Extraction and purification method of polygonum perfoliatum flavone extract

Abstract

The invention discloses an extraction and purification method of a polygonum perfoliatum flavone extract. The optimal extraction process is as follows: the ethanol concentration is 82.00%, the temperature is 90.29 DEG C, the liquid-material ratio is 32.78: 1mL / g, the extraction time is 1.5 hours, the extraction is performed twice, and the yield is 14.98 + / -0.11 mg / g; the concentration of a sample loading solution is 0.8 g / mL, the sample loading amount is 2.4 BV, 4.3 BV is used for washing and impurity removal, the eluent is 80% ethanol, and the volume is 4.3 BV. The purity of the flavone is 59.02 + / -2.23%, which is 1.37 times higher than that of a macroporous resin purified product (43.00 + / -2.55%), and 4.40 times higher than that of a crude extract (13.42 + / -1.04%).

Description

Technical Field

[0001] The invention relates to the technical field of extraction and purification of Radix Corydalis, in particular to a method for extracting and purifying a Radix Corydalis flavonoid extract. Background Art

[0002] Radix Glehniae is an annual herb of the Polygonaceae family. Radix Glehniae is a food, feed and medicine. It can not only be processed into dishes, but also used as poultry feed. It also has high medicinal value. Due to its extremely tenacious vitality, it was once regarded as a weed and classified as a harmful agricultural plant for control. In the traditional use of Radix Glehniae, the fresh product is mashed and the juice is applied to the affected area, which can be used to treat herpes zoster. Z verified the antiviral activity of Radix Glehniae against HSV-1 infection through in vitro and in vivo experiments. Related studies have shown that Radix Glehniae contains a variety of bioactive ingredients, such as flavonoids, phenylpropanoids, anthraquinones, terpenes and steroid compounds, which have antiviral, anti-inflammatory, liver-protecting, anti-cancer and antibacterial effects.

[0003] In order to maximize the yield of total flavonoids in Radix Corydalis, the water decoction method combined with orthogonal test was used to optimize the extraction process of total flavonoids, and the yield of flavonoids was only 7.21 mg / g; the microwave method was used to optimize the extraction process of total flavonoids in Radix Corydalis, and the average yield of total flavonoids was 5.69%, which enabled the flavonoid components of Radix Corydalis to be developed to a certain extent. However, microwave extraction has problems such as limited industrial application and operational safety, which restricts the large-scale development and utilization of Radix Corydalis. Since flavonoid compounds have strong pharmacological activity, but the yield in Radix Corydalis is low, it is very necessary to purify flavonoid compounds.

[0004] The invention optimizes the extraction conditions of total flavonoids from Radix Glehniae and further uses macroporous resin and macroporous resin combined with polyamide to purify the total flavonoids. The optimal parameters are determined by a small number of experiments, which has the advantages of flexibility and economy and is widely used in the field of traditional Chinese medicine extraction. Summary of the invention

[0005] The present invention aims to provide a method for extracting and purifying a flavonoid extract of Radix Corydalis and its application. The method can effectively increase the yield of total flavonoids in Radix Corydalis and maximize the anti-VZV activity and antioxidant activity of the purified Radix Corydalis flavonoids.

[0006] The technical solution of the present invention is a method for extracting and purifying a flavonoid extract of Radix Corydalis. The method for extracting and purifying a flavonoid extract of Radix Corydalis is carried out according to the following steps:

[0007] (1) Extraction of crude extract of Radix Angelicae Pubescentis flavonoids: grind Radix Angelicae Pubescentis to powder, pass through a No. 3 sieve, weigh 4-6 g of Radix Angelicae Pubescentis powder, add 75-90% ethanol solution and reflux extract at 80-100° C. twice, with the ratio of 82% ethanol solution to Radix Angelicae Pubescentis powder being 25-40 mL:1 g each time, each time for 1.5-2.5 hours, combine the two filtrates, concentrate the filtrate to a net medicinal material content of 1.0-1.2 g / mL, then dilute with distilled water to a content of 0.6-1.0 g / mL, centrifuge the extract at 4000 r / min for 8-12 min, take the filtrate and adjust the pH value to 2.5-3.5 with 1 mol / L hydrochloric acid, thus obtaining a crude extract of Radix Angelicae Pubescentis flavonoids, which is set aside;

[0008] (2) Preparation of mixed column: Take macroporous resin and polyamide respectively, soak them in anhydrous ethanol for 24 hours for activation, then wash them with distilled water until no residual ethanol is left, filter out the distilled water, weigh macroporous resin and polyamide in a mass ratio of 1:7-11, mix them well, and wet-coat them with distilled water. The diameter of the chromatography column is 1.2-1.8 cm, and the diameter-to-height ratio of the chromatography column is 1:6-10. The mixed column is obtained and set aside;

[0009] (3) Purification of Radix Angelicae Pubescentis flavonoids extract: 40-60 mL of the Radix Angelicae Pubescentis flavonoids crude extract was loaded onto a mixing column at a loading flow rate of 1.0-2.0 mL / min. After loading, the column was immediately washed with 80-100 mL of water to remove impurities, and then eluted with 80-100 mL of 70-90% ethanol solution. The 70-90% ethanol eluate was collected, prepared into a lyophilized powder, and stored at below -40°C to obtain the Radix Angelicae Pubescentis flavonoids purified product.

[0010] In the above step (1), the crude extract of Radix Angelicae Pubescentis flavonoids is extracted as follows: grind Radix Angelicae Pubescentis into powder, pass through a No. 3 sieve, weigh 4.5-5.5 g of Radix Angelicae Pubescentis powder, add 80-85% ethanol solution, extract twice in a constant temperature water bath at 85-95°C, the ratio of the amount of 82% ethanol solution added each time to Radix Angelicae Pubescentis powder is 30-35 mL:1 g, each time for 1.5-2 hours, combine the two filtrates, concentrate the filtrate to a net medicinal material content of 1.0-1.2 g / mL, then dilute with distilled water to a content of 0.7-0.9 g / mL, centrifuge the extract at 4000 r / min for 8-11 min, take the subsequent filtrate and adjust the pH value to 2.5-3.5 with 1 mol / L hydrochloric acid, so as to obtain the crude extract of Radix Angelicae Pubescentis flavonoids, which is set aside.

[0011] Specifically, in the aforementioned step (1), the extraction of the Radix Angelicae Pubescentis flavonoids crude extract is as follows: grind Radix Angelicae Pubescentis into powder, pass through a No. 3 sieve, weigh 5.0 g of Radix Angelicae Pubescentis powder, add 82% ethanol solution and extract twice in a constant temperature water bath at 90.29°C, the ratio of the amount of 82% ethanol solution added each time to Radix Angelicae Pubescentis powder is 32.78 mL:1 g, each time for 1.5 hours, combine the two filtrates, concentrate the filtrate to a net medicinal material content of 1.0 g / mL, then dilute with distilled water to a content of 0.8 g / mL, centrifuge the extract at 4000 r / min for 10 min, take the subsequent filtrate and adjust the pH value to 3 with 1 mol / L hydrochloric acid, thus obtaining the Radix Angelicae Pubescentis flavonoids crude extract for later use.

[0012] In the above step (2), the preparation of the mixed column is as follows: take macroporous resin and polyamide respectively, soak them in anhydrous ethanol for activation for 24 hours, then wash them with distilled water until there is no residual ethanol, then weigh the macroporous resin and polyamide according to a mass ratio of 1:8-10, mix them well and wet-coat them with distilled water on a chromatography column, the diameter of the chromatography column is 1.5 cm, and the diameter-to-height ratio of the chromatography column is 1:7-9, so as to obtain a mixed column for use.

[0013] Specifically, in the aforementioned step (2), the preparation of the mixed column is as follows: take the macroporous resin and polyamide respectively, soak them in anhydrous ethanol for activation for 24 hours, then wash them with distilled water until there is no residual ethanol, then weigh the macroporous resin and polyamide in a mass ratio of 1:9, mix them well and wet-coat them with distilled water on a chromatography column, the diameter of the chromatography column is 1.5 cm, and the diameter-to-height ratio of the chromatography column is 1:8, so as to obtain a mixed column for use.

[0014] In the above step (3), the purification of the Radix Angelicae Pubescentis flavonoids extract is as follows: 45-55 mL of the Radix Angelicae Pubescentis flavonoids crude extract is loaded onto a mixing column at a loading flow rate of 1.2-1.8 mL / min, and after loading, 85-95 mL of water is used to wash and remove impurities, and then 85-95 mL of 75-85% ethanol solution is used to elute, and the 75-85% ethanol eluate is collected, prepared into a lyophilized powder below -40°C, and stored to obtain the Radix Angelicae Pubescentis flavonoids purified product.

[0015] Specifically, in the aforementioned step (3), the purification of the Radix Angelicae Pubescentis flavonoids extract is as follows: 50 mL of the Radix Angelicae Pubescentis flavonoids crude extract is loaded onto a mixing column at a loading flow rate of 1.5 mL / min, and after loading, 90 mL of water is used to wash and remove impurities immediately, and then eluted with 90 mL of 80% ethanol solution, and the 80% ethanol eluate is collected, prepared into a lyophilized powder below -40°C, and stored to obtain the Radix Angelicae Pubescentis flavonoids purified product.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. The flavonoid purity of the purified flavonoid extract of Rhizoma Corydalis was 59.02±2.23% by using a mixed column of macroporous resin and polyamide 1:9, which was 1.37 times higher than that of the macroporous resin purified product (43.00±2.55%) and 4.40 times higher than that of the crude extract (13.42±1.04%). A total of 79 flavonoid compounds were identified by qualitative analysis of the purified flavonoids by HPLC-MS / MS. In addition, the purified flavonoids also showed good anti-VZV and antioxidant activities. Therefore, this study can provide technical support and theoretical basis for the further development and utilization of Rhizoma Corydalis resources.

[0018] 2. This study optimized the extraction process of total flavonoids from Radix Angelicae Pubescentis through single factor test, response surface analysis and quadratic regression equation model. In order to improve the purity of flavonoid extracts, a polyamide and macroporous resin mixed chromatography column was used for the first time to enrich and purify flavonoids in Radix Angelicae Pubescentis. HPLC-MS / MS was used to effectively analyze and identify the flavonoid components in the purified product. We further explored the biological activity of the purified flavonoids and found that the anti-VZV activity was significantly stronger than the existing clinical use of Radix Angelicae Pubescentis; in in vitro antioxidant tests, the purified flavonoids showed strong antioxidant capacity. This study provides theoretical and technical support for the further development and utilization of total flavonoids from Radix Angelicae Pubescentis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 :The effect curve of different single factors on flavonoid production;

[0020] Figure 2 : Contour lines and response surface plots between response variables in Box-Behnken (A: 3D response surface plot of the interaction between ethanol concentration and extraction temperature on yield; B: 3D response surface plot of the interaction between ethanol concentration and liquid-to-solid ratio on yield; C: 3D response surface plot of the interaction between extraction temperature and liquid-to-solid ratio on yield; D: Contour plot of the interaction between ethanol concentration and extraction temperature on yield; E: Contour plot of the interaction between ethanol concentration and liquid-to-solid ratio on yield; F: Contour plot of the interaction between extraction temperature and liquid-to-solid ratio on yield;);

[0021] Figure 3 : Curve diagram of single factor adsorption experiment of macroporous resin;

[0022] Figure 4 : Curve diagram of single factor desorption experiment of macroporous resin;

[0023] Figure 5 : Single factor investigation diagram of the combination of macroporous resin and polyamide;

[0024] Figure 6 : Single factor investigation curve diagram of macroporous resin and polyamide combined;

[0025] Figure 7 : Single factor investigation curve diagram of macroporous resin and polyamide combined;

[0026] Figure 8 : Total ion current chromatogram of purified Radix Codonopsis pilosulae flavonoids in positive ion mode;

[0027] Fig. 9 : Total ion current chromatogram of purified Radix Codonopsis pilosulae flavonoids in negative ion mode;

[0028] Fig.10 : Effects of each drug administration group on cell activity;

[0029] Fig.11 : Antioxidant activity of flavonoids from Radix Angelicae Pubescentis. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with the embodiments, but they are not intended to limit the present invention.

[0031] Example 1: Extraction and purification method of Radix Codonopsis pilosulae flavonoids extract

[0032] (1) Extraction of crude extract of Radix Angelicae Pubescentis flavonoids: Grind Radix Angelicae Pubescentis into powder, pass through a No. 3 sieve, weigh 5.0 g of Radix Angelicae Pubescentis powder, add 82% ethanol solution and extract twice in a constant temperature water bath at 90.29°C, each time adding 163.9 mL of 82% ethanol solution, each time for 1.5 h, combine the two filtrates, concentrate the filtrate to a pure medicinal material content of 1.0 g / mL, then add distilled water to dilute to a content of 0.8 g / mL, centrifuge the extract at 4000 r / min for 10 min, take the filtrate and adjust the pH value to 3 with 1 mol / L hydrochloric acid, thus obtaining a crude extract of Radix Angelicae Pubescentis flavonoids, which is set aside;

[0033] (2) Preparation of mixed column: Take macroporous resin and polyamide respectively, soak them in anhydrous ethanol for 24 hours for activation, then wash them with distilled water until no residual ethanol is left, filter out the distilled water, weigh macroporous resin and polyamide in a mass ratio of 1:9, mix them well, and wet-coat them with distilled water on a chromatography column. The diameter of the chromatography column is 1.5 cm, and the diameter-to-height ratio of the chromatography column is 1:8. Thus, a mixed column is obtained and set aside;

[0034] (3) Purification of Radix Angelicae Pubescentis flavonoids extract: 50 mL of the Radix Angelicae Pubescentis flavonoids crude extract was loaded onto a mixing column at a loading flow rate of 1.5 mL / min. Immediately after loading, the column was washed with 90 mL of water to remove impurities, and then eluted with 90 mL of 80% ethanol solution. The 80% ethanol eluate was collected, prepared into a lyophilized powder, and stored at below -40°C to obtain the Radix Angelicae Pubescentis flavonoids purified product.

[0035] Example 2: Extraction and purification method of Radix Codonopsis pilosulae flavonoids extract

[0036] (1) Extraction of crude extract of Radix Angelicae Pubescentis flavonoids: Grind Radix Angelicae Pubescentis into powder, pass through a No. 3 sieve, weigh 5.0 g of Radix Angelicae Pubescentis powder, add 80% ethanol solution and extract twice in a constant temperature water bath at 90°C, each time adding 150 mL of 82% ethanol solution, each time for 1.5 h, combine the two filtrates, concentrate the filtrate to a pure medicinal material content of 1.0 g / mL, then add distilled water to dilute to a content of 0.8 g / mL, centrifuge the extract at 4000 r / min for 10 min, take the filtrate and adjust the pH value to 3 with 1 mol / L hydrochloric acid, thus obtaining a crude extract of Radix Angelicae Pubescentis flavonoids, which is set aside;

[0037] (2) Preparation of mixed column: Take macroporous resin and polyamide respectively, soak them in anhydrous ethanol for 24 hours for activation, then wash them with distilled water until no residual ethanol is left, filter out the distilled water, weigh macroporous resin and polyamide in a mass ratio of 1:9, mix them well, and wet-coat them with distilled water on a chromatography column. The diameter of the chromatography column is 1.5 cm, and the diameter-to-height ratio of the chromatography column is 1:8. Thus, a mixed column is obtained and set aside;

[0038] (3) Purification of Radix Angelicae Pubescentis flavonoids extract: 50 mL of the Radix Angelicae Pubescentis flavonoids crude extract was loaded onto a mixing column at a loading flow rate of 1.5 mL / min. Immediately after loading, the column was washed with 90 mL of water to remove impurities, and then eluted with 90 mL of 80% ethanol solution. The 80% ethanol eluate was collected, prepared into a lyophilized powder, and stored at below -40°C to obtain the Radix Angelicae Pubescentis flavonoids purified product.

[0039] Example 3: Extraction and purification method of Radix Codonopsis pilosulae flavonoids extract:

[0040] (1) Extraction of crude extract of Radix Angelicae Pubescentis flavonoids: Grind Radix Angelicae Pubescentis into powder, pass through a No. 3 sieve, weigh 4 g of Radix Angelicae Pubescentis powder, add 90% ethanol solution and extract twice in a constant temperature water bath at 80°C, each time adding 100 mL of 90% ethanol solution, each time for 1.5 h, combine the two filtrates, concentrate the filtrate to a net medicinal material content of 1.0 g / mL, then add distilled water to dilute to a content of 0.6 g / mL, centrifuge the extract at 4000 r / min for 8 min, take the filtrate and adjust the pH value to 2.5 with 1 mol / L hydrochloric acid, thus obtaining a crude extract of Radix Angelicae Pubescentis flavonoids, which is set aside;

[0041] (2) Preparation of mixed column: Take macroporous resin and polyamide respectively, soak them in anhydrous ethanol for 24 hours for activation, then wash them with distilled water until no residual ethanol is left, filter out the distilled water, weigh macroporous resin and polyamide in a mass ratio of 1:7, mix them well, and wet-coat them with distilled water on a chromatography column. The diameter of the chromatography column is 1.8 cm, and the diameter-to-height ratio of the chromatography column is 1:6. The mixed column is obtained and set aside;

[0042] (3) Purification of Radix Angelicae Pubescentis flavonoids extract: 60 mL of the Radix Angelicae Pubescentis flavonoids crude extract was loaded onto a mixing column at a loading flow rate of 2.0 mL / min. Immediately after loading, the column was washed with 100 mL of water to remove impurities, and then eluted with 100 mL of 90% ethanol solution. The 90% ethanol eluate was collected, prepared into a lyophilized powder, and stored at below -40°C to obtain the Radix Angelicae Pubescentis flavonoids purified product.

[0043] Example 4: Extraction and purification method of Radix Codonopsis pilosulae flavonoids extract:

[0044] (1) Extraction of crude extract of Radix Angelicae Pubescentis flavonoids: Grind Radix Angelicae Pubescentis into powder, pass through a No. 3 sieve, weigh 6 g of Radix Angelicae Pubescentis powder, add 75% ethanol solution and extract twice in a constant temperature water bath at 100°C, each time adding 240 mL of 75% ethanol solution, each time for 2.5 hours, combine the two filtrates, concentrate the filtrate to a net medicinal material content of 1.2 g / mL, then add distilled water to dilute the content to 1.0 g / mL, centrifuge the extract at 4000 r / min for 12 min, take the filtrate and adjust the pH value to 3.5 with 1 mol / L hydrochloric acid, thus obtaining a crude extract of Radix Angelicae Pubescentis flavonoids, which is set aside;

[0045] (2) Preparation of mixed column: Take macroporous resin and polyamide respectively, soak them in anhydrous ethanol for 24 hours for activation, then wash them with distilled water until no residual ethanol is left, filter out the distilled water, weigh macroporous resin and polyamide in a mass ratio of 1:7, mix them well, and wet-coat them with distilled water. The diameter of the chromatography column is 1.2 cm, and the diameter-to-height ratio of the chromatography column is 1:6. The mixed column is obtained and set aside;

[0046] (3) Purification of Radix Angelicae Pubescentis flavonoids extract: 40 mL of the Radix Angelicae Pubescentis flavonoids crude extract was loaded onto a mixing column at a loading flow rate of 1.0 mL / min. Immediately after loading, the column was washed with 80 mL of water to remove impurities, and then eluted with 80 mL of 70% ethanol solution. The 70% ethanol eluate was collected, prepared into a lyophilized powder, and stored at below -40°C to obtain the Radix Angelicae Pubescentis flavonoids purified product.

[0047] Example 5: Extraction and purification method of Radix Codonopsis pilosulae flavonoids extract:

[0048] (1) Extraction of crude extract of Radix Angelicae Pubescentis flavonoids: Grind Radix Angelicae Pubescentis into powder, pass through a No. 3 sieve, weigh 4.5 g of Radix Angelicae Pubescentis powder, add 85% ethanol solution, extract twice in a constant temperature water bath at 85°C, add 230 mL of 85% ethanol solution each time, and extract for 2 hours each time. Combine the two filtrates, concentrate the filtrate to a pure medicinal material content of 1.1 g / mL, and then dilute it with distilled water to a content of 0.7 g / mL. Centrifuge the extract at 4000 r / min for 9 min, take the filtrate and adjust the pH value to 2.8 with 1 mol / L hydrochloric acid to obtain a crude extract of Radix Angelicae Pubescentis flavonoids, which is then used for later use.

[0049] (2) Preparation of mixed column: Take macroporous resin and polyamide respectively, soak them in anhydrous ethanol for 24 hours for activation, then wash them with distilled water until there is no residual ethanol, filter the distilled water, weigh macroporous resin and polyamide in a mass ratio of 1:10, mix them well, and wet-coat them with distilled water. The diameter of the chromatography column is 1.5 cm, and the diameter-to-height ratio of the chromatography column is 1:7. The mixed column is obtained and set aside;

[0050] (3) Purification of Radix Angelicae Pubescentis flavonoids extract: 45 mL of the Radix Angelicae Pubescentis flavonoids crude extract was loaded onto a mixing column at a loading flow rate of 1.8 mL / min. Immediately after loading, the column was washed with 85 mL of water to remove impurities, and then eluted with 95 mL of 85% ethanol solution. The 85% ethanol eluate was collected, prepared into a lyophilized powder, and stored at below -40°C to obtain the Radix Angelicae Pubescentis flavonoids purified product.

[0051] Example 6: Extraction and purification method of Radix Codonopsis pilosulae flavonoids extract:

[0052] (1) Extraction of crude extract of Radix Angelicae Pubescentis flavonoids: Grind Radix Angelicae Pubescentis into powder, pass through a No. 3 sieve, weigh 5.5 g of Radix Angelicae Pubescentis powder, add 80% ethanol solution and extract twice in a constant temperature water bath at 90°C, each time adding 135 mL of 80% ethanol solution, each time for 2 hours, combine the two filtrates, concentrate the filtrate to a pure medicinal material content of 1.2 g / mL, then add distilled water to dilute to a content of 0.9 g / mL, centrifuge the extract at 4000 r / min for 11 min, take the filtrate and adjust the pH value to 3 with 1 mol / L hydrochloric acid, thus obtaining a crude extract of Radix Angelicae Pubescentis flavonoids, which is set aside;

[0053] (2) Preparation of mixed column: Take macroporous resin and polyamide respectively, soak them in anhydrous ethanol for 24 hours for activation, then wash them with distilled water until no residual ethanol is left, filter out the distilled water, weigh macroporous resin and polyamide in a mass ratio of 1:9, mix them well, and wet-coat them with distilled water on a chromatography column. The diameter of the chromatography column is 1.5 cm, and the diameter-to-height ratio of the chromatography column is 1:9. Thus, a mixed column is obtained and set aside;

[0054] (3) Purification of Radix Angelicae Pubescentis flavonoids extract: 55 mL of the Radix Angelicae Pubescentis flavonoids crude extract was loaded onto a mixing column at a loading flow rate of 1.8 mL / min. Immediately after loading, the column was washed with 85 mL of water to remove impurities, and then eluted with 95 mL of 80% ethanol solution. The 80% ethanol eluate was collected, prepared into a lyophilized powder, and stored at below -40°C to obtain the Radix Angelicae Pubescentis flavonoids purified product.

[0055] Example 7: Extraction and purification method of Radix Codonopsis pilosulae flavonoids extract:

[0056] (1) Extraction of crude extract of Radix Angelicae Pubescentis flavonoids: Grind Radix Angelicae Pubescentis into powder, pass through a No. 3 sieve, weigh 5.0 g of Radix Angelicae Pubescentis powder, add 90% ethanol solution and extract twice in a constant temperature water bath at 95°C, each time adding 150 mL of 90% ethanol solution, each time for 1.5 h, combine the two filtrates, concentrate the filtrate to a pure medicinal material content of 1.2 g / mL, then add distilled water to dilute to a content of 0.8 g / mL, centrifuge the extract at 4000 r / min for 10 min, take the filtrate and adjust the pH value to 2.8 with 1 mol / L hydrochloric acid, thus obtaining a crude extract of Radix Angelicae Pubescentis flavonoids, which is set aside;

[0057] (2) Preparation of mixed column: Take macroporous resin and polyamide respectively, soak them in anhydrous ethanol for 24 hours for activation, then wash them with distilled water until there is no residual ethanol, filter the distilled water, weigh macroporous resin and polyamide in a mass ratio of 1:10, mix them well, and wet-coat them with distilled water. The diameter of the chromatography column is 1.5 cm, and the diameter-to-height ratio of the chromatography column is 1:7. The mixed column is obtained and set aside;

[0058] (3) Purification of Radix Angelicae Pubescentis flavonoids extract: 50 mL of the Radix Angelicae Pubescentis flavonoids crude extract was loaded onto a mixing column at a loading flow rate of 1.2 mL / min. Immediately after loading, the column was washed with 90 mL of water to remove impurities, and then eluted with 85 mL of 70% ethanol solution. The 70% ethanol eluate was collected, prepared into a lyophilized powder, and stored at below -40°C to obtain the Radix Angelicae Pubescentis flavonoids purified product.

[0059] Example 8: Extraction and purification method of Radix Codonopsis pilosulae flavonoids extract:

[0060] (1) Crude extraction of Radix Angelicae Pubescentis flavonoids extract: Weigh 5.5 g of Radix Angelicae Pubescentis powder, add 80% ethanol solution and extract twice in a constant temperature water bath at 85°C, each time adding 220 mL of 80% ethanol solution, concentrate the filtrate to a pure medicinal material content of 1.0 g / mL, then add distilled water to dilute to a content of 0.9 g / mL, centrifuge the extract at 4000 r / min for 10 min, take the filtrate and adjust the pH value to 3.0 with 1 mol / L hydrochloric acid, thus obtaining a crude Radix Angelicae Pubescentis flavonoids extract, which is set aside;

[0061] (2) Preparation of mixed column: Take macroporous resin and polyamide respectively, soak them in anhydrous ethanol for 24 hours for activation, then wash them with distilled water until no residual ethanol is left, filter out the distilled water, weigh macroporous resin and polyamide in a mass ratio of 1:7, mix them well, and wet-coat them with distilled water on a chromatography column. The diameter of the chromatography column is 1.5 cm, and the diameter-to-height ratio of the chromatography column is 1:9. Thus, a mixed column is obtained and set aside.

[0062] (3) Purification of Radix Angelicae Pubescentis flavonoids extract: 45 mL of the Radix Angelicae Pubescentis flavonoids crude extract was loaded onto a mixing column at a loading flow rate of 1.8 mL / min. Immediately after loading, the column was washed with 80 mL of water to remove impurities, and then eluted with 80 mL of 80% ethanol solution. The 80% ethanol eluate was collected, prepared into a lyophilized powder, and stored at below -40°C to obtain the Radix Angelicae Pubescentis flavonoids purified product.

[0063] The present invention has done a lot of experimental analysis, which is as follows:

[0064] 1. Validation of extraction process methodology

[0065] 1.1 Materials and Chemicals

[0066] Radix Angelicae Pubescentis (batch number: 230501) was purchased from Guiyang Daosheng Health Industry Co., Ltd. and identified by Associate Professor Yang Ye of the School of Pharmacy of Guizhou University of Traditional Chinese Medicine. Rutin (batch number: D13HB202516, purity ≥ 98%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd., 95% analytical pure (batch number: 20230424) was purchased from Chongqing Chuandong Group Co., Ltd., vitamin C (batch number: PS020110, purity 99.73%) was purchased from Chengdu Pusi Biotechnology Co., Ltd., macroporous adsorption resins (HPD-600, NKA-9, DM130, AB-8, D-101, HPD-100, HP-20) and polyamide (14-30 mesh, 30-60 mesh, 60-100 mesh, 100-200 mesh) were purchased from Beijing Solebow Technology Co., Ltd., and AlCl3 reagent was purchased from Tianjin Zhiyuan Chemical Reagent Co., Ltd.

[0067] 1.2 Determination of total flavonoids

[0068] Accurately weigh 10.15 mg of rutin reference substance, place it in a 50 mL volumetric flask, add a small amount of 70% ethanol to dissolve and dilute to the scale, shake well, and obtain a reference solution with a concentration of 0.203 mg / mL. Accurately draw 0 mL, 0.4 mL, 0.5 mL, 0.7 mL, 0.8 mL, 1 mL, 1.2 mL, and 1.3 mL of rutin reference solution and place them in 10 mL volumetric flasks, add AlCl3 solution for color development, add 70% ethanol to the scale line, shake well and place for 20 minutes. Take the corresponding reagent as blank and measure the absorbance at 405 nm. With rutin mass concentration (x) as the horizontal coordinate and absorbance (y) as the vertical coordinate, the standard curve Y=28.95X-0.0064, r=0.9994 is obtained. The extraction calculation formula of total flavonoids is C*V / m. C is the concentration of the extract (mg / mL), V is the volume of the extract (mL), and m is the mass of the Glehnia littoralis powder (mg).

[0069] 1.3 Single factor experimental design for flavonoid extraction

[0070] The effects of ethanol concentration (50%, 60%, 70%, 80%, 90%), extraction temperature (60, 70, 80, 90, 100°C), extraction time (0.5, 1, 1.5, 2, 2.5, 3h), liquid-to-solid ratio (5, 10, 20, 30, 40) and extraction times (1, 2, 3, 4 times) on flavonoids yield were studied.

[0071] 1.4 Response surface design (RSM) experiment

[0072] On the basis of single factor, Design-Expert V8.0.6.1 software was used to further optimize the effects of ethanol concentration, extraction temperature and solid-liquid ratio on flavonoid yield according to the three-factor and three-level response surface (Table 1).

[0073] Table 1 Response surface test factors and levels

[0074]

[0075] 1.5 Experimental Design Verification

[0076] The other factors were fixed at the optimal levels obtained in the single-factor experiment, and the experiment was carried out three times in parallel based on the optimal process parameters predicted by the RSM experiment.

[0077] 1.6 Single factor experimental results of flavonoids extraction from Radix Glehniae

[0078] 1.6.1 Ethanol concentration

[0079] like Figure 1 As shown in the results, when the ethanol concentration increased from 50% to 80%, the yield of total flavonoids gradually increased, and when the ethanol concentration reached 80%, the yield reached a peak of 11.50 mg / g. However, when the ethanol concentration exceeded 80%, the yield of flavonoids began to decrease with the increase in concentration; this may be because the high concentration of ethanol reduced the affinity of the polar groups in the flavonoid extract, making it impossible to extract it from the plant, and the high concentration of ethanol would dissolve other fat-soluble impurities. The single factor results showed that the optimal extraction solvent was 80% ethanol.

[0080] 1.6.2 Extraction temperature

[0081] like Figure 1 As shown in the figure, when the extraction temperature increases from 60℃ to 90℃, the yield of flavonoids increases significantly. However, when the extraction temperature exceeds 90℃, the yield of flavonoids decreases significantly. The reason for this result may be that the high extraction temperature will cause oxidation and degradation of flavonoid components. The results show that the optimal extraction temperature is 90℃.

[0082] 1.6.3 Liquid-to-solid ratio

[0083] Figure 1It shows that the flavonoid yield in Radix Angelicae Pubescentis first increases and then decreases with the increase of liquid-to-solid ratio. When the liquid-to-solid ratio increases from 10:1 to 30:1, the flavonoid yield gradually increases, reaching a peak at 30:1, with a yield of 13.22 mg / g. When the liquid-to-solid ratio exceeds 30:1, it may be because the excessive extraction solvent will extract more impurities while diluting the flavonoid concentration, so the flavonoid yield begins to decrease. Therefore, the optimal liquid-to-solid ratio is 30:1.

[0084] 1.6.4 Extraction time

[0085] like Figure 1 As shown in the figure, with the increase of extraction time (0.5-1.5h), the yield of flavonoids increased and reached a peak at 1.5h, with a yield of 14.05mg / g. However, when the extraction time exceeded 2.5h, the yield of flavonoids decreased. This may be due to the degradation of flavonoids caused by long-term heating. When the extraction time increased from 1.5h to 2.5h, the yield of flavonoids tended to stabilize. Considering the reduction of energy consumption, the extraction time was determined to be 1.5h.

[0086] 1.6.5 Number of extractions

[0087] Figure 1 The results show the effect of different extraction times on the yield of flavonoids. When the extraction times were increased to two, the yield of flavonoids reached a peak of 14.39 mg / g, so the optimal extraction times were determined to be two.

[0088] 1.7 Response surface optimization results of flavonoid extraction from Radix Glehniae

[0089] 1.7.1 Model fitting and significance test

[0090] The response surface test data (Table 2) were analyzed using Design Expert 8.0.6 software, and the multivariate quadratic regression equation of flavonoids yield (Y) on A, B, and C was obtained as Y = -49.57042 + 0.55575 * A + 0.94370 * B - 0.074033 * C +

[0091] 0.00420600*A*B+0.00292850*A*30+0.00482392*B*30-0.00628942*A 2 -0.00801134*B 2 -0.00917984*C 2 The regression model was subjected to variance analysis (Table 3). The P value of the response surface model was <0.0001 (P < 0.01), indicating that the model was statistically significant. The P value of the lack of fit term was 0.1946 (P > 0.05), indicating that the model had a high degree of fit. The coefficient of determination (R 2Pred) is 0.8433, which is consistent with the adjusted determination coefficient (R 2 Adj) is 0.9675, which is close to the predicted value and the experimental value. The coefficient of variation (CV, 1.21%) is less than 10%, indicating that the reliability and stability of the experimental results are good.

[0092] Table 2 Response surface design and results

[0093]

[0094]

[0095] Table 3 Analysis of variance of response surface test regression model

[0096]

[0097] 1.7.2 Model Validation

[0098] Table 2 and Table 3 and Figure 2 The results showed that the optimal process conditions for extracting total flavonoids from Radix Glehniae were as follows: ethanol concentration of 82.00%, temperature of 90.29℃, and liquid-to-solid ratio of 32.78:1mL / g. Under these conditions, three parallel experiments were carried out and the flavonoid yield was 14.98±0.11mg / g, which was not significantly different from the predicted total flavonoid yield of 14.61mg / g, verifying the accuracy and reliability of the model.

[0099] 2. Validation of purification process methodology

[0100] 2.1 Purification of flavonoid extracts by macroporous resin

[0101] 2.1.1 Selection of macroporous adsorption resin model

[0102] The static adsorption method was used to select the model of macroporous adsorption resin. About 1g (wet weight) of pretreated AB-8, D-101, HPD100, HPD600, HP20, NKA-9, and DM130 macroporous adsorption resins were taken, accurately weighed and placed in a 100mL stoppered conical flask, and 20mL of 0.2g / mL crude extract of Radix Corydalis flavonoids was added respectively. The resin was placed in a constant temperature oscillating water tank and shaken for 24h (25℃, 100r / min) at room temperature, filtered, and the absorbance of the filtrate was measured to calculate the specific adsorption amount and adsorption rate of the total flavonoids. The resin was washed twice with 50mL of distilled water and filtered. 20mL of 70% ethanol was accurately added for desorption, and the resin was placed in a constant temperature oscillating water tank and shaken for 24h (25℃, 100 times / min) to fully desorb and then taken out and filtered. This solution was used as the desorption solution. Accurately pipette an appropriate amount of desorption solution, measure its absorbance, and calculate the specific desorption amount and desorption rate of total flavonoids according to the following formula.

[0103] Specific adsorption capacity (mg / g) = (C0-C1) × V / W

[0104] Adsorption rate (%) = (C0-C1) / C0×100%

[0105] Specific desorption amount (mg / g) = C2V2 / W

[0106] Desorption rate (%) = C2 × V2 / (C0-C1) × V1 × 100%

[0107] Purity (%) = C2 × V2 × dilution factor / dry weight × 100%

[0108] Note: C 0 is the initial solubility (mg / mL) C 1 is the equilibrium solution concentration (mg / mL) C 2 is the mass concentration of the desorption solution (mg / mL)

[0109] V is the volume of the sample solution (mL) 1 V is the volume of adsorption liquid (mL) 2 is the volume of desorption liquid (mL) W is the weight of resin (g)

[0110] Table 4 Physical parameters of different types of macroporous resins

[0111]

[0112] 2.1.2 Effect of pH value of crude extract of flavonoids from Radix Glehniae on static adsorption

[0113] About 1 g (wet mass) of the pretreated D101 macroporous adsorption resin was accurately weighed and placed in a 100 mL stoppered conical flask. 20 mL of crude extract of Radix Angelicae Pubescentis flavonoids with a mass concentration of 0.2 g / mL and pH values ​​of 2, 3, 3.85, 4, 5, 6, 7, and 8 were adjusted with 1 mol / L hydrochloric acid and 1 mol / L sodium hydroxide, respectively. The mixture was shaken at a rate of 100 r / min in a water bath at 25 °C for 24 h, the total flavonoids mass concentration in the adsorption solution was determined, and the specific adsorption amount and adsorption rate were calculated.

[0114] 2.1.3 Dynamic adsorption and desorption of flavonoid extracts purified by macroporous resin—single factor experiment

[0115] About 7.8 g, 11.8 g, 16.5 g, 20.5 g and 23.8 g (wet weight, the diameter-to-height ratio of the chromatography column is 1:4, 1:6, 1:8, 1:10 and 1:12, respectively) of the pretreated macroporous adsorption resin were accurately weighed and slowly loaded into a chromatography column (1.5 cm*30 cm) by a wet method. Extracts of different column volumes and 3 different concentrations (equivalent to 0.3, 0.4, 0.45, 0.5, 0.6, 0.7 and 0.8 g / mL of crude drug amount) were added at different flow rates (0.5, 1, 1.5, 2 and 3 mL / min) and passed through the chromatography column, respectively. The effluents were collected and the absorbance was determined, leakage curves were drawn and the optimal loading concentration and loading flow rate were determined. Before dynamic desorption, in order to remove flavonoids and impurities not adsorbed by the resin, water of different column volumes was used for water washing and impurity removal to determine the optimal water washing amount. The ethanol solution with different column volumes of 50-90% was passed through the chromatography column at different flow rates (1, 1.5, 2, 3 mL / min), and the elution curve was drawn to determine the optimal ethanol concentration and flow rate for elution. The collected desorption solution was freeze-dried and its purity was determined.

[0116] 2.1.4 Screening results of macroporous resin models

[0117] The adsorption and desorption effects of macroporous adsorption resins are affected by the chemical structure, polarity, surface area and pore size of the components. In order to improve the enrichment effect of flavonoids from Radix Glehniae, 7 macroporous resins with different polarities were selected for investigation in this experiment (Table 5). The adsorption capacity of each resin for total flavonoids from Radix Glehniae is ranked as HPD-600>D-101>AB-8>HP-20>HPD-100>NKA-9>DM130, and the desorption capacity is ranked as D-101>HP-20>AB-8>HPD-100>NKA-9>DM130>HPD-600. Through a comprehensive comparison of the static adsorption and desorption capacities of 7 different resins, the non-polar D101 macroporous resin has the best effect, so D101 resin was selected for subsequent research.

[0118] Table 5 Adsorption and desorption rates of flavonoid extracts on different types of macroporous resins (n=3, )

[0119] Macroporous resin model Adsorption rate (%) Desorption rate (%) HPD-600 70.56±1.53 45.81±0.78 NKA-9 35.08±0.97 72.31±0.53 DM-130 27.56±0.10 67.91±0.91 AB-8 63.66±0.87 86.45±0.82 D-101 66.68±0.67 92.87±0.18 HPD-100 52.52±2.37 83.63±1.03 HP-20 56.27±0.03 87.20±0.74

[0120] 2.1.5 Single factor adsorption experimental results

[0121] Effects of different pH values ​​of crude flavonoid extracts from Radix Glehniae on the adsorption properties of total flavonoids Figure 3As shown. With the increase of pH value, the adsorption performance shows a trend of first increasing and then decreasing, and the adsorption performance is best when the pH value is 3. Too low pH value may cause flavonoids to form salts, and too high pH value will reduce the ability of flavonoids to form hydrogen bonds with the resin, thereby causing the adsorption performance to decrease. However, the pH value of the extract of Radix Glehniae itself is 3.85, which is speculated to be due to the fact that the flavonoids contained in the extract have many phenolic hydroxyl groups in their structure. Therefore, the extract with a pH value of 3 was selected for loading to obtain the best adsorption performance.

[0122] As the diameter-to-height ratio increases, the adsorption rate increases, while the specific adsorption amount decreases ( Figure 3 ). When the diameter-to-height ratio is 1:12, although the adsorption rate is the highest, the penetration of the sample solution is poor due to the high resin bed, and the resin at the bottom of the column is still white after the adsorption is complete, which causes a waste of resin. The adsorption rate of the diameter-to-height ratio of 1:10 is equivalent to that of 1:8, but the specific adsorption amount is reduced. After comprehensive comparison, the optimal diameter-to-height ratio is determined to be 1:8.

[0123] During the purification process, if the sample load is too small, the resin utilization rate will be low, resulting in waste and prolonging the experimental cycle; when the sample load is too large, the flavonoids cannot be completely adsorbed due to the limited internal space of the resin microspheres, reducing the purification effect. Figure 3 As shown in the figure, the total flavonoids mass concentration in the third effluent reaches one tenth of the total flavonoids mass concentration in the loading solution, i.e., the leakage point. Therefore, 30 mL (1.4 BV) is taken as the optimal loading volume.

[0124] like Figure 3 As shown in the figure, with the increase of the sample solution concentration, the adsorption rate of flavonoids showed an upward trend, and the specific adsorption amount showed a downward trend. It may be because with the increase of the sample solution concentration, the total amount of flavonoids in contact with the unit surface area of ​​the resin increased, so the adsorption rate increased; however, it may also cause the sample to leak and flow out directly without being adsorbed, resulting in a decrease in the specific adsorption amount. In order to balance the specific adsorption amount and the adsorption rate, 0.6g / mL was selected as the optimal sample solution concentration. When the concentration of the crude flavonoid extract of Radix Corydalis was 0.6g / mL, the adsorption rate was 91.03% and the specific adsorption amount was 32.26mg / g.

[0125] Figure 3 The effect of sample flow rate on the adsorption capacity of D101 resin is shown. As the sample flow rate increases, the adsorption capacity of the resin decreases. This may be because the sample volume flow rate is too fast, which will lead to insufficient contact between the flavonoid components in the extract and the macroporous resin, causing the specific adsorption amount and adsorption rate to show a downward trend. The flow rate change from 0.5mL / min to 1.0mL / min has little effect on the specific adsorption amount and adsorption rate of flavonoids. In order to shorten the experimental period, the sample volume flow rate is selected to be 1.5mL / min.

[0126] 2.1.6 Single factor elution test results

[0127] Depend on Figure 4 It can be seen that when the water washing amount is 20mL (0.95BV), the concentration of flavonoids in the water washing liquid is the lowest. After that, as the water washing amount increases, the concentration of flavonoids in the water washing liquid increases first and then decreases. It is speculated that as the water washing amount increases, the flavonoid glycosides adsorbed on the resin column will be eluted. Therefore, the optimal water washing amount is determined to be 0.95BV.

[0128] Figure 4 It shows that when the amount of eluent reaches 70mL, no flavonoid components are detected in the eluent even if the amount is increased further, indicating that the flavonoids adsorbed by the resin have been completely eluted; therefore, the optimal amount of eluent is determined to be 3.3BV.

[0129] The effect of different concentrations of ethanol on the desorption capacity was studied. Figure 4 ). With the increase of ethanol concentration, the desorption capacity of flavonoids first increased and then decreased, and the desorption capacity of 60% ethanol reached a peak. It is speculated that the ethanol concentration is too low, which cannot effectively compete for the adsorption sites on the resin, resulting in insufficient desorption capacity; while the ethanol concentration is too high, resulting in less solubility of flavonoid components and incomplete elution of flavonoids. Subsequently, 60% ethanol was used as the eluent.

[0130] Figure 4 The results show that the effect of different elution flow rates on the desorption capacity of flavonoids was investigated. With the increase of flow rate, the desorption capacity of flavonoids first increased and then decreased. When the flow rate was 1.0 mL / min, the desorption effect was the best, so 1.0 mL / min was used as the optimal elution flow rate in the future.

[0131] 2.1.7 Optimal Conditions Verification

[0132] The optimal conditions were obtained through single-factor experiments of adsorption and desorption. A D101 macroporous resin-filled chromatography column was selected, and the diameter-to-height ratio was set to 1:8. 1.5BV of crude extract solution with a pH value of 3 and a concentration of 0.6g / mL was loaded at a flow rate of 1.5mL / min. After adsorption for 1h, 0.95BV of distilled water was used to elute the flavonoids that were not adsorbed by the resin, followed by elution with 3.3BV of 60% ethanol at a rate of 1.0mL / min. The eluate was collected and freeze-dried, and the purity of the flavonoids was calculated. The experiment was repeated three times according to the above process, and the purity of the flavonoids in Radix Glehniae was 43.00±2.55%, which was 3.20 times higher than that of the crude extract (13.42±1.04%).

[0133] 2.2 Purification of flavonoid extracts using macroporous resin and polyamide

[0134] In order to further improve the purity of flavonoids, polyamide and macroporous resin were used to further purify the flavonoids of Radix Codonopsis pilosulae.

[0135] 2.2.1 Screening method of polyamide mesh

[0136] The model of macroporous adsorption resin was optimized by static adsorption method. About 1g (wet mass) of polyamide with different mesh sizes after pretreatment was taken, accurately weighed and placed in a 100mL stoppered conical flask, and 20mL of 0.2g / mL of Radix Glehniae extract was added respectively. It was placed in a constant temperature oscillating water tank and shaken for 24h (25℃, 100r / min) at room temperature, filtered, and the filtrate was taken to measure the absorbance, and the specific adsorption amount and adsorption rate of total flavonoids were calculated. The resin was washed twice with 50mL of distilled water and filtered. 20mL of 70% ethanol was accurately added for desorption, and it was placed in a constant temperature oscillating water tank and shaken for 24h (25℃, 100 times / min) to fully desorb, then taken out and filtered. This solution was used as the desorption solution. An appropriate amount of desorption solution was accurately taken, and its absorbance was measured to calculate the specific desorption amount and desorption rate of total flavonoids.

[0137] Screening results: Polyamides with different mesh sizes will affect the purification effect. Figure 5 It can be seen that the smaller the polyamide particle size, the worse the adsorption and desorption capacity of flavonoids from Radix Glehniae. Among them, the 14-30 mesh polyamide has the best adsorption and elution capacity for flavonoids. It is speculated that it may be because the pore size is too small, which easily leads to pore blockage, affecting the diffusion and adsorption of flavonoid molecules. Therefore, the 14-30 mesh polyamide was used for the subsequent purification of total flavonoids from Radix Glehniae.

[0138] 2.2.2 Comparison of the sequence of macroporous resin and polyamide

[0139] Precisely weigh 7.0 g of pretreated macroporous resin (MAR) and polyamide (PA) (dosage ratio 1:1, diameter-to-height ratio 1:8) and load the column in different orders. Load the crude extract of Radix Glehniae flavonoids with a pH of 3 and a crude drug content of 0.6 g / mL at a flow rate of 1 mL / min. Wash with 1 BV (21 mL) of water to remove impurities, then elute with 3.8 BV (80 mL) of 70% ethanol at a flow rate of 1 mL / min, and collect the eluate. Determine its A 405 , calculate the adsorption rate, desorption rate and purity of flavonoids, and determine the best combination order.

[0140] In order to obtain better enrichment and purification effects and improve purification efficiency, a chromatography column with two mixed fillers was used for purification.

[0141] First, the order of combining the two fillers was investigated, namely: (1) macroporous resin in the upper layer and polyamide in the lower layer (separated by filter paper in the middle); (2) macroporous resin in the lower layer and polyamide in the upper layer (separated by filter paper in the middle); (3) polyamide and macroporous resin were mixed and then loaded into the column.

[0142] Depend on Figure 5 It can be seen that the adsorption rates of the first, second and third schemes are 89.74%, 88.98% and 89.36% respectively; the desorption rates are 88.48%, 85.27% and 89.43% respectively; and the purities are 51.35%, 47.43% and 50.56% respectively. The first and third combined schemes have comparable purification effects on flavonoid extracts, both better than the second. Since the third scheme is simple to operate and considering the needs of subsequent industrial production, polyamide and macroporous resin are mixed and then loaded into the column for subsequent purification of flavonoids. It is speculated that this may be because the adsorption mechanisms of polyamide and macroporous resin are different. Polyamide mainly adsorbs flavonoids through hydrogen bonding, while macroporous resin adsorbs flavonoids through physical adsorption and molecular sieving. When the two are used together, they can complement each other and give full play to their own advantages. This combination method can more effectively adsorb and purify flavonoids.

[0143] 2.2.3 Purification of flavonoids by macroporous resin combined with polyamide—single factor investigation

[0144] 2.2.3.1 Single factor investigation method

[0145] The pretreated macroporous resin and polyamide were wet-filled into chromatography columns according to different dosage ratios (0:10, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, 10:0), with a fixed diameter-to-height ratio of 1:8. The crude extract of Radix Glehniae flavonoids with different column volumes, pH 3 and different concentrations (containing 0.6 g / mL of crude drug) were loaded at a flow rate of 1 mL / min to investigate the leakage curve, the optimal loading amount and loading concentration. After adsorption for 1 hour, the impurities were removed with distilled water of different column volumes to determine the optimal water washing amount. Different column volumes of 50%-90% ethanol solution were used for elution at a flow rate of 1 mL / min, the eluate was collected to determine the absorbance, the adsorption rate, desorption rate and purity of the total flavonoids were calculated, and the optimal eluent concentration and dosage were screened.

[0146] 2.2.3.2 Single factor experimental results

[0147] like Figure 6 As shown in the figure, the dosage ratio of macroporous resin to polyamide was investigated. Considering the adsorption rate, desorption rate and purity, the best purification effect of flavonoids was achieved when the dosage ratio of the two was 1:9. This may be because at the dosage ratio of 1:9, physical adsorption, molecular sieving effect and hydrogen bond adsorption were better balanced, achieving highly selective enrichment of flavonoids. Therefore, 1:9 was selected as the best dosage ratio of the two.

[0148] Then the loading amount of the polyamide and macroporous resin mixed column was investigated ( Figure 6), due to the limited adsorption capacity, as the volume of the sample solution increases, the content of flavonoid components in the column liquid increases. The total flavonoid mass concentration in the fifth effluent reaches one tenth of the total flavonoid mass concentration of the sample solution, which is the leakage point. Therefore, 50mL (2.4BV) is taken as the optimal sample volume.

[0149] Depend on Figure 7 It can be seen that with the increase of the loading solution concentration, the adsorption capacity of the mixed column for flavonoids first increases and then decreases. It is speculated that this may be because when the loading concentration is low, the mixed column contains sufficient adsorption sites, resulting in an increase in the adsorption capacity of flavonoid components; as the loading concentration increases, more impurities will compete for limited binding sites, resulting in a decrease in adsorption capacity. When the loading solution concentration is 0.8g / mL, the adsorption performance reaches a peak, so 0.8g / mL is taken as the optimal loading solution concentration.

[0150] In order to remove the unadsorbed flavonoids and impurities in the mixed column, the amount of water used for washing was investigated, such as Figure 7 As shown in the figure, with the increase of distilled water dosage, the concentration of flavonoids in the washing solution gradually decreased. When the washing dosage was 90 mL, the concentration of flavonoids in the washing solution was low and tended to be stable. Therefore, 90 mL (4.3 BV) was taken as the optimal washing dosage.

[0151] Figure 7 It shows that with the increase of eluent concentration, desorption first increases and then decreases. When the concentration of ethanol is 80%, the desorption rate reaches the peak, so 80% ethanol is determined as the best elution solvent for this mixed column. Figure 7 It can be seen that when the elution volume is 90mL, no flavonoids are detected in the eluent, indicating that the flavonoids are completely eluted from the mixed chromatography column. Therefore, the optimal elution volume is 4.3BV.

[0152] 2.2.4 Verification of optimal conditions

[0153] The pretreated macroporous resin and polyamide were used in a ratio of 1:9, mixed and slowly loaded into the chromatography column by wet method, so that the diameter-to-height ratio was 1:8. 2.4BV of 0.8g / mL crude extract of flavonoids from Radix Glehniae was loaded on the column. After adsorption for 1h, 4.3BV of distilled water was used to wash and remove impurities at a flow rate of 1.0mL / min, and then 4.3BV of 80% ethanol solution was used for elution. The eluate was collected and freeze-dried. The parallel test was performed three times to calculate the purity of flavonoids. After purification by the mixed column of polyamide and macroporous resin, the purity of flavonoids was 59.02±2.23%, which was 1.37 times higher than the purity of the macroporous resin purified product (43.00±2.55%) and 4.40 times higher than the crude extract (13.42±1.04%). This study was the first to use a mixed filler of macroporous resin and polyamide to achieve the enrichment and purification of flavonoids in Radix Glehniae. Compared with using a single filler, the mixed column can efficiently separate different components and obtain target compounds with higher purity. Jin et al. separated and purified the extract through polyamide and macroporous resin in turn. This method is cumbersome and prolongs the experimental cycle. We filled the two fillers in the same chromatography column, which is easy to operate and improves the purification efficiency, making it suitable for subsequent industrial production.

[0154] 3 Analysis of purified flavonoids by HPLC-MS / MS

[0155] 3.1 Test samples

[0156] Take an appropriate amount of the mixed sample powder (Example 1), add 1 mL of 70% methanol solution and 3 mm steel beads, place in a 2 mL centrifuge tube, grind with a fully automatic sample rapid grinder (70 Hz) for 3 min, take out, vortex for 10 min to mix; centrifuge the mixed sample solution at 12000 rpm for 10 min at a low temperature of 4 ° C. The supernatant is filtered through a 0.22 μm microporous membrane, and 100 μg / mL internal standard solution (2-chlorophenylalanine) is added to a solution with a concentration of 1 mg / L.

[0157] 3.2 Chromatographic conditions

[0158] A Zorbax Eclipse C18 column (1.8 μm*2.1 mm*100 mm) was used for full spectrum analysis. The separation conditions were: column temperature of 30°C, flow rate of 0.3 mL / min. The mobile phase composition was (A) 0.1% formic acid aqueous solution, (B) pure acetonitrile; the injection volume was 2 μL. The sample gradient elution program is shown in Table 6.

[0159] Table 6 HPLC-MS / MS gradient elution program

[0160] Time (min) Flow rate (mL / min) B% acetonitrile 0-2 0.3 5 2-6 0.3 30 6-7 0.3 30 7-12 0.3 78 12-14 0.3 78 14-17 0.3 95 17-20 0.3 95 20-21 0.3 5 21-25 0.3 5

[0161] 3.3 Mass spectrometry conditions

[0162] A Q-Exactive quadrupole Orbitrap mass spectrometer was used for mass spectrometry analysis. The mass spectrometry parameters were set as follows: positive and negative ion mode; spray voltage: 3.5 kV / -3 kV; sheath gas flow rate: 45 ARB; auxiliary gas: 15 arb; capillary temperature: 330 ° C; auxiliary temperature: 325 ° C; the sample was scanned in the following scanning modes: full scan (Full Scan, m / z 100-1500) and data-dependent secondary mass spectrometry scan (dd-MS2, TopN=5). The resolution was 120,000 (primary mass spectrometry) & 60,000 (secondary mass spectrometry). The normalized collision energy (NCE) was set to 12.5%, 25%, and 35% to obtain its MS / MS spectrum.

[0163] 3.4 Test results

[0164] The purified flavonoids were identified by HPLC-MS / MS, and the components of the purified flavonoids from Radix Glehniae were identified using databases such as Thermo mzCloud and Thermo mzValut based on the secondary mass spectrometry information. The total ion chromatograms of the purified flavonoids from Radix Glehniae in positive and negative ion modes are shown in the figure. Figure 8 and Fig. 9 As shown. A total of 79 flavonoid compounds were identified from Radix Corydalis, most of which existed in free form or glycoside form. Among them, there were 9 isoflavones, 7 dihydroflavonoids, 36 flavonols, 4 flavanols and 23 flavonoids. Liu et al. only identified 26 flavonoid compounds in Radix Corydalis.

[0165] 4 Analysis of anti-Varicella-zoster virus (VZV) activity

[0166] 4.1 Cell culture and virus recovery

[0167] Vero cells were purchased from Wuhan Saios Biotechnology Co., Ltd. When the Vero cells were cultured to a cell density of 75-80%, the complete medium in the cell culture plate was aspirated and replaced with a complete medium containing 2% fetal bovine serum. In a safety cabinet, 1 mL of complete medium was used to dissolve the VZV virus dry powder, and the dry powder solution was transferred to the previously prepared cells and shaken well, and then placed in a 37°C, 5% CO2 incubator for culture, and the cell lesions were observed. The virus was subcultured when the cell lesions were about 90%, and the virus digested with trypsin was added to the prepared Vero cells at a ratio of 1:3, and the virus was continued to be cultured and produced. In the safety cabinet, take the cells containing the virus with a lesion degree of more than 90%, aspirate the medium, wash once with PBS solution, and then use a cell scraper to hang the virus-infected cells, and then resuspend them with 2 mL of virus protection solution, mix well, and then transfer the resuspended cells to the virus cryopreservation tube and store them at -80°C. When in use, take out the frozen virus, thaw it at room temperature, centrifuge it at 300 rpm for 5 minutes, and take the supernatant as the cell-free virus suspension of VZV.

[0168] 4.2 Determination of the virus half tissue infectious dose (TCID50)

[0169] The density of Vero cells was 2×10 5 / mL of cell suspension was inoculated into a 96-well plate, and a 10-fold diluted virus was added to each well. Eight replicate wells were set up, and a cell control group was also set up. The 96-well plate was placed in a 37°C, 5% CO 2 Culture in a constant temperature incubator. Observe the cell pathological changes every day (whether there is non-specific cell death such as natural cell shedding and VZV-specific CPE: cell swelling, shrinkage, aggregation, and finally the appearance of multinucleated giant cells and shedding, etc.), observe for 6-7 days, change the medium every 3-4 days, and calculate the statistics. Calculate TCID50 according to the Reed-Muench method.

[0170] 4.3 Cytotoxicity assay

[0171] Inoculate the cell suspension in the culture plate and culture for 24 hours, with a cell density of about 8000 cells per well. Aspirate the culture medium from each well, add 100 μL of culture medium containing different concentrations of the sample to be tested to the culture plate, set up a blank control group (only culture medium) and a cell control group (cells and culture medium), and set up 3 replicate wells. Place the 96-well plate in an incubator and culture for 24 hours. Add 10 μL of CCK-8 reagent to each well, gently shake the 96-well plate to mix, and continue to culture in the incubator for 4 hours. Use an enzyme marker to detect the absorbance (OD value) at a wavelength of 450 nm, record the data, and calculate the cell survival rate.

[0172] 4.4 Anti-VZV experiment

[0173] CCK8 method: The cell suspension is inoculated in a culture plate, and the cell density is adjusted by a cell counting plate or a hemocytometer. The concentration of the experimental group is about 8000 cells per well according to the progress below the maximum non-toxic concentration, and cultured for 24 hours. The positive drug group, the virus control group, and the normal cell control group are set up in a 96-well plate for dilution. After 24 hours of culture, the culture medium is discarded and 100 μL of drug-free culture medium is added again. 10 μL of CCK-8 reagent is added to each well, and the culture plate is gently shaken to mix to avoid bubbles. The culture plate is placed in an incubator and cultured for 4 hours. The absorbance (OD value) at a wavelength of 450nm is detected using an enzyme marker and the data is recorded.

[0174] 4.5 Determination of TCDI50

[0175] As shown in Table 7, the CPE method was used to observe the morphology of cells infected with VZV, and the Reed-Muench method was used to determine the virus titer TCID50. The sampling data was repeated three times to ensure accuracy. The TCID50 of the varicella zoster virus was 10 -5.46 .

[0176] Table 7 TCDI50 of VZV determined by CPE method (n=3, )

[0177]

[0178]

[0179] 4.6 Determination of maximum non-toxic concentration

[0180] It is generally believed that if the absorbance of the compound on cells / the absorbance of the positive control is ≥ 70%, it is considered to have no obvious toxicity to the cells. The maximum non-toxic concentrations of acyclovir, purified flavonoids (Example 1) and fresh juice are 500 μg / mL, 250 μg / mL, and 250 μg / mL, respectively. Fig.10 shown.

[0181] 4.7 Anti-VZV Activity

[0182] When the mass concentration was 31.3 μg / mL, the cell survival rate of the acyclovir group was 66.32%, the cell survival rate of the flavonoids purified group was 43.28%, and the cell survival rate of the fresh juice group was 39.58%. The IC50 of acyclovir was 8.067 μg / mL, the IC50 of the flavonoids purified group was 40.10 μg / mL, and the IC50 of the fresh juice of Radix Angelicae Pubescentis was 56.30 μg / mL. Therefore, acyclovir has the strongest antiviral ability, followed by the Radix Angelicae Pubescentis purified group, and finally the Radix Angelicae Pubescentis fresh juice group. In the traditional application of Radix Angelicae Pubescentis, people crushed the fresh Radix Angelicae Pubescentis and applied it to the affected area, which can effectively cure herpes zoster. Therefore, this study compared the anti-VZV activity of the Radix Angelicae Pubescentis crude extract after purification with the traditional use. The results showed that the antiviral activity of the flavonoids purified was stronger than that of the fresh juice.

[0183] Study on the Antioxidant Activity of Flavonoids from 5-Centella asiatica

[0184] 5.1 Antioxidant activity analysis

[0185] 5.1.1 Determination of total reduction amount

[0186] Vitamin C, purified flavonoids (Example 1), crude extract and fresh juice of Radix Angelicae Pubescentis were freeze-dried and dissolved in anhydrous ethanol to prepare solutions of 0.10, 0.12, 0.14, 0.16, 0.18, 0.20, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 mg / mL, respectively. A total of 2 mL of purified extracts and crude extracts of different concentrations were measured in a test tube. 2.5 mL of 1% potassium ferricyanide solution and 2.5 mL of 0.2 mol / L phosphate buffer (pH=6.6) were added and mixed thoroughly. The mixture was placed in a 50°C water bath for 20 min, cooled, and then 2.5 mL of 10% trichloroacetic acid solution (TCA) was added and mixed thoroughly. Then centrifuged at a centrifugal speed of 4000 r / min for 10 min, 5 mL of supernatant in the test tube was taken, 4 mL of pure water was added, and 1 mL of 0.1% ferric chloride solution was added. The mixture was mixed thoroughly, reacted at room temperature for 15 min, and the absorbance was measured at 700 nm with pure water as blank. Each group was measured three times in parallel and the average value was taken.

[0187] 5.1.2 Results of determination of total reducing capacity of total flavonoids

[0188] For the determination of total reducing ability, the larger the absorbance value, the stronger the reducing ability of the sample. Fig.11As shown, when the mass concentration is 0.16 mg / mL, the absorbance of vitamin C is 2.84, the absorbance of the purified flavonoids (Example 1) is 0.79, the absorbance of the crude extract is 0.41, and the absorbance of the fresh juice is 0.17; the order of reducing ability is vitamin C>purified flavonoids>crude extract>fresh juice of Radix Angelicae Pubescentis, which is speculated to be due to the increase in the content of flavonoids after purification, so the antioxidant capacity is enhanced. The total flavonoids of Radix Angelicae Pubescentis have antioxidant activity, and the reducing ability reaches saturation at a concentration of 0.6 mg / mL.

[0189] 5.2DPPH free radical scavenging rate

[0190] (1) Vitamin C, purified flavonoids (Example 1), crude extract and fresh juice of Radix Angelicae Pubescentis were freeze-dried and dissolved in anhydrous ethanol to prepare solutions of 0.008, 0.01, 0.02, 0.04, 0.06, 0.08, 0.10, 0.12, 0.14, 0.16, 0.18, 0.20, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 mg / mL, respectively.

[0191] (2) Preparation of DPPH anhydrous ethanol solution: Take 3.95 mg DPPH, dissolve it in anhydrous ethanol and make up to 50 mL.

[0192] (3) Determination of DPPH free radical scavenging rate: 100 μL of the test solution of different concentrations was mixed with an equal volume of DPPH ethanol solution, 100 μL of the test solution of different concentrations was mixed with an equal volume of anhydrous ethanol, and 100 μL of anhydrous ethanol was mixed with an equal volume of DPPH ethanol solution (5 portions each), and the mixture was placed in a 96-well plate mixer for 1 min, and allowed to stand at room temperature and away from light for 30 min. The absorbance at 517 nm was measured with an ELISA reader, and the values ​​were A1, A2, and A0, respectively. Vitamin C was used as a positive control, and the DPPH free radical scavenging rate was calculated according to the formula.

[0193] DPPH free radical scavenging rate = 1-[(A1-A2) / A0]*100%

[0194] like Fig.11As shown in the figure, the antioxidant activity of each sample was compared by measuring the DPPH free radical scavenging rate. When the mass concentration was 0.12 mg / mL, the scavenging rate of vitamin C reached 97.22%, the scavenging rate of purified flavonoids reached 95.36%, the scavenging rate of crude extracts was 86.26%, and the scavenging rate of fresh juice was 15.00%. Among them, the IC50 of vitamin C was 8.90 μg / mL, the IC50 of purified flavonoids was 15.58 μg / mL, the IC50 of crude extracts was 40.51 μg / mL, and the IC50 of fresh juice was 809.90 μg / mL. The ranking of DPPH free radical scavenging ability of each sample is as follows: vitamin C>purified flavonoids>crude extracts>fresh juice. After purification, the crude extract significantly improved the scavenging ability of DPPH free radicals. The results show that the total flavonoids of Radix Corydalis have a good scavenging effect on DPPH free radicals.

[0195] 5.3 Determination of ABTS free radical scavenging ability of total flavonoids

[0196] (1) Vitamin C, purified flavonoids (Example 1), crude extract and fresh juice of Radix Angelicae Pubescentis were freeze-dried and dissolved in anhydrous ethanol to prepare solutions of 0.008, 0.01, 0.02, 0.04, 0.06, 0.08, 0.10, 0.12, 0.14, 0.16, 0.18, 0.20, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and 0.9 mg / mL, respectively.

[0197] (2) Preparation of ABTS solution and potassium persulfate solution: Weigh 0.3841 g of ABTS reagent and 3.7845 g of potassium persulfate, and prepare 7 mmol / L ABTS solution and 140 mmol / L potassium persulfate solution with distilled water.

[0198] (3) Preparation of ABTS free radical stock solution: Mix 5 mL of ABTS reagent with 88 μL of potassium persulfate solution and let stand at room temperature in the dark for more than 15 h.

[0199] (2) ABTS free radical working solution: dilute it with distilled water to the corresponding concentration so that its A value is 0.70±0.02 at a wavelength of 734nm.

[0200] (3) Determination of ABTS free radical scavenging rate: 100 μL of the test solution of different concentrations was mixed with an equal volume of ABTS free radical working solution, 100 μL of the test solution of different concentrations was mixed with an equal volume of anhydrous ethanol, and 100 μL of anhydrous ethanol was mixed with an equal volume of ABTS working solution (5 portions each), and the mixture was placed in a 96-well plate mixer for 1 min, and allowed to stand at room temperature and away from light for 30 min. The absorbance at 734 nm was measured with an ELISA reader, and the values ​​were A1, A2, and A0, respectively. Vitamin C was used as a positive control, and the calculation formula was as follows.

[0201] DPPH free radical scavenging rate = 1-[(A1-A2) / A0]*100%

[0202] Comparison of the ABTS free radical scavenging abilities of the samples Fig.11 As shown. In the concentration range of 0.02-0.9 mg / mL, the scavenging ability of each sample on the ABTS free radical extract gradually increased with the increase of concentration until it reached equilibrium. At a concentration of 0.10 mg / mL, the ABTS free radical scavenging rate of vitamin C was 99.53%, the scavenging rate of purified flavonoids was 93.06%, the scavenging rate of crude extracts was 54.54%, and the scavenging rate of crude extracts was 9.96%. The IC50 of vitamin C, purified flavonoids, crude extracts and fresh juice were 10.58 μg / mL, 13.40 μg / mL, 27.53 μg / mL, and 626.00 μg / mL, respectively. The antioxidant capacity of the samples ranked as follows: vitamin C> purified flavonoids> crude extracts> fresh juice. In summary, the total flavonoids of Radix Angelicae Pubescentis have a good scavenging effect on ABTS free radicals.

[0203] 6 Analytical methods

[0204] SPSS 26.0 was used to analyze all data. The results were expressed as mean ± standard deviation. P < 0.05 indicated that the data were statistically significant.

[0205] 7 Conclusion

[0206] In this study, the extraction and purification process of total flavonoids in Radix Angelicae Pubescentis was optimized by single factor test, response surface analysis and quadratic regression equation model. In order to improve the purity of flavonoids, a polyamide and macroporous resin mixed chromatography column was used for the first time to enrich and purify flavonoids in Radix Angelicae Pubescentis. HPLC-MS / MS was used to effectively analyze and identify the flavonoid components in the purified product. We further explored the biological activity of the purified flavonoids and found that the anti-VZV activity was significantly stronger than the existing clinical use of Radix Angelicae Pubescentis; in the in vitro antioxidant test, the purified flavonoids showed strong antioxidant capacity. This study provides theoretical and technical support for the further development and utilization of total flavonoids in Radix Angelicae Pubescentis.

Claims

1. A method for extracting and purifying a flavonoid extract of Radix Codonopsis pilosulae, characterized in that: The extraction and purification method of the Radix Codonopsis pilosulae flavonoids extract is carried out according to the following steps: (1) Extraction of crude extract of Radix Angelicae Pubescentis flavonoids: grind Radix Angelicae Pubescentis to powder, pass through a No. 3 sieve, weigh 4-6 g of Radix Angelicae Pubescentis powder, add 75-90% ethanol solution and reflux extract at 80-100° C. twice, with the ratio of 82% ethanol solution to Radix Angelicae Pubescentis powder being 25-40 mL:1 g each time, each time for 1.5-2.5 hours, combine the two filtrates, concentrate the filtrate to a net medicinal material content of 1.0-1.2 g / mL, then dilute with distilled water to a content of 0.6-1.0 g / mL, centrifuge the extract at 4000 r / min for 8-12 min, take the filtrate and adjust the pH value to 2.5-3.5 with 1 mol / L hydrochloric acid, thus obtaining a crude extract of Radix Angelicae Pubescentis flavonoids, which is set aside; (2) Preparation of mixed column: Take macroporous resin and polyamide respectively, soak them in anhydrous ethanol for 24 hours for activation, then wash them with distilled water until no residual ethanol is left, filter out the distilled water, weigh macroporous resin and polyamide in a mass ratio of 1:7-11, mix them well, and wet-coat them with distilled water. The diameter of the chromatography column is 1.2-1.8 cm, and the diameter-to-height ratio of the chromatography column is 1:6-10. The mixed column is obtained and set aside; (3) Purification of Radix Angelicae Pubescentis flavonoids extract: 40-60 mL of the Radix Angelicae Pubescentis flavonoids crude extract was loaded onto a mixing column at a loading flow rate of 1.0-2.0 mL / min. After loading, the column was immediately washed with 80-100 mL of water to remove impurities, and then eluted with 80-100 mL of 70-90% ethanol solution. The 70-90% ethanol eluate was collected, prepared into a lyophilized powder, and stored at below -40°C to obtain the Radix Angelicae Pubescentis flavonoids purified product.

2. The method for extracting and purifying the flavonoid extract of Radix Codonopsis pilosulae according to claim 1, characterized in that: In step (1), the extraction of the crude extract of Radix Angelicae Pubescentis flavonoids is as follows: grind Radix Angelicae Pubescentis into powder, pass through a No. 3 sieve, weigh 4.5-5.5 g of Radix Angelicae Pubescentis powder, add 80-85% ethanol solution, extract twice in a constant temperature water bath at 85-95°C, the ratio of the amount of 82% ethanol solution added each time to Radix Angelicae Pubescentis powder is 30-35 mL:1 g, each time for 1.5-2 hours, combine the two filtrates, concentrate the filtrate to a net medicinal material content of 1.0-1.2 g / mL, then dilute with distilled water to a content of 0.7-0.9 g / mL, centrifuge the extract at 4000 r / min for 8-11 min, take the subsequent filtrate and adjust the pH value to 2.5-3.5 with 1 mol / L hydrochloric acid, so as to obtain the crude extract of Radix Angelicae Pubescentis flavonoids, which is set aside.

3. The method for extracting and purifying the flavonoid extract of Radix Codonopsis pilosulae according to claim 2, characterized in that: In step (1), the extraction of the crude extract of Radix Angelicae Pubescentis flavonoids is as follows: grind Radix Angelicae Pubescentis into powder, pass through a No. 3 sieve, weigh 5.0 g of Radix Angelicae Pubescentis powder, add 82% ethanol solution and extract twice in a constant temperature water bath at 90.29°C, the ratio of the amount of 82% ethanol solution added each time to Radix Angelicae Pubescentis powder is 32.78 mL:1 g, each time for 1.5 hours, combine the two filtrates, concentrate the filtrate to a net medicinal material content of 1.0 g / mL, then dilute with distilled water to a content of 0.8 g / mL, centrifuge the extract at 4000 r / min for 10 min, take the subsequent filtrate and adjust the pH value to 3 with 1 mol / L hydrochloric acid to obtain the crude extract of Radix Angelicae Pubescentis flavonoids for later use.

4. The method for extracting and purifying the flavonoid extract of Radix Codonopsis pilosulae according to claim 1, characterized in that: In step (2), the preparation of the mixed column is as follows: take macroporous resin and polyamide respectively, soak them in anhydrous ethanol for activation for 24 hours, then wash them with distilled water until there is no residual ethanol, then weigh the macroporous resin and polyamide according to a mass ratio of 1:8-10, mix them well and wet-coat them with distilled water on a chromatography column, the diameter of the chromatography column is 1.5 cm, and the diameter-to-height ratio of the chromatography column is 1:7-9, so as to obtain a mixed column for use.

5. The method for extracting and purifying the flavonoid extract of Radix Codonopsis pilosulae according to claim 4, characterized in that: In step (2), the preparation of the mixed column is as follows: take macroporous resin and polyamide respectively, soak them in anhydrous ethanol for activation for 24 hours, then wash them with distilled water until there is no residual ethanol, then weigh the macroporous resin and polyamide in a mass ratio of 1:9, mix them well and wet-coat them with distilled water on a chromatography column, the diameter of the chromatography column is 1.5 cm, and the diameter-to-height ratio of the chromatography column is 1:8, so as to obtain a mixed column for use.

6. The method for extracting and purifying the flavonoid extract of Radix Codonopsis pilosulae according to claim 1, characterized in that: In step (3), the purification of the Radix Angelicae Pubescentis flavonoids extract is as follows: 45-55 mL of the Radix Angelicae Pubescentis flavonoids crude extract is loaded onto a mixing column at a loading flow rate of 1.2-1.8 mL / min, and after loading, 85-95 mL of water is used to wash and remove impurities, and then 85-95 mL of 75-85% ethanol solution is used to elute, and the 75-85% ethanol eluate is collected, prepared into a lyophilized powder below -40°C, and stored to obtain the Radix Angelicae Pubescentis flavonoids purified product.

7. The method for extracting and purifying the flavonoid extract of Radix Codonopsis pilosulae according to claim 6, characterized in that: In step (3), the purification of the Radix Angelicae Pubescentis flavonoids extract is as follows: 50 mL of the Radix Angelicae Pubescentis flavonoids crude extract is loaded onto a mixing column at a loading flow rate of 1.5 mL / min. After loading, 90 mL of water is used to wash and remove impurities, and then 90 mL of 80% ethanol solution is used to elute. The 80% ethanol eluate is collected and prepared into a lyophilized powder below -40°C and stored to obtain the Radix Angelicae Pubescentis flavonoids purified product.

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