A method for extracting flavonoid components from Scutellaria baicalensis

Abstract

The present invention provides a method for extracting flavonoid components in safflower, and relates to the technical field of drug extraction. The present invention mixes safflower with alkali solution for heating extraction to obtain an extract; the extract is mixed with acid, and precipitated under the condition of pH value of 1 to 3 to obtain a precipitated extract; the precipitated extract is dissolved and separated by preparative liquid chromatography to obtain Fr1 to Fr6 fractions; the Fr1 to Fr6 fractions are respectively concentrated to dryness to obtain luteolin-7-O-β-D-glucuronide, apigenin-7-O-β-D-glucuronide, diosmetin-7-O-β-D-glucuronide, cynarin, acacetin-7-O-glucuronide, and acacetin in sequence. The present invention quickly realizes the acquisition of high-purity monomers of the main flavonoid components in safflower, and the purity of each component is 98 to 99%.

Description

Technical Field

[0001] The invention relates to the technical field of drug extraction, in particular to a method for extracting flavonoid components from Scutellaria baicalensis. Background Art

[0002] Dracocephalum Moldavica L. is a Xinjiang specialty Uyghur medicine. Its Uyghur name is Badiranjbuya. It is an annual herbaceous plant of the Lamiaceae family. The whole herb is often used as medicine. Its medicinal material standards are included in the "Drug Standards of the Ministry of Health of the People's Republic of China·Uyghur Medicine Volume" and "Uyghur Medicine Records (Volume 1)". Dracocephalum Moldavica L. mainly contains flavonoids, volatile oils, phenylpropanoids, polysaccharides, amino acids, proteins and trace elements. The main components of the effective parts of the total flavonoids of Dracocephalum Moldavica L. are luteolin-7-O-β-D-glucuronide, apigenin-7-O-β-D-glucuronide, diosmetin-7-O-β-D-glucuronide, cynaroside, acacetin-7-O-glucuronide, acacetin, etc. The total flavonoids of Scutellaria baicalensis have certain therapeutic effects on many diseases, such as treating atherosclerosis, protecting myocardial ischemia-reperfusion injury, anti-inflammation, anti-oxidation, etc.

[0003] At present, there are few studies on the extraction and purification of flavonoid components of Scutellaria baicalensis at home and abroad. They mainly adopt alcohol extraction followed by traditional column chromatography for separation and purification. For example, a macroporous resin adsorption column is used to obtain total flavonoids from Scutellaria baicalensis, and then the total flavonoids are subjected to silica gel column chromatography to obtain a certain component. This method is costly, time-consuming, has poor reproducibility, and low purity. In addition, the poor solubility of some substances further increases the difficulty of separation, resulting in low yield, which is difficult to meet the needs of large-scale preparation of multiple components. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a method for extracting flavonoid components from Scutellaria baicalensis. The extraction method provided by the present invention has a simple process and can obtain high-purity flavonoid components (luteolin-7-O-β-D-glucuronide, apigenin-7-O-β-D-glucuronide, diosmetin-7-O-β-D-glucuronide, cynaroside, acacetin-7-O-glucuronide, acacetin) from Scutellaria baicalensis.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a method for extracting flavonoid components from radix scutellariae, comprising the following steps:

[0007] Mixing the fragrant blue orchid and the alkali solution and performing heating extraction to obtain an extract;

[0008] The extract is mixed with an acid, and precipitated at a pH value of 1 to 3 to obtain a precipitated extract;

[0009] The precipitate extract is dissolved and then separated by preparative liquid chromatography, wherein the conditions for the preparative liquid chromatography separation include: a C18 chromatographic column; mobile phases A and B, wherein the mobile phase A is acetonitrile and the mobile phase B is a formic acid aqueous solution, wherein the volume fraction of formic acid in the formic acid aqueous solution is 0.1%; gradient elution;

[0010] The elution program of the gradient elution is: 0-55min, the volume ratio of mobile phase A is 25%; 55-80min, the volume ratio of mobile phase A is 50%; collect the fractions at 9-10.5min, 11.5-12.5min, 15-17.5min, 42-46min, 47-52min, 68-70min, respectively, and record them as Fr1 fraction, Fr2 fraction, Fr3 fraction, Fr4 fraction, Fr5 fraction, Fr6 fraction respectively;

[0011] The Fr1 fraction, Fr2 fraction, Fr3 fraction, Fr4 fraction, Fr5 fraction and Fr6 fraction were concentrated to dryness to obtain luteolin-7-O-β-D-glucuronide, apigenin-7-O-β-D-glucuronide, diosgenin-7-O-β-D-glucuronide, cynaroside, acacetin-7-O-glucuronide and acacetin, respectively.

[0012] Preferably, the alkali solution is saturated lime water.

[0013] Preferably, the dosage ratio of the fragrant blue orchid to the alkali solution is 1g: (15-45)mL.

[0014] Preferably, the temperature of the heating extraction is 70-85° C., the number of extractions is 2-3 times, and the time of a single extraction is 1-3 hours.

[0015] Preferably, the acid is hydrochloric acid, and the concentration of the hydrochloric acid is 5 mol / L.

[0016] Preferably, the precipitation temperature is room temperature and the time is 3 to 9 hours.

[0017] Preferably, the solvent used for the dissolution is a mixed solvent of aqueous formic acid solution and acetonitrile, the volume fraction of the aqueous formic acid solution is 0.1%, and the volume ratio of the aqueous formic acid solution to acetonitrile is 75:25.

[0018] Preferably, the flow rate of the mobile phase is 5 to 20 mL / min.

[0019] Preferably, the column temperature of the chromatographic column is 35°C.

[0020] Preferably, after the concentration to dryness, the method further comprises recrystallizing the obtained components separately, wherein the recrystallization reagent is an alcohol aqueous solution, the alcohol in the alcohol aqueous solution is methanol or ethanol, and the volume fraction of the alcohol in the alcohol aqueous solution is 20-70%.

[0021] The invention provides a method for extracting flavonoid components in scutellaria baicalensis. The method comprises the following steps: mixing scutellaria baicalensis with an alkali solution and performing heating extraction to obtain an extract; mixing the extract with an acid and performing precipitation under the condition of a pH value of 1 to 3 to obtain a precipitated extract; dissolving the precipitated extract and performing preparative liquid chromatography separation to obtain Fr1 to Fr6 fractions; concentrating the Fr1 to Fr6 fractions to dryness respectively to obtain luteolin-7-O-β-D-glucuronide, apigenin-7-O-β-D-glucuronide, diosmetin-7-O-β-D-glucuronide, cynaroside, acacetin-7-O-glucuronide and acacetin in sequence. The present invention uses the method of alkali extraction, acid precipitation and preparative liquid chromatography separation to extract and separate and purify the fragrant blue orchid, and obtains 6 flavonoid components, namely luteolin-7-O-β-D-glucuronide, apigenin-7-O-β-D-glucuronide, diosmetin-7-O-β-D-glucuronide, cynarin, acacetin-7-O-glucuronide and acacetin. The present invention quickly realizes the acquisition of high-purity monomers of the main flavonoid components in the fragrant blue orchid, namely luteolin-7-O-β-D-glucuronide, apigenin-7-O-β-D-glucuronide, diosmetin-7-O-β-D-glucuronide, cynarin, acacetin-7-O-glucuronide and acacetin, and the purity of each component is 98-99%. In addition, the method of the present invention has low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The present invention is a schematic diagram of the extraction process of flavonoid components from safflower;

[0023] Figure 2 The figure is a bar graph showing the effects of the solid-liquid ratio (a), alkali extraction time (b), alkali extraction temperature (c), and alkali extraction times (d) on the quality of cynaroside extracted in Example 1;

[0024] Figure 3 is the response surface diagram of each factor in Example 1, Figure 3 A is the effect of alkali extraction time and solid-liquid ratio on the extraction rate of cynaroside, B is the effect of alkali extraction temperature and alkali extraction time on the extraction rate of cynaroside, and C is the effect of alkali extraction temperature and solid-liquid ratio on the extraction rate of cynaroside;

[0025] Figure 4 The figure is a curve diagram showing the effect of different pH values ​​on the precipitation quality in Example 2;

[0026] Figure 5 It is a curve diagram of the effect of different acid precipitation times on the precipitation quality in Example 2;

[0027] Figure 6 This is the HPLC chromatogram of the alkali-extraction and acid-precipitation extract in Example 3;

[0028] Figure 7 The liquid chromatogram of the preparative liquid phase separation in Example 3;

[0029] Figure 8 is a high performance liquid chromatogram of the standard solution of six components in Example 3;

[0030] Fig. 9 is the HPLC chromatogram of the blank solution in Example 3;

[0031] Fig.10 This is a HPLC chromatogram of the concentrated PEAK1 fraction obtained by the preparation and separation in Example 3;

[0032] Fig.11 This is a HPLC chromatogram of the concentrated PEAK2 fraction obtained by the preparation and separation in Example 3;

[0033] Fig.12 This is a HPLC chromatogram of the concentrated PEAK3 fraction obtained by the preparation and separation in Example 3;

[0034] Fig.13 This is a HPLC chromatogram of the concentrated PEAK4 fraction obtained by the preparation and separation in Example 3;

[0035] Fig.14 This is a HPLC chromatogram of the concentrated PEAK5 fraction obtained by the preparation and separation in Example 3;

[0036] Fig.15 This is the HPLC chromatogram of the concentrated PEAK6 fraction obtained by the preparation and separation in Example 3. DETAILED DESCRIPTION

[0037] The invention provides a method for extracting flavonoid components from radix scutellariae, comprising the following steps:

[0038] Mixing the fragrant blue orchid and the alkali solution and performing heating extraction to obtain an extract;

[0039] The extract is mixed with an acid, and precipitated at a pH value of 1 to 3 to obtain a precipitated extract;

[0040] The precipitate extract is dissolved and then separated by preparative liquid chromatography, wherein the conditions for the preparative liquid chromatography separation include: a C18 chromatographic column; mobile phases A and B, wherein the mobile phase A is acetonitrile and the mobile phase B is a formic acid aqueous solution, wherein the volume fraction of formic acid in the formic acid aqueous solution is 0.1%; gradient elution;

[0041] The elution program of the gradient elution is: 0-55min, the volume ratio of mobile phase A is 25%; 55-80min, the volume ratio of mobile phase A is 50%; collect the fractions at 9-10.5min, 11.5-12.5min, 15-17.5min, 42-46min, 47-52min, 68-70min, respectively, and record them as Fr1 fraction, Fr2 fraction, Fr3 fraction, Fr4 fraction, Fr5 fraction, Fr6 fraction respectively;

[0042] The Fr1 fraction, Fr2 fraction, Fr3 fraction, Fr4 fraction, Fr5 fraction and Fr6 fraction were concentrated to dryness to obtain luteolin-7-O-β-D-glucuronide, apigenin-7-O-β-D-glucuronide, diosgenin-7-O-β-D-glucuronide, cynaroside, acacetin-7-O-glucuronide and acacetin, respectively.

[0043] Figure 1 The present invention is a schematic diagram of the extraction process of flavonoid components in Scutellaria baicalensis.

[0044] The present invention mixes the fragrant blue orchid and alkali solution and performs heating extraction to obtain an extract. In the present invention, the alkali solution is preferably saturated lime water. In an embodiment of the present invention, the saturated lime water is specifically prepared by dissolving calcium oxide in water. In the present invention, the dosage ratio of the fragrant blue orchid and the alkali solution is preferably 1g: (15-45)mL, more preferably 1g: (25-40)mL, and further preferably 1g: (35-40)mL.

[0045] In the present invention, the temperature of the heating extraction is preferably 70-85°C, more preferably 75-85°C, the number of extractions is preferably 2-3 times, more preferably 2 times, the time of a single extraction is preferably 1-3h, more preferably 3h, and the extracts are combined after 2-3 extractions.

[0046] After obtaining the extract, the present invention mixes the extract with an acid, and precipitates under the condition of a pH value of 1 to 3 to obtain a precipitated extract. In the present invention, the acid is preferably hydrochloric acid, and the concentration of the hydrochloric acid is preferably 5 mol / L; the amount of hydrochloric acid added is based on adjusting the pH value of the extract to 1 to 3. In the present invention, the pH value is preferably 1 to 2, and more preferably 1. In the present invention, the precipitation temperature is preferably room temperature, and the precipitation time is preferably 3 to 9 hours, and more preferably 6 hours.

[0047] The present invention performs alkali extraction and acid precipitation on the flavonoids in the flavonoids, and the flavonoids in the flavonoids can be extracted while increasing their solubility by using the method. In the present invention, the alkali extraction and acid precipitation method can remove components such as protein and cellulose, perform pretreatment for subsequent material separation and purification, and reduce corresponding impurities.

[0048] After obtaining the precipitate extract, the present invention dissolves the precipitate extract and performs preparative liquid chromatography separation to obtain luteolin-7-O-β-D-glucuronide, apigenin-7-O-β-D-glucuronide, diosmetin-7-O-β-D-glucuronide, cynaroside, acacetin-7-O-glucuronide, and acacetin.

[0049] In the present invention, the solvent used for the dissolution is preferably a mixed solvent of aqueous formic acid solution and acetonitrile, the volume fraction of the aqueous formic acid solution is preferably 0.1%, and the volume ratio of the aqueous formic acid solution to acetonitrile is preferably 75:25. In the present invention, the concentration of the sample solution obtained by the dissolution is preferably 1 to 30 mg / mL. In the present invention, the dissolution is preferably carried out under shaking conditions.

[0050] In the present invention, the conditions for the preparative liquid chromatography separation include: the chromatographic column is a C18 chromatographic column; the mobile phases are mobile phase A and mobile phase B, the mobile phase A is acetonitrile, the mobile phase B is a formic acid aqueous solution, the volume fraction of formic acid in the formic acid aqueous solution is 0.1%; gradient elution.

[0051] In the present invention, the specification of the C18 chromatographic column is preferably 20 mm ID×250 mm, the model of the C18 chromatographic column is preferably COSMOSILpacked Column, 5C18-MS-II, and the column temperature of the chromatographic column is preferably 35°C.

[0052] In the present invention, the detection wavelength corresponding to the preparative liquid chromatography separation is preferably 254nm or 330nm, more preferably 330nm. In the present invention, the elution procedure of the gradient elution is: 0-55min, the volume ratio of mobile phase A is 25%; 55-80min, the volume ratio of mobile phase A is 50%; collect the flow fractions of 9-10.5min, 11.5-12.5min, 15-17.5min, 42-46min, 47-52min, 68-70min respectively, and record them as Fr1 flow fraction (also can be recorded as PEAK1 flow fraction in the embodiment of the present invention), Fr2 flow fraction, Fr3 flow fraction, Fr4 flow fraction, Fr5 flow fraction, Fr6 flow fraction; the flow rate of the mobile phase is preferably 5-20mL / min, more preferably the flow rate is 10mL / min. In the present invention, the injection volume of the gradient elution is preferably 10μL.

[0053] In the present invention, if a high-purity finished product is required, the mid-peak fraction can be collected as much as possible; if a large amount of 6 components need to be collected, the same peak fractions obtained by multiple separations can be combined.

[0054] Preparative liquid chromatography is a separation and purification technology that uses a preparative chromatographic column to extract the target compound from a mixture. Compared with other chromatographic separation methods, such as thin layer chromatography, preparative liquid chromatography has the characteristics of high column efficiency and fast separation speed.

[0055] After obtaining the Fr1 fraction, the Fr2 fraction, the Fr3 fraction, the Fr4 fraction, the Fr5 fraction and the Fr6 fraction, the present invention concentrates the Fr1 fraction, the Fr2 fraction, the Fr3 fraction, the Fr4 fraction, the Fr5 fraction and the Fr6 fraction to dryness, respectively, to obtain luteolin-7-O-β-D-glucuronide, apigenin-7-O-β-D-glucuronide, diosgenin-7-O-β-D-glucuronide, cynaroside, acacetin-7-O-glucuronide and acacetin in sequence. The present invention has no special requirements for the concentration method, and a method well known to those skilled in the art can be used. In the present invention, after the concentration to dryness, the purity of each component obtained is 98-99%.

[0056] After the concentration to dryness, the present invention also preferably recrystallizes the obtained components separately. In the present invention, the reagent for the recrystallization is preferably an alcohol aqueous solution, the alcohol in the alcohol aqueous solution is preferably methanol or ethanol, and the volume fraction of the alcohol in the alcohol aqueous solution is preferably 20-70%. After the recrystallization, the six components of luteolin-7-O-β-D-glucuronide, apigenin-7-O-β-D-glucuronide, diosgenin-7-O-β-D-glucuronide, cynaroside, acacetin-7-O-glucuronide, and acacetin are obtained in the form of pale yellow or white powder crystals with a purity of more than 98%.

[0057] In order to further illustrate the present invention, the extraction method of flavonoid components in the fragrant blue orchid provided by the present invention is described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present invention.

[0058] The Xiangqinglan medicinal materials in each embodiment were purchased from Haozhou Shan'antang Chinese Medicine Pieces Co., Ltd., acetonitrile and methanol were chromatographically pure, and water was ultrapure water.

[0059] Example 1

[0060] Alkaline extraction of the Herba Cinerariae: Take the Herba Cinerariae slices and heat them with saturated lime water for extraction.

[0061] The quality of cynaroside in the extract obtained by alkaline extraction was used as the evaluation index to investigate the effects of solid-liquid ratio, alkaline extraction time, alkaline extraction temperature and alkaline extraction times on the extraction of six flavonoid components.

[0062] 1.1 Solid-liquid ratio: Take 2g of medicinal powder, weigh accurately, and extract once at a water bath temperature of 75℃ for 1h. The effects of different solid-liquid ratios of 1g:15mL, 1g:25mL, 1g:35mL, and 1g:45mL on the extraction of cynaroside were investigated. The mass of cynaroside was determined by three parallel experiments and the average value was taken. The results are shown in Table 1. Figure 2 Middle (a).

[0063] Depend on Figure 2 As can be seen in (a), the solid-liquid ratio tends to be stable when it is 1g:35mL, so a solid-liquid ratio of 1g:35mL is selected to continue the subsequent experiments.

[0064] 1.2 Alkali extraction time: Take 2g of medicinal powder, weigh accurately, and extract once in a water bath at 75℃. The solid-liquid ratio is 1g:35mL. The effect of different extraction times of 0.5, 1, 3, and 5h on the extraction of cynaroside was investigated. The experiment was repeated 3 times in parallel to determine the quality of cynaroside. The average value was taken. The results are shown in Figure 2 Middle (b).

[0065] Depend on Figure 2 As can be seen in (b), the extraction quality tends to be stable when the alkaline extraction time is 3 hours, so the extraction time of 3 hours is selected.

[0066] 1.3 Alkali extraction temperature: Take 2g of medicinal powder, accurately weigh, solid-liquid ratio 1g:35mL, extract once, extraction time 3h, investigate the effect of different extraction temperatures 25℃, 50℃, 75℃, 100℃ on the extraction of cynaroside, parallel experiments 3 times, determine the quality of cynaroside, take the average value, the results are shown in Figure 2 Middle (c).

[0067] Depend on Figure 2 As can be seen in (c), the mass of cynaroside is the largest when the extraction temperature is 75°C, so the extraction temperature of 75°C is selected.

[0068] 1.4 Number of alkaline extractions: 2 g of medicinal powder was taken and accurately weighed, with a solid-liquid ratio of 1 g:35 mL, an extraction time of 3 h, and an extraction temperature of 75 °C. The effects of different extraction times of 1, 2, and 3 on the extraction of cynaroside were investigated. The quality of cynaroside was determined by performing the experiment 3 times in parallel, and the average value was taken. The results are shown in Figure 2 Middle (d).

[0069] Depend on Figure 2 As can be seen in (d), when the extraction times are 2, the total extraction mass of cynaroside 2 times accounts for 83.63% of the total extraction mass of 3 times. For economic reasons, 2 extraction times were selected.

[0070] The Box-Behnken response surface optimization of the extraction process and the result verification are as follows:

[0071] (1) Response surface experimental design and results: Design Expert 12 software was used for response surface design. Based on the single factor experiment, the extraction rate of cynaroside in cynaroside was used as the response value. The Box-Behnken response surface method was used to design 17 groups of experiments with three factors and three levels (-1, 0, 1) of solid-liquid ratio (A), alkali extraction time (B), and alkali extraction temperature (C). The experimental design and results are shown in Tables 1 and 2. The extraction rate of cynaroside in cynaroside was calculated as follows:

[0072]

[0073] Table 1 Factor levels of Box-Behnken response surface experiment

[0074]

[0075]

[0076] Table 2 Box-Behnken response surface experimental design and results

[0077]

[0078] Note: In Table 2, A-solid-to-liquid ratio (g / mL); B-extraction time (h); C-extraction temperature (°C).

[0079] (2) Model establishment and significance test

[0080] The above results were fitted by multiple regression using Design Expert 12 software, and the equation was Y = 0.3376 + 0.0097A-0.0026B + 0.0194C-0.0015AB + 0.0487AC-0.0262BC-0.0323A 2 -0.0587B 2+0.0444C 2 , analysis of variance is shown in Table 3. It can be seen that the model is extremely significant (P < 0.01), and the lack of fit term is not significant (P > 0.05), indicating that its fitting degree is high and the error is small. 2 =0.9671, indicating a strong correlation; Adj R 2 (0.9248) and Pred R 2 (0.6643) is within the acceptable range, and the difference is close to 0.4, indicating that the model can well correspond to the changes in extraction rate caused by factor changes. Factors C, AC, BC, A 2 , B 2 , C 2 There was a significant effect (P < 0.05), while A, B, and AB had no significant effect (P > 0.05).

[0081] Table 3 Analysis of variance of multiple regression model

[0082]

[0083] (3) Response surface analysis

[0084] Figure 3 Response surface diagram for each factor.

[0085] analyze Figure 3 The response surface diagram shows that when the alkali extraction temperature is fixed, the interaction between the alkali extraction time and the solid-liquid ratio is Figure 3 In A, when the alkali extraction time is constant, the extraction rate increases first and then decreases as the solid-liquid ratio changes; when the solid-liquid ratio is constant, the extraction rate increases first and then decreases as the alkali extraction time changes. This result shows that the interaction between the solid-liquid ratio and the alkali extraction time is significant. When the solid-liquid ratio is fixed, the interaction between the alkali extraction temperature and the alkali extraction time is Figure 3 In B, when the alkali extraction temperature is constant, the extraction first increases and then decreases with the change of alkali extraction time; when the alkali extraction time is constant, the extraction gradually increases with the change of alkali extraction temperature. This result shows that the interaction between alkali extraction time and alkali extraction temperature is significant. When the alkali extraction time is fixed, the interaction between alkali extraction temperature and solid-liquid ratio is Figure 3 In C, when the alkali extraction temperature is constant, the extraction rate increases slightly and then decreases with the change of the solid-liquid ratio; when the solid-liquid ratio is 1:40, the extraction rate gradually increases with the change of the alkali extraction temperature. This result shows that there is a certain interaction between the solid-liquid ratio and the alkali extraction temperature.

[0086] (4) Optimal process verification experiment

[0087] The optimal process obtained by Design-expert 12 software is: material-liquid ratio 1g:40mL, alkali extraction time 3h, alkali extraction temperature 85℃, alkali extraction times 2 times, and the predicted extraction rate is 0.427%. According to the above optimized process, 120g of material was added for three batches of verification tests, and the average extraction rate was measured to be 0.472%, which is close to the predicted value, indicating that the model is stable and reliable.

[0088] Example 2

[0089] Take 120g of Xiangqinglan slices, prepare the extract according to the optimal extraction parameters determined in Example 1 (solid-liquid ratio 1g:40mL, alkali extraction time 3h, alkali extraction temperature 85℃, alkali extraction times 2 times), divide it into 5 parts, adjust the pH value of the solution with 5mol / L hydrochloric acid solution, and measure it with a pH meter to investigate the effect of different pH values ​​on the quality of the precipitate obtained by acid precipitation. The results of the effect of different pH values ​​on the quality of the precipitate are shown in Figure 1. Figure 4 As shown, it can be seen that the precipitation mass is the largest when pH = 1.

[0090] Take 120g of medicinal materials, prepare the extract according to the optimal extraction parameters determined in Example 1 (material-liquid ratio 1g:40mL, alkali extraction time 3h, alkali extraction temperature 85℃, alkali extraction times 2 times), divide it into 5 parts, adjust the solution pH to 1 with 5mol / L hydrochloric acid solution, and examine the precipitation quality after 0.5h, 1h, 3h, 6h, 12h, and 24h. The results are as follows Figure 5 As shown, the precipitation mass reaches the maximum value at 6h.

[0091] Example 3

[0092] The main flavonoid components, luteolin-7-O-β-D-glucuronide, apigenin-7-O-β-D-glucuronide, diosgenin-7-O-β-D-glucuronide, cynaroside, acacetin-7-O-glucuronide, and acacetin, are extracted and separated from the fragrant blue orchid, and the steps are as follows:

[0093] (1) Extraction: Take the slices of Radix Achyranthis Bidentatae and heat them at 85°C for 2 times with saturated lime water. The single extraction time is 3 h, and the solid-liquid ratio is 1 g:40 mL. Filter and combine the extracts, adjust the pH value of the extracts to 1 with hydrochloric acid, and precipitate for 6 h to obtain the alkaline-extraction and acid-precipitation extracts.

[0094] (2) preparing a sample solution: accurately weigh an appropriate amount of the alkali-extracted and acid-precipitated extract in (1), add a solvent to dissolve, shake thoroughly, and prepare a sample solution with a concentration range of 15 mg / mL. The solvent is a mixed solvent of 0.1% formic acid water and acetonitrile, and the volume ratio of 0.1% formic acid water to acetonitrile is 75:25;

[0095] (3) Preparative liquid chromatography separation: the mobile phases are mobile phase A and mobile phase B, mobile phase A is acetonitrile, and mobile phase B is 0.1% formic acid aqueous solution; a COSMOSIL packed Column, 5C18-MS-II chromatographic column (20 mm ID×250 mm) is used, the column temperature is 35° C., and the mobile phase (volume ratio: mobile phase A: 25%, mobile phase B: 75%) is injected at a flow rate of 10 mL / min. After the liquid completely infiltrates the column and the detector baseline is stable, the sample solution in (2) is injected, and the ultraviolet absorption wavelength is set to 330 nm. Gradient elution: 0-55 min: the volume ratio of mobile phase A is 25%, and the volume ratio of mobile phase B is 75%; 55-80 min: the volume ratio of mobile phase A is 50%, and the volume ratio of mobile phase B is 50%. After loading the sample, a chromatogram was collected and fractions were collected according to the UV peaks: 9-10.5 min, PEAK1; 11.5-12.5 min, PEAK2; 15-17.5 min, PEAK3; 42-46 min, PEAK4; 47-52 min, PEAK5; 68-70 min, PEAK6.

[0096] (4) The PEAK1 to PEAK6 fractions collected in step (3) are concentrated to dryness in sequence to obtain six flavonoid component monomers, namely, luteolin-7-O-β-D-glucuronide, apigenin-7-O-β-D-glucuronide, diosgenin-7-O-β-D-glucuronide, cynaroside, acacetin-7-O-glucuronide, and acacetin.

[0097] HPLC determination of 6 flavonoid components in the intermediate product:

[0098] Chromatographic conditions: Waters E2695 high performance liquid chromatograph (Waters Corporation, USA), chromatographic column: Zorbax SB-C18 column (150×4.6 mm, 5 μm); mobile phase: acetonitrile-0.1% formic acid aqueous solution, gradient elution: 0-10 min, 22%-25% acetonitrile; 10-25 min, 25% acetonitrile; 25-35 min, 25-80% acetonitrile; 35-40 min, 80% acetonitrile; detection wavelength 330 nm; flow rate: 1.0 mL / min; column temperature: 35°C, injection volume: 10 μL.

[0099] Preparation of reference substance solution: Accurately weigh 2.5 mg of the six reference substances, place in a 25 mL volumetric flask, add 70% methanol to dissolve and dilute to the scale, shake well, and obtain 100 μg / mL reference substance stock solution.

[0100] The 6-component standard solution was run under the determined HPLC conditions. The HPLC chromatogram is as follows: Figure 8 As shown ( Fig. 9is the HPLC chromatogram of the blank solution), in which the peak times and peak shapes of the six components were all good.

[0101] Figure 6 The HPLC chromatogram of the alkali-extraction and acid-precipitation extract obtained in Example 3. The transfer rate in Example 3 is 85% (the transfer rate in the example refers to the percentage of the mass of cynaroside in the intermediate product obtained by alkali-extraction and acid-precipitation to the mass of cynaroside in the extracted medicinal material, wherein the mass of cynaroside in the extracted medicinal material is measured by three times of ethanol ultrasonic extraction).

[0102] Figure 7 This is a liquid chromatogram of Example 3 using preparative liquid separation.

[0103] Figure 10 to Figure 15 The HPLC chromatograms after concentration of each fraction obtained by the preparation and separation in Example 3. The purity of each component after concentration was above 97%, specifically, the purity of luteolin-7-O-β-D-glucuronide was 98.52%, the purity of apigenin-7-O-β-D-glucuronide was 98.26%, the purity of diosgenin-7-O-β-D-glucuronide was 97.63%, the purity of cynaroside was 97.85%, the purity of acacetin-7-O-glucuronide was 97.34%, and the purity of acacetin was 98.95%.

[0104] Example 4

[0105] The extraction conditions were as follows: extraction times: 2 times, extraction temperature: 85° C., extraction time: 2 h, solid-liquid ratio: 1 g:40 mL, acid precipitation pH: 1, acid precipitation time: 6 h, transfer rate: 83%, and subsequent separation operation was performed using the preparative liquid phase according to the method and conditions of Example 3, and the purity of each component was above 97%.

[0106] Example 5

[0107] The extraction conditions were as follows: extraction times: 2 times, extraction temperature: 65° C., extraction time: 2 h, solid-liquid ratio: 1 g:20 mL, acid precipitation pH: 2, acid precipitation time: 6 h, transfer rate: 67%, and subsequent separation operation was performed using the preparative liquid phase according to the method and conditions of Example 3, and the purity of each component was above 97%.

[0108] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for extracting flavonoids from Scutellaria baicalensis, characterized in that: The following steps are involved: Mixing the fragrant blue orchid and alkali solution and performing heating extraction to obtain an extract; the alkali solution is saturated lime water, and the amount ratio of the fragrant blue orchid to the alkali solution is 1g: (15-45)mL; The extract is mixed with an acid, and precipitated at a pH value of 1 to 3 to obtain a precipitated extract; the acid is hydrochloric acid, and the concentration of the hydrochloric acid is 5 mol / L; The precipitate extract is dissolved and then separated by preparative liquid chromatography, wherein the conditions for the preparative liquid chromatography separation include: a C18 chromatographic column; mobile phases A and B, wherein the mobile phase A is acetonitrile and the mobile phase B is a formic acid aqueous solution, wherein the volume fraction of formic acid in the formic acid aqueous solution is 0.1%; gradient elution; The elution program of the gradient elution is: 0-55min, the volume ratio of mobile phase A is 25%; 55-80min, the volume ratio of mobile phase A is 50%; collect the fractions at 9-10.5min, 11.5-12.5min, 15-17.5min, 42-46min, 47-52min, 68-70min, respectively, and record them as Fr1 fraction, Fr2 fraction, Fr3 fraction, Fr4 fraction, Fr5 fraction, Fr6 fraction respectively; The Fr1 fraction, the Fr2 fraction, the Fr3 fraction, the Fr4 fraction, the Fr5 fraction and the Fr6 fraction are concentrated to dryness to obtain luteolin-7-O-β-D-glucuronide, apigenin-7-O-β-D-glucuronide, diosmetin-7-O-β-D-glucuronide, cynaroside, acacetin-7-O-glucuronide and acacetin in sequence; The solvent used for the dissolution is a mixed solvent of aqueous formic acid solution and acetonitrile, the volume fraction of the aqueous formic acid solution is 0.1%, and the volume ratio of the aqueous formic acid solution to acetonitrile is 75:

25.

2. The extraction method according to claim 1, characterized in that The temperature of the heating extraction is 70-85° C., the number of extractions is 2-3 times, and the time of a single extraction is 1-3 hours.

3. The extraction method according to claim 1, characterized in that The precipitation temperature is room temperature and the precipitation time is 3 to 9 hours.

4. The extraction method according to claim 1, characterized in that The flow rate of the mobile phase is 5 to 20 mL / min.

5. The extraction method according to claim 1, characterized in that The column temperature of the chromatographic column is 35°C.

6. The extraction method according to claim 1, characterized in that After the concentration to dryness, the method further comprises recrystallizing the obtained components respectively, wherein the recrystallization reagent is an alcohol aqueous solution, the alcohol in the alcohol aqueous solution is methanol or ethanol, and the volume fraction of the alcohol in the alcohol aqueous solution is 20-70%.

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