Method for extracting flavonoid components in dracocephalum moldavica l.

Abstract

A method for extracting flavonoid components in Dracocephalum moldavica L., comprising the following steps: mixing Dracocephalum moldavica L. and an alkaline solution for heating extraction; under the condition that the pH value is 1-3, mixing an obtained extract solution with acid for precipitation; dissolving an obtained precipitated extract and then carrying out preparative liquid chromatography separation; and respectively concentrating obtained Fr1-Fr6 fractions until the fractions are dry, so as to sequentially obtain luteolin-7-O-β-D-glucuronide, apigenin-7-O-β-D-glucuronide, diosmetin-7-O-β-D-glucuronide, tilianin, acacetin-7-O-glucuronide, and acacetin.

Description

A method for extracting flavonoids from Scutellaria baicalensis

[0001] This application claims priority to a Chinese patent application filed with the Patent Office of China on January 23, 2024, with application number CN202410099619.6 and invention name “A method for extracting flavonoid components from Orchidaceae”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present application relates to the technical field of drug extraction, and in particular to a method for extracting flavonoid components from Herba Citri Reticulatae. Background Art

[0003] Dracocephalum Moldavica L., a Xinjiang specialty Uyghur medicine known as Badiranjbuya, is an annual herbaceous plant of the Lamiaceae family, often used as a whole herb. Its medicinal material standards are documented in the "Drug Standards of the Ministry of Health of the People's Republic of China: Uyghur Medicine Volume" and the "Uyghur Pharmacy (Volume 1)." Dracocephalum Moldavica L. primarily contains flavonoids, volatile oils, phenylpropanoids, polysaccharides, amino acids, proteins, and trace elements. The main components of the total flavonoid fraction of Dracocephalum Moldavica include 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 total flavonoids of Scutellaria baicalensis have certain therapeutic effects on many diseases, such as treating atherosclerosis, protecting myocardial ischemia-reperfusion injury, anti-inflammation, and anti-oxidation.

[0004] At present, there are few studies on the extraction and purification of flavonoid components from Scutellaria baicalensis at home and abroad. The main method is to use 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 flavonoid 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.

[0005] Application Contents

[0006] In view of this, the purpose of this application is to provide a method for extracting flavonoids from Scutellaria baicalensis. The extraction method provided in this application is simple in process and can obtain high-purity flavonoids (luteolin-7-O-β-D-glucuronide, apigenin-7-O-β-D-glucuronide, diosmetin-7-O-β-D-glucuronide, cynarin, acacetin-7-O-glucuronide, acacetin) from Scutellaria baicalensis.

[0007] In order to achieve the above application objectives, this application provides the following technical solutions:

[0008] The present application provides a method for extracting flavonoids from Scutellaria baicalensis, comprising the following steps:

[0009] Mixing the orchid and alkali solution and performing heating extraction to obtain an extract;

[0010] mixing the extract with an acid, and performing precipitation at a pH value of 1 to 3 to obtain a precipitated extract;

[0011] The precipitate extract is dissolved and then subjected to preparative liquid chromatography separation, 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%; and gradient elution;

[0012] The gradient elution program is as follows: from 0 to 55 min, the volume ratio of mobile phase A is 25%; from 55 to 80 min, the volume ratio of mobile phase A is 50%; fractions at 9 to 10.5 min, 11.5 to 12.5 min, 15 to 17.5 min, 42 to 46 min, 47 to 52 min, and 68 to 70 min are collected and recorded as fraction Fr1, fraction Fr2, fraction Fr3, fraction Fr4, fraction Fr5, and fraction Fr6, respectively;

[0013] The Fr1 fraction, Fr2 fraction, Fr3 fraction, Fr4 fraction, Fr5 fraction and Fr6 fraction were respectively concentrated to dryness to obtain luteolin-7-O-β-D-glucuronide, apigenin-7-O-β-D-glucuronide, diosgenin-7-O-β-D-glucuronide, cynarin, acacetin-7-O-glucuronide and acacetin, respectively.

[0014] Preferably, the alkali solution is saturated lime water.

[0015] Preferably, the usage ratio of the fragrant blue orchid and the alkali solution is 1g: (15-45)mL.

[0016] 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.

[0017] Preferably, the acid is hydrochloric acid, and the concentration of the hydrochloric acid is 5 mol / L.

[0018] Preferably, the precipitation temperature is room temperature and the precipitation time is 3 to 9 hours.

[0019] 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.

[0020] Preferably, the flow rate of the mobile phase is 5 to 20 mL / min.

[0021] Preferably, the column temperature of the chromatographic column is 35°C.

[0022] Preferably, after concentrating 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%.

[0023] The present application provides a method for extracting flavonoid components from Herba Lycopersicum odoratum, comprising the following steps: mixing Herba Lycopersicum odoratum with an alkali solution and performing heating extraction to obtain an extract; mixing the extract with an acid and performing precipitation at 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 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 application uses the method of alkali extraction, acid precipitation and preparative liquid chromatography separation to extract and separate and purify the 6 flavonoid components of 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 application quickly achieves the high-purity monomer acquisition of the main flavonoid components in 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 application is low in cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG1 is a schematic diagram of the extraction process of flavonoid components from the present application;

[0025] FIG2 is a bar graph showing the effects of the material-liquid ratio (a), alkali extraction time (b), alkali extraction temperature (c), and alkali extraction times (d) on the quality of cynarin extracted in Example 1;

[0026] Figure 3 is a response surface diagram of each factor in Example 1, in which A is the effect of alkali extraction time and solid-liquid ratio on the extraction rate of cynarin, B is the effect of alkali extraction temperature and alkali extraction time on the extraction rate of cynarin, and C is the effect of alkali extraction temperature and solid-liquid ratio on the extraction rate of cynarin;

[0027] FIG4 is a graph showing the effect of different pH values ​​on the quality of the precipitate in Example 2;

[0028] FIG5 is a graph showing the effect of different acid precipitation times on precipitation quality in Example 2;

[0029] FIG6 is a high performance liquid chromatogram of the alkali-extracted and acid-precipitated extract in Example 3;

[0030] FIG7 is a liquid chromatogram of the preparative liquid phase separation in Example 3;

[0031] FIG8 is a high performance liquid chromatogram of a standard solution of six components in Example 3;

[0032] FIG9 is a high performance liquid chromatogram of the blank solution in Example 3;

[0033] FIG10 is a HPLC chromatogram of the concentrated PEAK1 fraction obtained from the preparative separation in Example 3;

[0034] FIG11 is a HPLC chromatogram of the concentrated PEAK2 fraction obtained from the preparative separation in Example 3;

[0035] FIG12 is a HPLC chromatogram of the concentrated PEAK3 fraction obtained from the preparative separation in Example 3;

[0036] FIG13 is a HPLC chromatogram of the concentrated PEAK4 fraction obtained from the preparative separation in Example 3;

[0037] FIG14 is a HPLC chromatogram of the concentrated PEAK5 fraction obtained from the preparative separation in Example 3;

[0038] FIG15 is a HPLC chromatogram of the concentrated PEAK6 fraction obtained by preparative separation in Example 3. DETAILED DESCRIPTION

[0039] The present application provides a method for extracting flavonoids from Scutellaria baicalensis, comprising the following steps:

[0040] Mixing the orchid and alkali solution and performing heating extraction to obtain an extract;

[0041] mixing the extract with an acid, and performing precipitation at a pH value of 1 to 3 to obtain a precipitated extract;

[0042] The precipitate extract is dissolved and then subjected to preparative liquid chromatography separation, 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%; and gradient elution;

[0043] The gradient elution program is as follows: from 0 to 55 min, the volume ratio of mobile phase A is 25%; from 55 to 80 min, the volume ratio of mobile phase A is 50%; fractions at 9 to 10.5 min, 11.5 to 12.5 min, 15 to 17.5 min, 42 to 46 min, 47 to 52 min, and 68 to 70 min are collected and recorded as fraction Fr1, fraction Fr2, fraction Fr3, fraction Fr4, fraction Fr5, and fraction Fr6, respectively;

[0044] The Fr1 fraction, Fr2 fraction, Fr3 fraction, Fr4 fraction, Fr5 fraction and Fr6 fraction were respectively concentrated to dryness to obtain luteolin-7-O-β-D-glucuronide, apigenin-7-O-β-D-glucuronide, diosgenin-7-O-β-D-glucuronide, cynarin, acacetin-7-O-glucuronide and acacetin, respectively.

[0045] FIG1 is a schematic diagram of the extraction process of flavonoid components from Scutellaria baicalensis in this application.

[0046] In the present application, the mixture of fennel and alkali solution is heated and extracted to obtain an extract. In the present application, the alkali solution is preferably saturated lime water. In the embodiment of the present application, the saturated lime water is specifically prepared by dissolving calcium oxide in water. In the present application, the dosage ratio of the fennel and alkali solution is preferably 1g: (15-45) mL, more preferably 1g: (25-40) mL, and further preferably 1g: (35-40) mL.

[0047] In the present application, 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 1-3 hours, more preferably 3 hours, and the extracts are combined after 2-3 extractions.

[0048] After obtaining the extract, the present application mixes the extract with an acid and performs precipitation at a pH of 1 to 3 to obtain a precipitated extract. In the present application, 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 application, the pH value is preferably 1 to 2, more preferably 1. In the present application, the precipitation temperature is preferably room temperature, and the precipitation time is preferably 3 to 9 hours, more preferably 6 hours.

[0049] The present invention performs alkaline extraction and acid precipitation on the flavonoids in the flavonoids, and the flavonoids in the flavonoids can be extracted by this method while increasing their solubility. In the present invention, the alkaline extraction and acid precipitation method can remove components such as protein and cellulose, perform pretreatment for subsequent separation and purification of substances, and reduce corresponding impurities.

[0050] After obtaining the precipitated extract, the present application dissolved the precipitated extract and performed preparative liquid chromatography separation to obtain luteolin-7-O-β-D-glucuronide, apigenin-7-O-β-D-glucuronide, diosgenin-7-O-β-D-glucuronide, cynarin, acacetin-7-O-glucuronide, and acacetin.

[0051] In the present application, the solvent used for the dissolution is preferably a mixed solvent of aqueous formic acid and acetonitrile, the volume fraction of the aqueous formic acid is preferably 0.1%, and the volume ratio of the aqueous formic acid to acetonitrile is preferably 75:25. In the present application, the concentration of the sample solution obtained by the dissolution is preferably 1 to 30 mg / mL. In the present application, the dissolution is preferably performed under shaking conditions.

[0052] In the present application, 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.

[0053] In the present application, the specification of the C18 chromatographic column is preferably 20 mm ID×250 mm, the model of the C18 chromatographic column is preferably COSMOSIL packed Column, 5C18-MS-II, and the column temperature of the chromatographic column is preferably 35°C.

[0054] In the present application, the detection wavelength corresponding to the preparative liquid chromatography separation is preferably 254nm or 330nm, more preferably 330nm. In the present application, 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 of 9-10.5min, 11.5-12.5min, 15-17.5min, 42-46min, 47-52min, and 68-70min respectively, and record them as Fr1 fraction (also referred to as PEAK1 fraction in the embodiment of the present application), Fr2 fraction, Fr3 fraction, Fr4 fraction, Fr5 fraction, and Fr6 fraction respectively; the flow rate of the mobile phase is preferably 5-20mL / min, more preferably a flow rate of 10mL / min. In the present application, the injection volume of the gradient elution is preferably 10μL.

[0055] In this application, 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.

[0056] Preparative liquid chromatography (HPLC) is a separation and purification technique that uses preparative columns to extract target compounds from mixtures. Compared to other chromatographic separation methods, such as thin-layer chromatography (TLC), HPLC boasts high column efficiency and rapid separation speed.

[0057] After obtaining the Fr1 fraction, the Fr2 fraction, the Fr3 fraction, the Fr4 fraction, the Fr5 fraction, and the Fr6 fraction, the present application 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, diosmetin-7-O-β-D-glucuronide, cynarin, acacetin-7-O-glucuronide, and acacetin, in sequence. The present application has no particular requirements for the concentration method, and methods well known to those skilled in the art can be used. In the present application, after the concentration to dryness, the purity of each component obtained is 98-99%.

[0058] After the concentration to dryness, the present application also preferably recrystallizes each of the obtained components separately. In the present application, the recrystallization reagent 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, cynarin, acacetin-7-O-glucuronide, and acacetin are obtained in the form of light yellow or white powder crystals with a purity of more than 98%.

[0059] In order to further illustrate the present application, the extraction method of flavonoid components from the fragrant blue orchid provided in the present application is described in detail below with reference to examples, but they should not be understood as limiting the scope of protection of the present application.

[0060] The medicinal materials of Xiangqinglan 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.

[0061] Example 1

[0062] Perform alkaline extraction on the Herba Lycoris Radiatae: Take the Herba Lycoris Radiatae slices and heat them with saturated lime water for extraction.

[0063] The quality of cynarin in the extract obtained by alkaline extraction was used as the evaluation index to investigate the effects of material-liquid ratio, alkaline extraction time, alkaline extraction temperature and alkaline extraction times on the extraction of six flavonoid components.

[0064] 1.1 Material-liquid ratio: 2 g of medicinal material powder was accurately weighed and extracted once in a water bath at 75°C for 1 h. The effects of different material-liquid ratios of 1 g:15 mL, 1 g:25 mL, 1 g:35 mL, and 1 g:45 mL on the extraction of cynarin were investigated. The experiments were repeated three times in parallel, and the mass of cynarin was determined. The average value was taken. The results are shown in Figure 2 (a).

[0065] As can be seen from Figure 2 (a), the solid-liquid ratio tends to be stable when it is 1g:35mL, so the solid-liquid ratio of 1g:35mL was selected to continue the subsequent experiments.

[0066] 1.2 Alkali extraction time: 2 g of medicinal material powder was accurately weighed and extracted once in a water bath at 75°C with a solid-liquid ratio of 1 g:35 mL. The effects of different extraction times of 0.5, 1, 3, and 5 h on the extraction of cynarin were investigated. Three parallel experiments were performed to determine the mass of cynarin and take the average value. The results are shown in Figure 2 (b).

[0067] As can be seen from Figure 2 (b), the extraction quality tends to be stable when the alkaline extraction time is 3 h, so the extraction time of 3 h was selected.

[0068] 1.3 Alkali extraction temperature: 2 g of medicinal material powder was accurately weighed, with a solid-liquid ratio of 1 g:35 mL. Extraction was performed once for 3 h. The effects of different extraction temperatures (25°C, 50°C, 75°C, and 100°C) on the extraction of cynarin were investigated. Three parallel experiments were conducted to determine the mass of cynarin and take the average value. The results are shown in Figure 2 (c).

[0069] As can be seen from Figure 2 (c), the mass of cynarin is the largest when the extraction temperature is 75°C, so the extraction temperature of 75°C is selected.

[0070] 1.4 Number of alkaline extractions: 2 g of medicinal material powder was 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 1, 2, and 3 extractions on the extraction of cynarin were investigated. Three parallel experiments were performed, and the mass of cynarin was determined. The average value was taken. The results are shown in Figure 2 (d).

[0071] As can be seen from Figure 2 (d), when the extraction times are 2, the total extraction mass of cynarin in 2 times accounts for 83.63% of the total extraction mass in 3 times. For economic reasons, the extraction times were selected to be 2 times.

[0072] The Box-Behnken response surface optimization of the extraction process and the results verification are as follows:

[0073] (1) Response surface design and results: Design Expert 12 software was used to perform response surface design. Based on the single-factor experiment, the extraction rate of cyperin from cyperin was used as the response value. The Box-Behnken response surface methodology was used to design 17 experimental groups with three factors and three levels (-1, 0, 1) for 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 cyperin from cyperin was calculated as follows:

[0074] Table 1 Factor levels in Box-Behnken response surface experiment

[0075] Table 2 Box-Behnken response surface design and results Note: In Table 2, A-solid-to-liquid ratio (g / mL); B-extraction time (h); C-extraction temperature (°C).

[0076] (2) Model establishment and significance test

[0077] The above results were subjected to multiple regression fitting 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).

[0078] Table 3 Analysis of variance of multiple regression model

[0079] (3) Response surface analysis

[0080] Figure 3 is the response surface diagram of each factor.

[0081] Analysis of the response surface plot in Figure 3 shows that when the alkali extraction temperature is fixed, the interaction between alkali extraction time and solid-liquid ratio is shown in Figure 3A. When the alkali extraction time is constant, the extraction rate initially increases and then decreases with changes in the solid-liquid ratio. When the solid-liquid ratio is constant, the extraction rate initially increases and then decreases with changes in the alkali extraction time. This result indicates a significant interaction between the solid-liquid ratio and alkali extraction time. When the solid-liquid ratio is fixed, the interaction between alkali extraction temperature and alkali extraction time is shown in Figure 3B. When the alkali extraction temperature is constant, the extraction rate initially increases and then decreases with changes in the alkali extraction time. When the alkali extraction time is constant, the extraction rate gradually increases with changes in the alkali extraction temperature. This result indicates a significant interaction between the alkali extraction time and alkali extraction temperature. When the alkali extraction time is fixed, the interaction between alkali extraction temperature and solid-liquid ratio is shown in Figure 3C. When the alkali extraction temperature is constant, the extraction rate slightly increases and then decreases with changes in the solid-liquid ratio. When the solid-liquid ratio is 1:40, the extraction rate gradually increases with changes in the alkali extraction temperature. This result indicates a certain interaction between the solid-liquid ratio and alkali extraction temperature.

[0082] (4) Optimal process verification experiment

[0083] The optimal process obtained by Design-expert 12 software is: material-liquid ratio 1g:40mL, alkaline extraction time 3h, alkaline extraction temperature 85℃, and alkaline extraction times 2 times. 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.

[0084] Example 2

[0085] Take 120g of Xiangqinglan slices and prepare an 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). The extract is divided into 5 equal parts and the pH value of the solution is adjusted with 5mol / L hydrochloric acid solution. The pH meter is used to measure 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 4. It can be seen that the precipitate quality is the largest when pH=1.

[0086] 120 g of the medicinal material was used to prepare an extract according to the optimal extraction parameters determined in Example 1 (material-liquid ratio 1 g:40 mL, alkali extraction time 3 h, alkali extraction temperature 85°C, and alkali extraction times 2 times). The extract was divided into five equal portions and the pH of the solution was adjusted to 1 with 5 mol / L hydrochloric acid solution. The precipitation quality was tested after 0.5 h, 1 h, 3 h, 6 h, 12 h, and 24 h. The results are shown in Figure 5. The precipitation quality reached its maximum at 6 h.

[0087] Example 3

[0088] The main flavonoid components 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 are extracted and separated from the radix serratae. The steps are as follows:

[0089] (1) Extraction: Take the slices of Radix Angelicae Dahuricae and heat them at 85℃ with saturated lime water for 2 times. The single extraction time is 3 hours and the material-liquid ratio is 1g:40mL. Filter and combine the extracts. Adjust the pH value of the extracts to 1 with hydrochloric acid and precipitate for 6 hours to obtain the alkaline-extraction and acid-precipitation extracts.

[0090] (2) Preparation of sample solution: Accurately weigh an appropriate amount of the alkali-extracted and acid-precipitated extracts in (1), add 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;

[0091] (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 (20mm ID×250mm) 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 soaks 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%, the volume ratio of mobile phase B is 75%; 55-80 min: the volume ratio of mobile phase A is 50%, 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.

[0092] (4) The PEAK1 to PEAK6 fractions collected in step (3) were concentrated to dryness in sequence to obtain 6 flavonoid monomers: luteolin-7-O-β-D-glucuronide, apigenin-7-O-β-D-glucuronide, diosgenin-7-O-β-D-glucuronide, cynarin, acacetin-7-O-glucuronide, and acacetin.

[0093] HPLC determination of 6 flavonoid components in the intermediate product:

[0094] Chromatographic conditions: Waters E2695 high performance liquid chromatograph (Waters, 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.

[0095] Preparation of reference solution: Accurately weigh 2.5 mg of each of the six reference components and place them in a 25 mL volumetric flask. Dissolve and dilute to the mark with 70% methanol and shake well to obtain a 100 μg / mL reference stock solution.

[0096] The 6-component standard solution was run under the determined liquid chromatography conditions. The HPLC chromatogram is shown in FIG8 ( FIG9 is the HPLC chromatogram of the blank solution). The peak times and peak shapes of the 6 components were all good.

[0097] FIG6 is a 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 examples refers to the percentage of the mass of cynarin in the intermediate product obtained by alkali-extraction and acid-precipitation to the mass of cynarin in the extracted medicinal material, where the mass of cynarin in the extracted medicinal material is measured by three ethanol ultrasonic extractions).

[0098] FIG7 is a liquid chromatogram of the preparative liquid phase separation in Example 3.

[0099] Figures 10 to 15 are HPLC chromatograms of the concentrated fractions obtained from the preparative separation in Example 3. The purity of each component after concentration was over 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 diosmetin-7-O-β-D-glucuronide was 97.63%, the purity of cynarin was 97.85%, the purity of acacetin-7-O-glucuronide was 97.34%, and the purity of acacetin was 98.95%.

[0100] Example 4

[0101] The extraction conditions were as follows: 2 extractions, 85° C., 2 h of extraction time, a solid-liquid ratio of 1 g:40 mL, acid precipitation pH=1, 6 h of acid precipitation time, and a transfer rate of 83%. Subsequently, separation was performed using preparative liquid phase according to the method and conditions of Example 3, and the purity of each component was above 97%.

[0102] Example 5

[0103] The extraction conditions were as follows: 2 extractions, an extraction temperature of 65° C., an extraction time of 2 h, a solid-liquid ratio of 1 g:20 mL, an acid precipitation pH of 2, an acid precipitation time of 6 h, a transfer rate of 67%, and subsequent separation operations using the preparative liquid phase according to the method and conditions of Example 3. The purity of each component was above 97%.

[0104] The above description is merely a preferred embodiment of the present application and does not constitute any form of limitation to the present application. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present application, and such improvements and modifications shall also be considered within the scope of protection of the present application.

Claims

1. A method for extracting flavonoid components from Dracocephalum moldavica, characterized in that, It includes the following steps: Mix Dracocephalum moldavica and an alkaline solution and heat for extraction to obtain an extract solution; Mix the said extract solution with an acid and carry out precipitation under the condition that the pH value is 1 - 3 to obtain a precipitate extract; Dissolve the said precipitate extract and carry out preparative liquid chromatography separation, and respectively collect the fractions at 9 - 10.5 min, 11.5 - 12.5 min, 15 - 17.5 min, 42 - 46 min, 47 - 52 min, 68 - 70 min, and respectively record them as Fr1 fraction, Fr2 fraction, Fr3 fraction, Fr4 fraction, Fr5 fraction, Fr6 fraction; The conditions for the said preparative liquid chromatography separation include: the chromatographic column is a C18 chromatographic column; The mobile phase is mobile phase A and mobile phase B. The mobile phase A is acetonitrile, and the mobile phase B is an aqueous formic acid solution. The volume fraction of formic acid in the aqueous formic acid solution is 0.1%; gradient elution; The elution program for the said gradient elution is: 0 - 55 min, the volume ratio of mobile phase A is 25%; 55 - 80 min, the volume ratio of mobile phase A is 50%; Respectively concentrate the Fr1 fraction, Fr2 fraction, Fr3 fraction, Fr4 fraction, Fr5 fraction and Fr6 fraction to dryness, and successively obtain luteolin - 7 - O - β - D - glucuronide, apigenin - 7 - O - β - D - glucuronide, diosmetin - 7 - O - β - D - glucuronide, silybin, acacetin - 7 - O - glucuronide, acacetin.

2. The extraction method according to claim 1, wherein The said alkaline solution is saturated lime water.

3. The extraction method according to claim 1 or 2, characterized in that, The dosage ratio of Dracocephalum moldavica to the alkaline solution is 1 g : (15 - 45) mL.

4. The extraction method according to claim 3, characterized in that, The dosage ratio of Dracocephalum moldavica to the alkaline solution is 1 g : (25 - 40) mL.

5. The extraction method according to claim 1, characterized in that, The temperature for the said heating extraction is 70 - 85°C, the extraction times are 2 - 3 times, and the time for single - extraction is 1 - 3 h.

6. The extraction method according to claim 5, wherein The temperature for the said heating extraction is 75 - 85°C.

7. The extraction method according to claim 1, wherein The said acid is hydrochloric acid, and the concentration of the hydrochloric acid is 5 mol / L.

8. The extraction method according to claim 1 or 7, characterized in that The temperature for the said precipitation is room temperature, and the time is 3 - 9 h.

9. The extraction method according to claim 8, characterized in that The time for the said precipitation is 6 h.

10. The extraction method according to claim 1, wherein The solvent used for the said 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.

11. The extraction method according to claim 1, wherein The flow rate of the said mobile phase is 5 - 20 mL / min.

12. The extraction method according to claim 1, wherein The specification of the said C18 chromatographic column is 20 mm I.D×250 mm, and the model of the C18 chromatographic column is COSMOSIL packed Column, 5C18 - MS - II.

13. The extraction method according to claim 1 or 12, characterized in that, The column temperature of the said chromatographic column is 35°C.

14. The extraction method according to claim 1, wherein After concentrating to dryness, it further includes respectively carrying out recrystallization on the obtained components. The reagent for the recrystallization 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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