A method for preparing HSCCC of cynaroside

Abstract

The present invention provides a HSCCC preparation method of cynarin, and relates to the technical field of preparation of Chinese medicine extracts. The preparation method comprises the steps of: extracting the raw material of cynarin with an alcohol solvent or purifying it with a macroporous resin to obtain a total flavonoid extract of cynarin, and dissolving it with a mobile phase to obtain a sample solution; configuring a high-speed countercurrent chromatography solvent: the chromatographic solvent is divided into two components, A and B, the component A is selected from at least one of methanol, ethanol, and water, and the component B is selected from at least one of n-hexane, cyclohexane, and ethyl acetate, and the volume ratio of component A to component B is 9-1:1-9; using high-speed countercurrent chromatography for elution analysis, collecting the flow fraction after qualitative detection, and recrystallizing to obtain cynarin. The preparation method can improve the yield and purity of cynarin, reduce the difficulty of separation, and improve reproducibility.

Description

Technical Field

[0001] The invention belongs to the technical field of preparation of traditional Chinese medicine extracts, and particularly relates to a method for preparing HSCCC of cynaroside. Background Art

[0002] Tilianin is a flavonoid monomer compound extracted from the dried aerial part of the plant medicine Dracocephalum moldovica L. Studies have found that tilianin has biological activities such as anti-oxidative stress, inhibition of myocardial cell apoptosis, myocardial protection, and anti-atherosclerosis, and can be used to prepare drugs for the treatment of cardiovascular diseases. Although tilianin, as a flavonoid compound, is present in a high content in Dracocephalum moldovica L, there are still few studies on its separation and purification. At present, the main separation and purification method is to use traditional column chromatography, such as using a macroporous resin adsorption chromatographic column to obtain total flavonoids from Dracocephalum moldovica L, and then subjecting the total flavonoids to silica gel column chromatography to obtain tilianin.

[0003] Chinese invention patent CN101781345B discloses a method for preparing cynarin, that is, first extracting cynarin with an organic solvent, and then performing silica gel column chromatography, the mobile phase adopts a composite eluent mixed with components A and B, the component A is selected from at least one of methanol, ethanol, acetone, isopropyl ketone, butanol, and the component B is selected from at least one of chloroform, ethyl acetate, dichloromethane, and dichloroethane; the volume ratio of component A to component B is 90:10-10:90, and gradient elution is performed to obtain an eluent; the eluents of the same components are combined and concentrated to obtain cynarin. However, the separation and purification method of the traditional chromatographic column is costly, time-consuming, and has poor reproducibility. In addition, due to the poor solubility of cynarin, the separation difficulty is further increased, resulting in a low yield. The maximum yield of cynarin in the invention is 78.2%, which cannot be mass-produced and is difficult to meet the needs of efficient, high-yield, and large-scale preparation of cynarin.

[0004] High-Speed ​​Counter-Current Chromatography (HSCCC) is a continuous and efficient liquid-liquid distribution chromatography technology. Its working principle is to establish a special unidirectional fluid dynamic equilibrium for the two-phase solvent system in the spiral tube through high-speed rotation, and use the different distribution coefficients of the sample in the two-phase solvent system to achieve the purpose of separation. The biggest advantage of high-speed counter-current chromatography technology is that both the stationary phase and the mobile phase are liquids, and sample loss such as inactivation and irreversible adsorption can be avoided during the elution process. It has unique advantages such as high recovery rate, large preparation volume, low economic cost, and good reproducibility. It has been widely used in biomedicine research and development, natural product separation, chemical industry, life sciences, and food.

[0005] Wang Chaoyue et al. (Large-scale separation of baicalin and wogonosidefrom ScutellariaBaicalensisGeorgi by the combination of pH-zone-refining andconventional counter-current chromatography) applied high-speed countercurrent chromatography to the separation and purification of scutellaria root from the Chinese herbal medicine scutellaria, using a two-phase solvent system consisting of n-butanol-ethyl acetate-water, and ethyl acetate-acetic acid-hydrochloric acid for CCC. 186.7 mg of baicalin with a purity of 95.3% was successfully separated from 500 mg of crude scutellaria extract by HSCCC in one step; further secondary separation gave 64.3 mg of baicalin with a purity of 98.2%.

[0006] In the existing preparation methods of cynaroside, there is no separation and preparation of cynaroside using high-speed countercurrent chromatography. In order to solve the above problems and realize high-yield and large-scale industrial production of cynaroside, the present invention provides a HSCCC preparation technology of cynaroside, which is applied to the separation and preparation of cynaroside, improves the yield and reproducibility of cynaroside, and reduces the cost. Summary of the invention

[0007] The present invention aims at the problems existing in the prior art and provides a HSCCC preparation method of cynaroside, which can effectively solve the problems of low yield, low reproducibility, high separation difficulty, long time consumption and high cost in the preparation of cynaroside, and realizes the production of cynaroside with high yield while retaining high purity and activity.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0009] The present invention provides a method for preparing HSCCC of cynaroside, comprising the following steps:

[0010] (1) Sample extraction: Take the raw material of Scutellaria baicalensis, extract it with an alcohol solvent or purify it with a macroporous resin column to obtain the total flavonoid extract of Scutellaria baicalensis;

[0011] (2) Preparation of a high-speed countercurrent chromatography solvent system: After component A and component B are mixed, the components are separated into layers, the lower layer is the lower phase, and the upper liquid is the upper phase; the component A is selected from at least one of methanol, ethanol, and water, and the component B is selected from at least one of n-hexane, cyclohexane, and ethyl acetate;

[0012] (3) Sample solution preparation: using the lower phase or the upper phase as a solvent to dissolve the total flavonoids extract of Scutellaria baicalensis to obtain a sample solution;

[0013] (4) Countercurrent chromatography (CCC) preparation procedure:

[0014] Elution mode: normal phase elution or reverse phase elution;

[0015] Stationary phase: lower phase or upper phase;

[0016] (5) injecting samples, acquiring chromatograms, collecting fractions, and obtaining cynaroside after recrystallization.

[0017] Preferably, in step (1), the raw material of the Herba Cibotii is selected from at least one of the Herba Cibotii medicinal materials and the raw material of the aerial parts of the Herba Cibotii plant.

[0018] Preferably, in step (1), the crushing passes through a 40-mesh sieve and has a particle size of about 0.42 mm.

[0019] Preferably, in step (1), the alcohol solvent is selected from at least one of ethanol, methanol, isopropanol and n-butanol.

[0020] More preferably, the alcohol solvent is selected from at least one of ethanol and methanol.

[0021] More preferably, the concentration of the alcohol solvent is 40%-99.99%.

[0022] More preferably, the alcohol solvent is 60% ethanol.

[0023] Preferably, in step (1), the extraction temperature is 40-60°C.

[0024] Further preferably, the extraction temperature is 60°C.

[0025] Preferably, in step (1), the eluent for the macroporous resin column purification is a 10-90% ethanol solution.

[0026] Preferably, in step (1), the upper phase and the lower phase are subjected to ultrasound and degassing for later use, and the duration of ultrasound is 20-40 min.

[0027] Preferably, in step (2), in the high-speed countercurrent chromatography solvent system, component A is a mixture of methanol and water or a mixture of ethanol and water.

[0028] Further preferably, in the high-speed countercurrent chromatography solvent system, component A is a mixture of methanol and water or a mixture of ethanol and water; when component A is a mixture of methanol and water, the volume ratio thereof is 1-3:3-8; when component A is a mixture of ethanol and water, the volume ratio thereof is 1-3:1-5.

[0029] More preferably, in the high-speed countercurrent chromatography solvent system, component A is a mixture of methanol and water or a mixture of ethanol and water; when component A is a mixture of methanol and water, the volume ratio thereof is 1-2:3-8; when component A is a mixture of ethanol and water, the volume ratio thereof is 1-2:3-5.

[0030] More preferably, in the high speed countercurrent chromatography solvent system, component A is a mixture of ethanol and water in a volume ratio of 1-2:3-5.

[0031] Preferably, in step (2), in the high-speed countercurrent chromatography solvent system, component B is a mixture of n-hexane and ethyl acetate or a mixture of cyclohexane and ethyl acetate.

[0032] Further preferably, in the high-speed countercurrent chromatography solvent system, component B is a mixture of n-hexane and ethyl acetate or a mixture of cyclohexane and ethyl acetate; when component B is a mixture of n-hexane and ethyl acetate, the volume ratio thereof is 1:4-9; when component B is a mixture of cyclohexane and ethyl acetate, the volume ratio thereof is 1:4-9.

[0033] More preferably, in the high speed countercurrent chromatography solvent system, component B is a mixture of n-hexane and ethyl acetate in a volume ratio of 1:4-9.

[0034] More preferably, in the high speed countercurrent chromatography solvent system, component B is a mixture of n-hexane and ethyl acetate in a volume ratio of 1:4.

[0035] Preferably, in step (2), in the high-speed countercurrent chromatography solvent system, the volume ratio of component A to component B is 9-1:1-9.

[0036] Further preferably, in the high speed countercurrent chromatography solvent system, the volume ratio of component A to component B is 7-3:3-7.

[0037] More preferably, the volume ratio of component A to component B is 6-5:5-6.

[0038] Preferably, in step (3), the concentration of the sample solution is 1-30 mg / mL.

[0039] Further preferably, the concentration of the sample solution is 5-20 mg / mL.

[0040] Preferably, in step (4), in the countercurrent chromatography preparation procedure, the HSCCC column needs to be filled with the stationary phase before starting the countercurrent chromatography.

[0041] Preferably, in step (4), in the countercurrent chromatography preparation procedure, the rotation speed is set to 600-2000 rpm and the flow rate is set to 1-200 mL / min.

[0042] More preferably, the rotation speed is 1000-1800 rpm and the flow rate is 1-5 mL / min.

[0043] More preferably, the rotation speed is 1600-1800 rpm; the flow rate is 1-2 mL / min.

[0044] Preferably, in step (4), the mobile phase is the opposite phase of the stationary phase, that is, when the stationary phase is the lower phase, the mobile phase is the upper phase; when the stationary phase is the upper phase, the mobile phase is the lower phase.

[0045] In step (4), the stationary phase and the mobile phase reach a hydrodynamic equilibrium in the column, that is, the sample is injected when the stationary phase no longer loses.

[0046] Preferably, in step (4), in the countercurrent chromatography preparation procedure, the ultraviolet absorption wavelength is set to 254-330 nm.

[0047] Further preferably, the ultraviolet absorption wavelength is 254 nm or 330 nm.

[0048] Preferably, in step (5), the solvent used for the recrystallization is methanol-water solution or ethanol-water solution.

[0049] Further preferably, the concentration of the solvent used in the recrystallization is 20-70%.

[0050] The present invention analyzes the components of the collected products and determines that they are cynaroside through HPLC qualitative analysis. The structural formula of cynaroside is:

[0051]

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] 1. The present invention successfully and efficiently prepares high-purity cynaroside from Scutellaria baicalensis through high-speed countercurrent chromatography preparation technology, and compared with the current preparation process of cynaroside components, the separation difficulty is reduced and the yield of cynaroside is high.

[0054] 2. The present invention achieves high-yield and high-purity separation of cynaroside by high-speed countercurrent chromatography preparation technology, a solvent system of specific components, and setting a reasonable chromatographic procedure. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 is a high-speed countercurrent chromatogram of the total flavonoids extract of the Herba Lysimachiae of Example 1 of the present invention;

[0056] Figure 2 is the qualitative analysis HPLC detection chromatogram of Example 1 of the present invention;

[0057] Figure 3 It is the HPLC detection chromatogram of the cynaroside standard solution of the present invention;

[0058] Figure 4 It is the HPLC detection chromatogram of cynaroside of Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0059] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is merely an exemplary description of the scope of protection claimed in this application, and those skilled in the art can make various changes and modifications to the invention of this application based on the disclosed content, which should also fall within the scope of protection claimed in this application.

[0060] The present invention is further described below by way of specific examples. Unless otherwise specified, the various chemical reagents used in the examples of the present invention are obtained through conventional commercial channels.

[0061] In the following embodiments, the Herba Cynanchii medicinal materials were purchased from Xinjiang Yongze Chinese Herbal Medicine Technology Development Co., Ltd. with the item number 20200411; the Herba Cynanchii reference substance was purchased from Chengdu Ruifensidedan Biotechnology Co., Ltd. with the item number X-015; in Example 8, the Herba Cynanchii total flavonoids were provided by the Xinjiang Uygur Autonomous Region Institute of Pharmacology.

[0062] The steps for HPLC detection of cynaroside purity are as follows:

[0063] (1) Prepare the solution. Take 1 ml of the target component solution (the fraction of PEAK2), blow dry the solution with nitrogen in a 40°C water bath, then add 1 ml of 70% ethanol water to reconstitute it, and then dilute the reconstituted solution 5-50 times with 70% ethanol water for later use;

[0064] (2) Chromatographic conditions: chromatographic column: Agilent C18 4.6 mm × 150 mm, 5 μm; mobile phase: acetonitrile (A)-0.1% formic acid water (B) gradient elution (0-10 min, A: 40%; 10-20 min, A: 40→80%; 20-25%, A: 80%); column temperature: 35°C; detection wavelength: 330 nm; flow rate: 1 ml / min; injection volume: 5-10 μl.

[0065] (3) Turn on the machine, set the condition parameters, change the mobile phase and remove the bubbles;

[0066] (4) Inject the standard solution first, then the sample solution, and inject each solution twice;

[0067] (5) Collect data and record peak area, retention time, and theoretical plate number;

[0068] (6) Clean the pipes and chromatographic column, then shut down the machine.

[0069] The yield of cynarin was calculated according to the following formula: Cynarin yield = M PEAK2 / M 总黄酮 , where M PEAK2 is the mass of peak 2 fraction (citrinin) after rotary evaporation and drying, M 总黄酮 It is the mass of total flavonoids extracted from the plant of Scutellaria baicalensis.

[0070] Example 1

[0071] (1) Sample extraction: Take the decoction pieces of the herbal medicine of the herb of the herb of the herb of the herb of the herb, grind them through a 20-mesh sieve, and the particle size is about 0.42 mm. Then, extract them by heating with a 60% ethanol solution at 60° C. After the extract is concentrated, it is spray-dried to obtain the herb of the herb of the herb of the herb of the herb;

[0072] Preparation of high-speed countercurrent chromatography solvent system: Component A is ethanol and water, and component B is n-hexane and ethyl acetate; the solvent system is n-hexane-ethyl acetate-ethanol-water (HEEW)—1:4:1:5, the upper phase is the stationary phase, and the lower phase is the mobile phase;

[0073] (2) Sample solution preparation: The following phase (mobile phase) is used as a solvent to dissolve the total flavonoid extract of Scutellaria baicalensis to prepare a sample solution with a concentration of 10 mg / mL for injection;

[0074] (3) High-speed countercurrent chromatography (HSCCC) preparation procedure: reverse phase elution, the HSCCC column is filled with the stationary phase (upper phase), countercurrent chromatography is started, the rotation speed is 1800 rpm, the flow rate is 1 mL / min, the lower phase is introduced as the mobile phase, and the stationary phase and the mobile phase are allowed to reach hydrodynamic equilibrium in the column before injection; the UV absorption wavelength is set to 330 nm; the injection volume is 0.5 mL;

[0075] (4) Collecting chromatograms and collecting fractions: Detection, collection of chromatograms, chromatograms such as Figure 1 As shown, two peak fractions were taken and the retention time and UV wavelength scanning chart were compared with those of cynaroside reference substance by HPLC for qualitative analysis and purity detection. Figure 2 The results show that Figure 1 PEAK2 in the assay is the target product cynaroside.

[0076] The ultraviolet absorption peak (PEAK2) was collected, and the collected fractions were concentrated to dryness, and then re-dissolved in 70% ethanol and tested by HPLC. The results showed that the purity of cynarin was 97.4%. After recrystallization with 50% methanol-water, cynarin was precipitated to obtain light yellow or white powder crystals with a purity of 99.5%. The yield of cynarin was 13.5%.

[0077] Example 2

[0078] (1) Sample extraction: same as in Example 1, obtaining a total flavonoid extract of Scutellaria baicalensis;

[0079] Preparation of high-speed countercurrent chromatography solvent system: Component A is ethanol and water, and component B is n-hexane and ethyl acetate; the solvent system is n-hexane-ethyl acetate-ethanol-water (HEEW)—1:4:2:3, the upper phase is the stationary phase, and the lower phase is the mobile phase;

[0080] (2) Sample solution preparation: The following phase (mobile phase) is used as a solvent to dissolve the total flavonoid extract of Scutellaria baicalensis to prepare a sample solution with a concentration of 5 mg / mL for injection;

[0081] (3) High-speed countercurrent chromatography (HSCCC) preparation procedure: reverse phase elution, the HSCCC column is filled with the stationary phase (upper phase), countercurrent chromatography is started, the rotation speed is 1600 rpm, the flow rate is 1.5 mL / min, the lower phase is introduced as the mobile phase, and the stationary phase and the mobile phase are allowed to reach hydrodynamic equilibrium in the column before injection; the UV absorption wavelength is set to 330 nm; the injection volume is 0.5 mL;

[0082] (4) Collecting fractions: Collect the second ultraviolet absorption peak (PEAK2), concentrate the collected fractions to dryness, and detect them by HPLC after redissolution. The results show that the purity of cynaroside is 93.4%. After recrystallization with 50% methanol-water, cynaroside is precipitated to obtain light yellow or white powder crystals with a purity of 99.3%. The yield of cynaroside is 13.3%.

[0083] Example 3

[0084] (1) Sample extraction: same as in Example 1, obtaining a total flavonoid extract of Scutellaria baicalensis;

[0085] Preparation of high-speed countercurrent chromatography solvent system: Component A is methanol and water, and component B is n-hexane and ethyl acetate; the solvent system is n-hexane-ethyl acetate-methanol-water (HEMW)—1:4:1:5, the upper phase is the mobile phase, and the lower phase is the stationary phase;

[0086] (2) Sample solution preparation: The above phase (mobile phase) is used as a solvent to dissolve the total flavonoids extract of Scutellaria baicalensis to prepare a sample solution with a concentration of 5 mg / mL for injection;

[0087] (3) High-speed countercurrent chromatography (HSCCC) preparation procedure: reverse phase elution, the HSCCC column is filled with the stationary phase (lower phase), countercurrent chromatography is started, the rotation speed is 1600 rpm, the flow rate is 2 mL / min, the upper phase is introduced as the mobile phase, and the stationary phase and the mobile phase are allowed to reach hydrodynamic equilibrium in the column before injection; the UV absorption wavelength is set to 330 nm; the injection volume is 0.5 mL;

[0088] (4) Collecting fractions: Collect the second ultraviolet absorption peak (PEAK2), concentrate the collected fractions to dryness, and use HPLC to detect after redissolution. The results show that the purity of cynarin is 90.4%. After recrystallization with 50% methanol-water, cynarin is precipitated to obtain light yellow or white powder crystals with a purity of 98.3%. The yield of cynarin is 13.7%.

[0089] Example 4

[0090] (1) Sample extraction: same as in Example 1, obtaining a total flavonoid extract of Scutellaria baicalensis;

[0091] Preparation of high-speed countercurrent chromatography solvent system: Component A is methanol and water, and component B is n-hexane and ethyl acetate; the solvent system is n-hexane-ethyl acetate-methanol-water (HEMW)—1:4:2:3, the upper phase is the stationary phase, and the lower phase is the mobile phase;

[0092] (2) Sample solution preparation: The following phase (mobile phase) is used as a solvent to dissolve the total flavonoid extract of Scutellaria baicalensis to prepare a sample solution with a concentration of 15 mg / mL for injection;

[0093] (3) High-speed countercurrent chromatography (HSCCC) preparation procedure: reverse phase elution, the HSCCC column is filled with the stationary phase (upper phase), countercurrent chromatography is started, the rotation speed is 1600 rpm, the flow rate is 2 mL / min, the lower phase is introduced as the mobile phase, and the stationary phase and the mobile phase are allowed to reach hydrodynamic equilibrium in the column before injection; the UV absorption wavelength is set to 330 nm; the injection volume is 1.0 mL;

[0094] (4) Collecting fractions: Collect the second ultraviolet absorption peak (PEAK2), concentrate the collected fractions to dryness, and use HPLC to detect after redissolution. The results show that the purity of cynaroside is 83.2%. After recrystallization with 50% methanol-water, cynaroside is precipitated to obtain light yellow or white powder crystals with a purity of 98.8%. The yield of cynaroside is 13.4%.

[0095] Example 5

[0096] (1) Sample extraction: same as in Example 1, obtaining a total flavonoid extract of Scutellaria baicalensis;

[0097] Preparation of high-speed countercurrent chromatography solvent system: Component A is methanol and water, and component B is n-hexane and ethyl acetate; the solvent system is n-hexane-ethyl acetate-methanol-water (HEMW)—1:9:2:8, the upper phase is the stationary phase, and the lower phase is the mobile phase;

[0098] (2) Sample solution preparation: The following phase (mobile phase) is used as a solvent to dissolve the total flavonoid extract of Scutellaria baicalensis to prepare a sample solution with a concentration of 20 mg / mL for injection;

[0099] (3) High-speed countercurrent chromatography (HSCCC) preparation procedure: reverse phase elution, the HSCCC column is filled with the stationary phase (upper phase), countercurrent chromatography is started, the rotation speed is 1600 rpm, the flow rate is 1 mL / min, the lower phase is introduced as the mobile phase, and the stationary phase and the mobile phase are allowed to reach hydrodynamic equilibrium in the column before injection; the UV absorption wavelength is set to 330 nm; the injection volume is 0.5 mL;

[0100] (4) Collecting fractions: Collect the second ultraviolet absorption peak (PEAK2), concentrate the collected fractions to dryness, and perform HPLC detection after redissolution. The results show that the purity of cynaroside is 89.6%. After recrystallization with 50% methanol-water, cynaroside is precipitated to obtain light yellow or white powder crystals with a purity of 99.1%. The yield of cynaroside is 13.1%.

[0101] Example 6

[0102] Different from Example 5, the high-speed countercurrent chromatography solvent system is different: component A is methanol and water, component B is cyclohexane and ethyl acetate; the solvent system is cyclohexane-ethyl acetate-methanol-water (CEMW) -1:9:2:8, the upper phase is the stationary phase, and the lower phase is the mobile phase.

[0103] The remaining steps and parameters are the same as those in Example 5.

[0104] The second ultraviolet absorption peak (PEAK2) was collected, and the collected fractions were concentrated to dryness. After redissolution, HPLC detection was performed, and the results showed that the purity of cynarin was 95.2%. After recrystallization with 50% methanol-water, cynarin was precipitated to obtain light yellow or white powder crystals with a purity of 97.3%. The yield of cynarin was 12.1%.

[0105] Example 7

[0106] The difference from Example 1 is that the sample extraction method is different:

[0107] Take the decoction pieces of the herb of the herb of the herb of the herb of the herb, grind them into a particle size of about 0.42 mm, extract them with a 40% methanol solution heated at 60°C, purify them with a macroporous resin column, elute them with 50% methanol, concentrate the extract, and spray dry them to obtain the herb of the herb of the herb of the herb of the herb;

[0108] The remaining steps and parameters are the same as those in Example 1.

[0109] The second ultraviolet absorption peak (PEAK2) was collected, and the collected fractions were concentrated to dryness. After redissolution, HPLC detection was performed, and the results showed that the purity of cynarin was 96.1%. After recrystallization with 50% methanol-water, cynarin was precipitated to obtain light yellow or white powder crystals with a purity of 99.2%. The yield of cynarin was 11.4%.

[0110] Example 8

[0111] Exploration of solvent system: The distribution coefficient (K value) of the high-speed countercurrent chromatography solvent system used in Examples 1-6 was analyzed.

[0112] (1) HPLC analysis conditions: 1.1 Preparation of standard solution

[0113] 50ug / ml cynaroside standard stock solution and 100ug / ml total flavonoids sample solution of Cynaropicrin (dissolved in 70% methanol) were stored at 4°C for use.

[0114] 1.2 Instruments and reagents

[0115] Waters 2695 high performance liquid chromatograph;

[0116] Methanol (analytical grade, chromatographic grade), formic acid (chromatographic grade), and water were ultrapure water.

[0117] 1.3 Investigation and determination of chromatographic conditions

[0118] The liquid elution conditions of cynaroside standard solution with acetonitrile-water and acetonitrile-0.1% formic acid water as the mobile phase in different proportions were investigated, and the determined liquid chromatography conditions were: chromatographic column: Agilent C18 4.6mm×150mm, 5μm; mobile phase: acetonitrile (A)-0.1% formic acid water (B) gradient elution (0-10min, A: 40%; 10-20min, A: 40→80%; 20-25%, A: 80%); column temperature: 35℃; detection wavelength: 330 nm; flow rate: 1ml / min; injection volume: 10μl.

[0119] The HPLC chromatogram was run under the determined HPLC conditions. Figure 3 As shown, the peak time of the standard substance cynaroside is 8.648 min, and both the peak time and peak shape are good.

[0120] (2) Determination of the distribution coefficient K value

[0121] The distribution coefficient is also called the K value (Partition coefficient, K value), which refers to the ratio of the equilibrium concentration of the solute in the upper phase and the lower phase when the distribution equilibrium is reached at a certain temperature and pressure. The K value plays a decisive role in the countercurrent chromatography separation process. If the K value of the compound is too small, the peak of the compound will be at the front of the solvent. If the K value is too large, the separation will be significantly reduced. It is generally believed that the K value of the solute in the solvent system is more suitable between 0.5-2. Explore the HPLC analysis conditions of cynarin → The sample is dissolved in two phases of equal volume, and impurities are removed by high-speed centrifugation → Take equal volumes of upper and lower phase solutions and perform HPLC analysis → By measuring the sample peak area A of the upper and lower phases U and AL To calculate the K value, it can be expressed by the following equation:

[0122] K U / L = U / L

[0123] Under the above HPLC conditions, the solvent systems with suitable K values ​​are shown in Table 1 (K values ​​of cynarin in different solvent systems). The results show that the K value of cynarin is most suitable when the volume ratio of n-hexane-ethyl acetate-ethanol-water (HEEW) system is 1:4:1:5 and 1:4:2:3, and replacing ethanol with methanol or n-hexane with cyclohexane has little effect on the K value, and both can be used as effective solvent systems for the separation of cynarin.

[0124] Table 1

[0125]

[0126] Comparative Example 1

[0127] Separation and Purification of Cynaropicrin by Polyamide Column Chromatography

[0128] (1) Sample extraction: Take the decoction pieces of the herbal medicine of the herb ...

[0129] (2) Sample preparation: Accurately measure the total flavonoids extract powder of the Herba Lysimachiae, dissolve it in 70% ethanol water to prepare a suspension, and then perform rotary evaporation at 60°C to obtain an extract.

[0130] (3) Column chromatography separation: Wet-pack the polyamide column (3.6 cm × 40 cm), and rinse the resin column with an eluent (90%-10% ethanol-water) after stabilization. Load the extract sample in (2) onto the pretreated polyamide resin column, and elute with a gradient of 90%-10% ethanol-water, and collect the fractions of the gradient eluent.

[0131] (4) The fractions of the different gradient eluents in (3) were injected into HPLC to detect the purity of cynaroside, and the purity of cynaroside in the total flavonoids of Scutellaria baicalensis was 60%, and the yield was 9.8%. The HPLC test results are as follows Figure 4 shown.

[0132] Comparative Example 2

[0133] The difference from Example 1 is that the components of the high-speed countercurrent chromatography solvent system are different: component A is ethanol and water, component B is chloroform and ethyl acetate; the solvent system is chloroform-ethyl acetate-ethanol-water -1:4:1:5, the upper phase is the stationary phase, and the lower phase is the mobile phase; the remaining steps and parameters are the same as in Example 1.

[0134] The second ultraviolet absorption peak (PEAK2) was collected, and the collected fractions were concentrated to dryness. After redissolution, HPLC detection was performed, and the results showed that the purity of cynarin was 65.6%. After recrystallization with 50% methanol-water, cynarin was precipitated to obtain light yellow or white powder crystals with a purity of 80.6%. The yield of cynarin was 6.5%.

[0135] Comparative Example 3

[0136] Different from Example 3, the ratio of the high-speed countercurrent chromatography solvent system is different: component A is methanol and water, component B is n-hexane and ethyl acetate; the solvent system is n-hexane-ethyl acetate-methanol-water (HEMW) - 3:2:3:2, the upper phase is the stationary phase, and the lower phase is the mobile phase;

[0137] The remaining steps and parameters are the same as those in Example 3.

[0138] The second ultraviolet absorption peak (PEAK2) was collected, and the collected fractions were concentrated to dryness. After redissolution, HPLC detection was performed, and the results showed that the purity of cynaroside was 78.0%. After recrystallization with 50% methanol-water, cynaroside was precipitated to obtain light yellow or white powder crystals with a purity of 90.1% and cynaroside 7.3%.

[0139] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing HSCCC of cynaroside, characterized in that: The following steps are involved: (1) Sample extraction: Take the raw material of Scutellaria baicalensis, extract it with alcohol solvent or purify it with macroporous resin column to obtain the total flavonoids extract of Scutellaria baicalensis; (2) Preparation of high-speed countercurrent chromatography solvent system: After component A and component B are mixed, they are separated into layers, the lower layer is the lower phase, and the upper liquid is the upper phase; Component A is a mixture of methanol and water in a volume ratio of 1-3:3-8, or a mixture of ethanol and water in a volume ratio of 1-3:1-5; Component B is a mixture of n-hexane and ethyl acetate in a volume ratio of 1:4-9, or a mixture of cyclohexane and ethyl acetate in a volume ratio of 1:4-9; The volume ratio of component A to component B is 9-1:1-9; (3) Sample solution preparation: using the lower phase or the upper phase as a solvent to dissolve the total flavonoids extract of Scutellaria baicalensis to obtain a sample solution; (4) Countercurrent chromatography preparation procedure: Elution mode: normal phase elution or reverse phase elution; Stationary phase: lower phase or upper phase; (5) Injecting the sample, acquiring the chromatogram, collecting the fractions, and obtaining cynaroside after recrystallization.

2. The preparation method according to claim 1, characterized in that: In step (1), the alcohol solvent is selected from at least one of ethanol, methanol, isopropanol and n-butanol.

3. The preparation method according to claim 1, characterized in that: In step (1), the temperature of the alcohol solvent extraction is 40-60°C.

4. The preparation method according to claim 1, characterized in that: When component A is a mixture of methanol and water, the volume ratio is 1-2:3-8; when component A is a mixture of ethanol and water, the volume ratio is 1-2:3-5.

5. The preparation method according to claim 1, characterized in that: In step (3), the concentration of the sample solution is 1-30 mg / mL.

6. The preparation method according to claim 1, characterized in that: In step (4), in the countercurrent chromatography preparation procedure, the rotation speed is set to 600-2000 rpm and the flow rate is set to 1-200 mL / min.

7. The preparation method according to claim 6, characterized in that: The rotation speed is 1000-1800 rpm, and the flow rate is 1-5 mL / min.

8. The preparation method according to claim 1, characterized in that: In step (4), in the countercurrent chromatography preparation procedure, the ultraviolet absorption wavelength is set to 254-330 nm.

9. The preparation method according to claim 1, characterized in that: In step (5), the solvent used for the recrystallization is methanol-water solution or ethanol-water solution with a concentration of 20-70%.

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