolet detector with a detection wavelength of 330nm, and the main chromatographic peak fraction in the preparative chromatogram is collected. The chromatographic peak fraction is dried under reduced pressure to obtain a purity greater than 98 % of the white powdery reference substance.

The conditions of the step (1) and the step (2) and the step (3) of drying under reduced pressure all refer to a vacuum degree of 0.06~0.09MPa and a temperature of 50~70°C.

The size of the silica gel column in the step (2) is 30mm~60mm×60mm~100mm.

The chloroform-methanol-water mixed solvent in the step (2) is obtained by uniformly mixing chloroform, methanol, and water in a volume ratio of 8:2:0.2~7:3:0.5.

In the described step (3), the operating parameters of hydrophilic liquid phase preparation chromatographic separation refer to chromatographic column column length 250mm, diameter 20mm, hydrophilic preparation column stationary phase is 10 μ mXAmide or Xion, and mobile phase is the acetonitrile of volume fraction 80%～90%. Aqueous solution, the injection volume is 2.0~3.0mL, and the flow rate is 15mL/min.

Compared with the prior art, the present invention has the following advantages:

1. The process of the present invention is simple and the recovery rate is high.

In the first step, the microporous resin is used to remove impurities, and the crude product of rosmarinic acid phenolic acid components with a purity of about 40% can be obtained. At the same time, the microporous resin has a good decolorization effect. The second step adopts silica gel column enrichment, which can be enriched to the crude product of rosmarinic acid with a purity of 75%~85%; the third step adopts the preparation of hydrophilic liquid phase preparative chromatography, which can obtain high-purity reference substance, and the yield is relatively high. High (>90%).

2. The technical means adopted by the present invention are very suitable for large-scale production.

Raw material requirements are not high, and generally wild or planted sage, sage or sage can be extracted, which is easy to prepare materials in batches; the first step is to use a microporous resin column to remove impurities, and the resin column can Recycling and recycling, the average price is low, and it is easy to scale; the second step is silica gel column enrichment, and the filler is cheap and easy to obtain; the third step of refining is prepared by hydrophilic liquid chromatography, which is a fast isocratic method and is also very suitable for scale Production.

Description of drawings

The specific implementation manners of the present invention will be further described in detail below in conjunction with the accompanying drawings.

Figure 1 is the HPLC analysis spectrum (330nm) of the crude rosmarinic acid in Example 1 of the present invention.

Fig. 2 is the HPLC preparation spectrum (330nm) of the crude rosmarinic acid in Example 1 of the present invention.

Fig. 3 is the chromatographic purity analysis (330nm) of rosmarinic acid prepared by hydrophilic liquid phase in Example 1 of the present invention.

detailed description

Embodiment 1 prepares the method for rosmarinic acid chemical reference substance from three kinds of alpine sage plants, comprising the following steps:

⑴Microporous resin (MCI) impurity removal:

200.0g of the brownish-red extract-like alcoholic extract of Salvia villosa produced by a conventional method is dissolved in an ethanol solution with a volume fraction 10% of its mass 10 times, and is filtered to obtain filtrate A, and the filtrate A is placed on a microporous resin column For separation, first remove impurities by eluting with 5 times column volume of aqueous solution, then carry out step gradient elution with 5 times column volume, 30% methanol solution and 80% methanol solution, and collect 30% methanol water eluate. The methanol water eluate was dried under reduced pressure at a vacuum degree of 0.06 MPa and a temperature of 70° C. to obtain 36.4 g of crude phenolic acid components.

in:

The ratio of the ethanol extract of Sage villosa to the microporous resin is 1g: 10mL.

⑵ Silica gel column enrichment:

After the crude phenolic acid components were separated by silica gel column chromatography, they were eluted with 5 times the column volume of chloroform-methanol-water mixed solvent, and the fractions were collected in 500 mL portions and detected by thin-layer chromatography, wherein the developing agent was chloroform-methanol-water Methanol-water mixed solvent, the color developer is concentrated sulfuric acid ethanol solution with a volume concentration of 10%, and the fractions with Rf value of 0.18~0.22 are combined; After pressing and drying, crude rosmarinic acid (16.9 g, Figure 1) was obtained as light yellow powder.

in:

The size of the silica gel column is 30mm x 60mm.

Chloroform-methanol-water mixed solvent is obtained by mixing chloroform, methanol, and water at a volume ratio of 8:2:0.2 (mL/mL).

⑶ Hydrophilic liquid phase preparative chromatographic purification:

The crude rosmarinic acid was dissolved in methanol solution with a volume fraction of 80% twice its mass, and then the sample concentration was prepared to be 120.0 mg/mL, and filtered through a 0.45 μm microporous membrane to obtain filtrate B, which was subjected to hydrophilic Liquid phase preparative chromatographic separation is detected by an ultraviolet detector with a detection wavelength of 330nm, and the main chromatographic peak fraction in the preparative chromatogram is collected. The chromatographic peak fraction is dried under reduced pressure at a vacuum degree of 0.06MPa and a temperature of 70°C to obtain a purity greater than 98% of the white powder control substance (see Figure 2, Figure 3).

in:

The working parameters of the hydrophilic liquid phase preparative chromatographic separation refer to the column length of 250mm and diameter of 20mm, the stationary phase of the hydrophilic preparative column is 10μmXAmide, the mobile phase is acetonitrile-water solution with a volume fraction of 80%, the injection volume is 2.0mL, and the flow rate is It is 15mL/min.

Embodiment 2 prepares the method for rosmarinic acid chemical reference substance from three kinds of alpine sage plants, comprising the following steps:

⑴Microporous resin (MCI) impurity removal:

100.0g of the reddish-brown extract-like alcoholic extract of Salvia ganxie prepared by a conventional method is dissolved in an ethanol solution with a volume fraction 30% of 5 times of its mass, and is filtered to obtain filtrate A, and the microporous resin column on the filtrate A is For separation, first remove impurities by eluting with 2 times column volume of aqueous solution, then use 2 times column volume, 50% methanol solution and 60% methanol solution to carry out step gradient elution, and collect 50% methanol water eluate, the The methanol water eluate was dried under reduced pressure at a vacuum degree of 0.09 MPa and a temperature of 50° C. to obtain 17.6 g of crude phenolic acid components.

in:

The ratio of the alcohol extract of Clary sage to microporous resin is 1g:10mL.

⑵ Silica gel column enrichment:

After the crude phenolic acid components were separated by silica gel column chromatography, they were eluted with 3 times the column volume of chloroform-methanol-water mixed solvent, and the fractions were collected in 500 mL portions and detected by thin-layer chromatography, wherein the developer was chloroform- Methanol-water mixed solvent, the chromogen is concentrated sulfuric acid ethanol solution with a volume concentration of 10%, and the fractions with an Rf value of 0.18~0.22 are combined; Press and dry to obtain 7.9 g of crude rosmarinic acid in the form of light yellow powder.

in:

The size of the silica gel column is 60mm x 100mm.

Chloroform-methanol-water mixed solvent is obtained by mixing chloroform, methanol, and water at a volume ratio of 7:3:0.5 (mL/mL).

⑶ Hydrophilic liquid phase preparative chromatographic purification:

The crude product of rosmarinic acid was dissolved in a 100% methanol solution with a volume fraction 5 times its mass, and then the sample concentration was prepared to be 150.0 mg/mL, and filtered through a 0.45 μm microporous membrane to obtain filtrate B, which was subjected to hydrophilic Liquid phase preparative chromatographic separation is detected by an ultraviolet detector with a detection wavelength of 330nm, and the main chromatographic peak fraction in the preparative chromatogram is collected. The chromatographic peak fraction is dried under reduced pressure at a vacuum degree of 0.09MPa and a temperature of 50°C to obtain a purity greater than 98% white powdery reference substance 6.6g.

in:

The working parameters of the hydrophilic liquid phase preparative chromatographic separation refer to the column length of 250mm and diameter of 20mm, the stationary phase of the hydrophilic preparative column is 10μmXion, the mobile phase is acetonitrile-water solution with a volume fraction of 90%, the injection volume is 3.0mL, and the flow rate is It is 15mL/min.

Embodiment 3 prepares the method for rosmarinic acid chemical reference substance from three kinds of alpine sage plants, comprising the following steps:

⑴Microporous resin (MCI) impurity removal:

500.0g of the brownish-red extract-like Sage Kangding ethanol extract prepared by a conventional method is dissolved in an ethanol solution with a volume fraction 20% of 8 times its mass, and is filtered to obtain filtrate A, which is separated by a microporous resin column , first eluted with 3 times column volume of aqueous solution to remove impurities, then carried out step gradient elution with 3 times column volume, methanol solution with a volume fraction of 40%, and 70% methanol solution, and collected 40% methanol water eluate, the methanol The water eluate was dried under reduced pressure at a vacuum degree of 0.08 MPa and a temperature of 60° C. to obtain 86.2 g of crude phenolic acid components.

in:

The ratio of Kangding sage alcohol extract to microporous resin is 1g: 10mL.

⑵ Silica gel column enrichment:

After the crude phenolic acid component was separated by silica gel column chromatography, it was eluted with 4 times the column volume of chloroform-methanol-water mixed solvent, and the fractions were collected in 500 mL portions and detected by thin-layer chromatography, wherein the developing agent was chloroform- Methanol-water mixed solvent, the chromogenic agent is concentrated sulfuric acid ethanol solution with a volume concentration of 10%, and the fractions with an Rf value of 0.18~0.22 are combined; Press and dry to obtain 36.4 g of crude rosmarinic acid in the form of light yellow powder.

in:

The size of the silica gel column is 40mm x 80mm.

Chloroform-methanol-water mixed solvent is obtained by mixing chloroform, methanol, and water at a volume ratio of 7.5:2.5:0.35 (mL/mL).

⑶ Hydrophilic liquid phase preparative chromatographic purification:

The crude product of rosmarinic acid was dissolved in a methanol solution with a volume fraction of 3 times its mass and a volume fraction of 90%, and then the sample concentration was prepared to be 100.0 mg/mL, and filtered through a 0.45 μm microporous membrane to obtain filtrate B, which was subjected to hydrophilic Liquid phase preparative chromatographic separation is detected by an ultraviolet detector with a detection wavelength of 330nm, and the main chromatographic peak fraction in the preparative chromatogram is collected. The chromatographic peak fraction is dried under reduced pressure at a vacuum degree of 0.08MPa and a temperature of 60°C to obtain a purity greater than 98% white powdery reference substance 29.3g.

in:

The working parameters of the hydrophilic liquid phase preparative chromatographic separation refer to the column length of 250mm and diameter of 20mm, the stationary phase of the hydrophilic preparative column is 10μmXion, the mobile phase is acetonitrile-water solution with a volume fraction of 85%, the injection volume is 2.5mL, and the flow rate is It is 15mL/min.

Claims (1)

1. the method for preparing rosmarinic acid chemical reference substance from three kinds of alpine sage plants, comprising the following steps: ⑴Microporous resin impurity removal: A kind of ethanol extract in the brown-red extract-like clary sage, ganxi sage or Kangding sage prepared by conventional methods is used in a volume fraction of 10 to 30% of 5 to 10 times its mass. Dissolved in ethanol solution, filtered to obtain filtrate A, the filtrate A was separated on a microporous resin column, first eluted with 2 to 5 times the column volume of aqueous solution to remove impurities, and then 2 to 5 times the column volume, with a volume fraction of 30 to 80% Carry out step gradient elution with methanol solution, collect 30%~50% methanol water eluate, the methanol water eluate is dried under reduced pressure to obtain the crude product of phenolic acid components; The ratio of an ethanol extract from Herba Grass or Sage Continus to the microporous resin is 1g:10mL; the conditions for the decompression drying all refer to a vacuum degree of 0.06-0.09MPa and a temperature of 50-70°C; ⑵ Silica gel column enrichment: After the crude product of the phenolic acid components was separated by silica gel column chromatography, it was eluted with 3 to 5 times the column volume of chloroform-methanol-water mixed solvent, and the fractions were collected in 500 mL portions and detected by thin-layer chromatography. The reagent is a mixed solvent of chloroform-methanol-water, the chromogenic agent is a concentrated sulfuric acid ethanol solution with a volume concentration of 10%, and the fractions with an Rf value of 0.18 to 0.22 are combined; the fractions with a Rf value of 0.18 to 0.22 are dried under reduced pressure to obtain Crude rosmarinic acid in the form of light yellow powder; the conditions for the decompression drying all refer to a vacuum degree of 0.06~0.09MPa and a temperature of 50~70°C; the size of the silica gel column is 30mm~60mm×60mm~100mm ; The chloroform-methanol-water mixed solvent is obtained by uniformly mixing chloroform, methanol, and water in a volume ratio of 8:2:0.2~7:3:0.5; ⑶ Hydrophilic liquid phase preparative chromatographic purification: The crude product of rosmarinic acid is dissolved in methanol solution with a volume fraction of 2 to 5 times its mass and is 80% to 100%, and then the concentration of the prepared sample is 100.0 to 150.0 mg/mL, and filtered through a 0.45 μm microporous membrane to obtain Filtrate B, the filtrate B is separated by hydrophilic liquid phase preparative chromatography, detected by an ultraviolet detector with a detection wavelength of 330nm, and the main chromatographic peak fraction in the preparative chromatogram is collected. The chromatographic peak fraction is dried under reduced pressure to obtain a purity greater than 98 % white powdery reference substance; the conditions of decompression drying all refer to a vacuum degree of 0.06~0.09MPa, and a temperature of 50~70°C; the working parameters of the hydrophilic liquid phase preparation chromatographic separation refer to chromatographic columns The column length is 250 mm, the diameter is 20 mm, the stationary phase of the hydrophilic preparation column is 10 μm XAmide or Xion, the mobile phase is acetonitrile-water solution with a volume fraction of 80%-90%, the injection volume is 2.0-3.0 mL, and the flow rate is 15 mL/min.