Baicalein and wogonin separation method based on phenylboronic acid functionalized solid-phase carrier

Abstract

The invention discloses a method for separating baicalein and wogonin based on a phenylboronic acid functionalized solid-phase carrier. The method comprises the following steps: (1) preparing a scutellaria baicalensis extracting solution; (2) carrying out pretreatment on the extracting solution, degrading baicalin and wogonoside, and removing macromolecular impurities; (3) diluting the treated extracting solution into an alkaline solution, adding the phenylboronic acid functionalized solid-phase carrier, and carrying out selective adsorption; (4) controlling the adsorption time to be 0.5-5 hours and the adsorption temperature to be 20-50 DEG C; (5) after separation and adsorption, putting the phenylboronic acid functionalized solid-phase carrier into an acid solution for desorption to obtain baicalein; (6) repeating the steps (3) to (5) until the wogonin concentration in the alkaline solution meets the requirement; and (7) respectively purifying and recovering baicalein and wogonin from the acidic solution and the alkaline solution. According to the method, baicalein and wogonin can be efficiently distinguished, a large amount of organic solvents are prevented from being used, the separation process is simple and convenient, the cost is low, environment friendliness is achieved, and the separation purity is high.

Description

Technical Field

[0001] The present invention relates to the field of separation of natural active substances, and particularly to a method for separating baicalein and wogonin based on a phenylboronic acid-functionalized solid support. Background Art

[0002] Baicalein and wogonin are two main flavonoid active ingredients in the traditional Chinese medicine Scutellaria baicalensis, and have a wide range of pharmacological effects. Baicalein has various biological activities such as anti-inflammatory, antioxidant, anti-tumor, and antiviral, while wogonin shows significant pharmacological effects in aspects such as anti-tumor, neuroprotection, and immunomodulation. Since these two components have important application values in the fields of drug research and development and health products, the efficient separation and purification of baicalein and wogonin from Scutellaria baicalensis have become a research hotspot.

[0003] However, the chemical structures of baicalein and wogonin are highly similar, both belonging to flavonoid compounds, and they coexist in Scutellaria baicalensis extracts. This structural similarity makes traditional separation methods face many challenges. Currently, the commonly used separation methods mainly include column chromatography, liquid-liquid extraction, and high-performance liquid chromatography (HPLC), etc. Although these methods can achieve the separation of baicalein and wogonin to a certain extent, there are still the following problems: (1) Large solvent consumption: Traditional methods usually require the use of a large amount of organic solvents (such as methanol, ethanol, acetonitrile, etc.), which not only increases the separation cost but also burdens the environment. (2) Low separation efficiency: Due to the structural similarity of baicalein and wogonin, the separation efficiency of traditional methods is low, and it is difficult to achieve high-purity separation. (3) Complicated operation: Methods such as column chromatography and HPLC require complicated operation steps and expensive equipment, and are difficult to be applied to large-scale industrial production.

[0004] Under pH-controllable conditions, a phenylboronic acid-functionalized solid support can achieve the efficient separation of baicalein and wogonin by regulating its selective adsorption of flavonoid substances with different numbers of hydroxyl groups. However, there is currently no report on the application of phenylboronic acid-functionalized solid supports in the separation of baicalein and wogonin. The existing separation methods still mainly rely on traditional technologies, and it is difficult to meet the requirements of industrial production for high efficiency, low cost, and environmental protection. Therefore, developing an efficient separation method based on phenylboronic acid-functionalized solid supports has important theoretical significance and practical application value. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for separating baicalein and wogonin based on a phenylboronic acid-functionalized solid support, so as to be able to separate baicalein and wogonin efficiently, at low cost, and in an environmentally friendly manner.

[0006] To solve the above technical problems, the technical solutions adopted by the present invention are as follows: A method for separating baicalein and wogonin based on a phenylboronic acid-functionalized solid support, comprising the following steps: (1) Prepare a baical extract; (2) Pretreat the extract to degrade baicalin and wogonin and remove macromolecular impurities; (3) Dilute the pretreated extract into an alkaline solution, add a phenylboronic acid-functionalized solid support, and perform selective adsorption; (4) Control the adsorption time to be 0.5 - 5 hours and the adsorption temperature to be 20 - 50 °C; (5) Separate the phenylboronic acid-functionalized solid support after adsorption, put it into an acidic solution for desorption, and obtain baicalein; (6) Repeat steps (3) to (5) until the concentration of wogonin in the alkaline solution reaches the requirement; (7) Purify and recover baicalein and wogonin from the acidic solution and the alkaline solution respectively.

[0007] Further optimized, the method for preparing the baical extract in step (1) includes extraction using methanol, ethanol, or a mixed solution of them and water.

[0008] Further optimized, the pretreatment method in step (2) includes degrading baicalin and wogonin into baicalein and wogonin using acid, base, or enzymatic method, and using macroporous adsorption resin to remove macromolecular impurities and non-flavonoid components.

[0009] Further optimized, the phenylboronic acid-functionalized solid support in step (3) is a solid support with a phenylboronic acid functional group, including but not limited to phenylboronic acid-functionalized materials based on TentaGel NH 2 resin, PS-DVB resin, Sepharose resin, magnetic beads, nanoparticles, and polymer microspheres.

[0010] Further optimized, the alkaline solution in step (3) is one or more of phosphate buffer, methanol, ethanol, acetonitrile, acetone, isopropanol, or a mixed solution of phosphate buffer and any one or more of methanol, ethanol, acetonitrile, acetone, isopropanol in different ratios.

[0011] Further optimized, the acidic solution in step (5) is one or more of acetate buffer, citrate buffer, hydrochloric acid solution, methanol, ethanol, acetonitrile, acetone, isopropanol, or a mixed solution of acetate buffer, citrate buffer, hydrochloric acid solution and any one or more of methanol, ethanol, acetonitrile, acetone, isopropanol in different ratios.

[0012] Further optimized, in step (6), the adsorption and desorption operations are repeated until the purity of wogonin in the alkaline solution reaches 80-90%.

[0013] Further optimized, the purification and recovery method in step (7) includes extraction, concentration and drying, and the final purity of baicalein is not less than 90%, and the purity of wogonin is not less than 80%.

[0014] Further optimized, the phenylboronic acid-functionalized solid-phase carrier preferentially adsorbs baicalein under the condition of pH 7.0-9.0, and desorbs under the condition of pH 3.0-6.0.

[0015] Further optimized, the phenylboronic acid-functionalized solid-phase carrier can be recycled and still maintains stable adsorption performance after multiple cycles.

[0016] The beneficial effects of adopting the above technical solutions are as follows: The present invention utilizes the specific binding of the phenylboronic acid-functionalized solid-phase carrier to the polyphenolic hydroxyl groups of baicalein, and can efficiently distinguish between baicalein and wogonin. Baicalein contains multiple phenolic hydroxyl groups and forms a strong bond with the phenylboronic acid group, while wogonin contains only one phenolic hydroxyl group and has a weak bond. By regulating the pH conditions, the high-selectivity separation of baicalein and wogonin is realized, and the separation efficiency is significantly improved. Moreover, by optimizing the adsorption and desorption conditions, the present invention can obtain high-purity baicalein and wogonin, meeting the requirements for high-purity active ingredients in the fields of drugs and health products. The present invention can complete the separation by adjusting the pH under aqueous phase conditions, greatly reducing the use of organic solvents, reducing the separation cost, and conforming to the development trend of green chemistry. The method of the present invention has simple operation steps, does not require complex equipment, and the adsorption and desorption processes are easy to control, suitable for large-scale industrial production. The phenylboronic acid-functionalized solid-phase carrier can be recycled, further reducing the production cost. Description of the Drawings

[0017] Figure 1 It is a mechanism diagram of a method for separating baicalein and wogonin based on a phenylboronic acid-functionalized solid-phase carrier; Figure 2 It is a schematic structural diagram of a solid-phase carrier with a phenylboronic acid functional group; Figure 3 It is the HPLC result diagram in Example 1, where A is the mixed diagram of baicalein and wogonin; B is the detection diagram of the mixed solution after the mixture and the phenylboronic acid-functionalized resin are adsorbed; C is the detection diagram of the solution after the phenylboronic acid-functionalized resin is desorbed in an acidic solution with pH = 4; Figure 4 It is the HPLC diagram of wogonin in Example 3; Figure 5 It is the HPLC diagram of baicalein in Example 3; Figure 6It is the HPLC chromatogram of baicalein in Example 4; Figure 7 It is the HPLC chromatogram of wogonin in Example 4. Detailed implementation manners

[0018] The following examples illustrate the present invention in detail. In the description of the following examples, specific details such as specific system structures and technologies are proposed for the purpose of illustration rather than limitation, so as to thoroughly understand the embodiments of the present application. However, those skilled in the art should clearly understand that the present application can also be implemented in other embodiments without these specific details. In other cases, detailed descriptions of well-known systems, devices, circuits, and methods are omitted to avoid unnecessary details from interfering with the description of the present application. It should be understood that when used in the specification of the present application and the appended claims, the term "comprising" indicates the presence of the described features, wholes, steps, operations, elements, and / or components, but does not exclude the presence or addition of one or more other features, wholes, steps, operations, elements, components, and / or their combinations. It should also be understood that the term "and / or" used in the specification of the present application and the appended claims refers to any combination and all possible combinations of one or more of the related listed items, and includes these combinations.

[0019] In addition, in the description of the specification of the present application and the appended claims, the terms "first", "second", "third", etc. are only used for distinguishing descriptions and cannot be understood as indicating or implying relative importance. The reference to "one embodiment" or "some embodiments" etc. described in the specification of the present application means that a specific feature, structure, or characteristic described in connection with this embodiment is included in one or more embodiments of the present application. Thus, the statements "in one embodiment", "in some embodiments", "in other some embodiments", "in still other embodiments", etc. that appear in different places in this specification do not necessarily all refer to the same embodiment, but mean "one or more but not all embodiments", unless otherwise specifically emphasized in other ways. The terms "comprising", "including", "having", and their variants all mean "including but not limited to", unless otherwise specifically emphasized in other ways.

[0020] The present invention provides a method for separating baicalein and wogonin based on a phenylboronic acid-functionalized solid phase carrier, and the separation mechanism is as Figure 1 shown, and the schematic structural diagram of the solid phase carrier with a phenylboronic acid functional group is as Figure 2 shown.

[0021] Example 1: Verification of the specific adsorption ability of the phenylboronic acid-functionalized solid phase carrier to baicalein Prepare 0.2 g each of baicalein and wogonin with known concentrations, and dissolve them separately in a 50% ethanol / PBS mixed solution with pH 7.5. Subsequently, mix them in equal volume ratios to form a 5 ml mixed solution. Mix this mixed solution with 1 g of pre-washed and dried TentaGe1 NH 2 functionalized resin and stir at a speed of 300 revolutions per minute at room temperature for 1.5 hours to ensure sufficient adsorption. Subsequently, separate the adsorbed resin from the solution by filtration, and perform desorption using a 50% acetic acid / ethanol solution with pH 4.0. Collect the desorbed solution, and repeat this process until baicalein in the filtrate is completely separated. Analyze the contents of baicalein and wogonin in the original mixed solution and the desorbed solution using high performance liquid chromatography (HPLC, eluted with 90% methanol / water). Finally, the HPLC results show that the content of baicalein in the desorbed solution is as high as 92%( Figure 3 ), and finally 0.176 g of baicalein is obtained with a recovery rate of 88%.

[0022] Example 2: Verify that the phenylboronic acid-functionalized solid phase carrier can be recycled for the adsorption and desorption of baicalein Prepare 0.2 g of baicalein with a known concentration and dissolve it in a 10 ml mixed solution of 50% ethanol / PBS with pH 7.5. Mix this mixed solution with 100 mg of pre-washed and dried phenylboronic acid-functionalized TentaGe1 NH 2 functionalized resin and stir at a speed of 300 revolutions per minute at room temperature for 1.5 hours to ensure sufficient adsorption. Then stir for desorption in a 50% PBS / ethanol solution with pH 4. Repeat the above adsorption and desorption process, and analyze the change in the content of baicalein in the desorbed solution using high performance liquid chromatography (HPLC). Investigate the effect of the number of recycling times on the adsorption capacity. The results show that after 8 cycles, the content of baicalein in the desorbed solution only decreases slightly, indicating that the resin has good reusability (Table 1). In addition, its adsorption performance does not change significantly at 50 °C, proving its excellent thermal stability. Through this example, it is proved that the adsorption resin has high recovery ability, good reusability and stable adsorption characteristics in the treatment of mixed solutions of baicalein and wogonin.

[0023] Table 1 Results of the cyclic adsorption experiment of phenylboronic acid-functionalized resin Number of cycles Adsorption capacity (baicalein mg / 100mg) 1 12.15 2 11.88 3 11.26 4 10.95 5 10.04 6 9.89 7 9.84 8 9.78 Example 3: Practical separation application of baicalein and wogonin in Scutellaria baicalensis extract After crushing 10 g of dry Scutellaria baicalensis root, it was extracted with 70% ethanol, baicalin and wogonoside were degraded by enzymatic hydrolysis, and the extract was treated with PS-DVB macroporous resin to remove polysaccharides and macromolecular impurities. The pH of the extract was adjusted to 8.5, 1 g of phenylboronic acid-functionalized resin (TentaGel NH2) was added, stirred for 1.5 hours, adsorbed at room temperature, and the desorption condition was a mixed solution of 50% PBS and methanol at pH 4.0, and this process was repeated. The contents of baicalein and wogonin in the original mixed solution and the desorbed solution were analyzed by high performance liquid chromatography (HPLC). Finally, the purity of baicalein was 92.2% and the purity of wogonin was 81.5%. ( Figures 4 - 5 ) Example 4: Practical separation and application of baicalein and wogonin in Scutellaria baicalensis extract 5 g of dry Scutellaria baicalensis root was pretreated as in Example 3, preliminarily purified using Sepharose macroporous adsorption resin (1:10 ratio), then phenylboronic acid-functionalized resin (PS-DVB) was added at pH 8.0, stirred for 2 hours, baicalein was adsorbed, and desorbed using a 65% PBS / ethanol mixed solution at pH 4.0. Finally, the purity of baicalein was 92.8% and the purity of wogonin was 80.7%. ( Figures 6 - 7 ) Example 5: General HPLC conditions for detection in Examples 2 - 4 Quantitative analysis of baicalein and wogonin was carried out by high performance liquid chromatography (HPLC), and separation was performed using a C18 reversed-phase column (250 mm × 4.6 mm, 5 µm) equipped with a UV detector. The mobile phase was methanol (B) and water (containing 0.1% phosphoric acid, A), and gradient elution was used. The specific program was as follows: 10% B from 0 - 5 minutes, increased to 60% B gradient from 5 - 15 minutes, 60% B remained constant from 15 - 25 minutes, increased to 80% B gradient from 25 - 30 minutes, and restored to 10% B and maintained equilibrium from 30 - 35 minutes. The flow rate was 1.0 ml / min, the column temperature was 25°C, the injection volume was 10 µl, and the detection wavelength was 280 nm. Standard samples of baicalein and wogonin were dissolved in methanol at a concentration of 1 mg / ml respectively. The samples were filtered through a 0.22 µm filter membrane and then injected into the HPLC system. Standard curves were plotted based on the standard samples. The retention time of baicalein was approximately 10 - 12 minutes and that of wogonin was 18 - 20 minutes. The content of the sample peak area was calculated through the standard curve, and its purity was quantitatively analyzed.

[0024] The present invention utilizes the specific binding between a phenylboronic acid-functionalized solid support and the polyphenolic hydroxyl groups of baicalein to efficiently distinguish baicalein from wogonin. Baicalein contains multiple phenolic hydroxyl groups and forms a strong binding with phenylboronic acid groups, while wogonin contains only one phenolic hydroxyl group and has a weak binding. By regulating the pH conditions, the high-selectivity separation of baicalein and wogonin is achieved, and the separation efficiency is significantly improved. Moreover, by optimizing the adsorption and desorption conditions, the present invention can obtain high-purity baicalein and wogonin, meeting the requirements for high-purity active ingredients in the fields of pharmaceuticals and health products. The separation of the present invention can be completed by adjusting the pH under aqueous conditions, greatly reducing the use of organic solvents and lowering the separation cost, which is in line with the development trend of green chemistry. The method of the present invention has simple operation steps, does not require complex equipment, and the adsorption and desorption processes are easy to control, making it suitable for large-scale industrial production. The phenylboronic acid-functionalized solid support can be recycled, further reducing the production cost.

[0025] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A method for separating baicalin and wogonin based on a phenylboronic acid functionalized solid phase carrier, characterized in that: The following steps are involved: (1) preparing Scutellaria baicalensis extract; (2) Pre-treating the extract to degrade baicalin and wogonin and remove macromolecular impurities; (3) diluting the treated extract into an alkaline solution and adding a phenylboronic acid functionalized solid support for selective adsorption; (4) Control the adsorption time to 0.5-5 hours and the adsorption temperature to 20-50°C; (5) separating the adsorbed phenylboronic acid functionalized solid phase carrier and placing it in an acidic solution for desorption to obtain baicalin; (6) Repeat steps (3) to (5) until the concentration of wogonin in the alkaline solution reaches the required level; (7) Purify and recover baicalein and wogonin from acidic solution and alkaline solution, respectively.

2. The method according to claim 1, characterized in that The method for preparing the scutellaria baicalensis extract in step (1) comprises extracting with methanol, ethanol or a mixed solution thereof with water.

3. The method according to claim 1, characterized in that The pretreatment method in step (2) includes using acid, alkali or enzyme method to degrade baicalin and wogonin into baicalein and wogonin, and using macroporous adsorption resin to remove macromolecular impurities and non-flavonoid components.

4. The method according to claim 1, characterized in that: The phenylboronic acid functionalized solid phase carrier in step (3) is a solid phase carrier with phenylboronic acid functional groups, including but not limited to TentaGel NH2 resin, PS-DVB resin, Sepharose resin, magnetic beads, nanoparticles and polymer microspheres as matrix phenylboronic acid functionalized materials.

5. The method according to claim 1, characterized in that The alkaline solution in step (3) is one or more of phosphate buffer, methanol, ethanol, acetonitrile, acetone, and isopropanol, or a mixed solution of phosphate buffer and any one or more of methanol, ethanol, acetonitrile, acetone, and isopropanol in different proportions.

6. The method according to claim 1, characterized in that The acidic solution in step (5) is one or more of acetate buffer, citric acid buffer, hydrochloric acid solution, methanol, ethanol, acetonitrile, acetone, and isopropanol, or a mixed solution of acetate buffer, citric acid buffer, hydrochloric acid solution and any one or more of methanol, ethanol, acetonitrile, acetone, and isopropanol in different proportions.

7. The method according to claim 1, characterized in that In step (6), the adsorption and desorption operations are repeated until the purity of wogonin in the alkaline solution reaches 80-90%.

8. The method according to claim 1, characterized in that The purification and recovery method in step (7) comprises extraction, concentration and drying, and the purity of baicalin finally obtained is not less than 90%, and the purity of wogonin is not less than 80%.

9. The method according to claim 1, characterized in that: The phenylboronic acid functionalized solid phase carrier preferentially adsorbs baicalein under the condition of pH 7.0-9.0, and desorbs it under the condition of pH 3.0-6.

0.

10. The method according to claim 1, characterized in that The phenylboronic acid functionalized solid phase carrier can be recycled and still maintains stable adsorption performance after multiple cycles.

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