Method for increasing contents of baicalin and wogonoside in scutellaria baicalensis extract

Abstract

The invention discloses a method for increasing the contents of baicalin and wogonoside in a scutellaria baicalensis extract. The method comprises the following steps: (1) preparing a scutellaria baicalensis extracting solution; (2) loading a sample; (3) collecting an eluent; and optionally (4) acid precipitation and purification: preparing the total eluate into a solution with a certain mass concentration, adding acid to adjust the pH value, storing at a proper temperature, standing in a refrigerator, filtering, separating and precipitating, washing the precipitate with water and ethanol until the pH value is neutral, volatilizing the ethanol until no alcohol smell exists, and drying in a drying oven until the weight is constant to obtain the scutellaria baicalensis extract. Wherein the active ingredients comprise baicalin and wogonoside. According to the method, purification is carried out successively through macroporous adsorption resin and optional acid precipitation, the content and the yield of wogonoside can be guaranteed, meanwhile, the yield and the content of wogonoside can be effectively increased, and the method is stable and reliable and can provide ideas for actual quality improvement and process development.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to a method for increasing the contents of baicalin and wogonin in a scutellaria baicalensis extract. Background Art

[0002] Scutellaria baicalensis is the dried root of Scutellaria baicalensis Georgi, a plant of the Labiatae family. It has the effects of clearing away heat and dampness, purging fire and detoxifying, stopping bleeding, and calming the fetus. It is widely distributed in Heilongjiang, Jilin, Liaoning, Hebei and other places in my country.

[0003] Scutellaria baicalensis extract contains multiple active ingredients such as baicalin and wogonin, but the current preparation methods of Scutellaria baicalensis extract have the following shortcomings: 1. The existing methods are cumbersome and complicated, with a long production cycle and large product losses; 2. The cost is high, easy to cause pollution, and a large amount of organic solvents are required; 3. The content of active ingredients such as wogonin is not high. Therefore, it is of great significance to study a method to increase the content of baicalin and wogonin in Scutellaria baicalensis extract. Summary of the invention

[0004] Based on this, the present invention provides a method for increasing the content of baicalin and wogonin in a Scutellaria baicalensis extract, the method comprising the following steps:

[0005] (1) Preparing a scutellaria baicalensis extract: weighing an appropriate amount of scutellaria baicalensis powder, performing reflux extraction with water at a first liquid-to-solid ratio, centrifuging and obtaining a supernatant to obtain a first extract; performing reflux extraction with alcohol at a second liquid-to-solid ratio on the obtained medicinal residue, centrifuging and obtaining a supernatant, and recovering ethanol until there is no alcohol taste to obtain a second extract; combining the first extract and the second extract to obtain the scutellaria baicalensis extract;

[0006] (2) Loading: Accurately weigh an appropriate volume of Scutellaria baicalensis extract, dilute with water to obtain a macroporous resin loading solution, and pass the macroporous resin loading solution through a pretreated macroporous resin adsorption column at a certain flow rate so that the macroporous resin loading solution is adsorbed on the macroporous resin adsorption column;

[0007] (3) collecting the eluate: using water and 50% to 70% ethanol solution to elute at a certain flow rate, collecting the eluate and combining them to obtain a total eluate; and

[0008] Optionally, (4) acid precipitation purification: after the total eluate is prepared into a solution of a certain mass concentration, acid is added to adjust the pH, the solution is stored at an appropriate temperature, and then placed in a refrigerator to stand, the precipitate is filtered and separated, and the precipitate is washed with water and ethanol until the pH is neutral, and after the ethanol is evaporated until there is no alcohol smell, the precipitate is placed in an oven and dried to constant weight to obtain the Scutellaria baicalensis extract;

[0009] Among them, the active ingredients include baicalin and wogonin.

[0010] Furthermore, in step (1), the preparation method of the radix scutellariae medicinal material powder comprises the following steps: weighing an appropriate amount of radix scutellariae medicinal slices, drying them in an oven to constant weight, and then crushing them with a traditional Chinese medicine grinder, collecting them, and sieving them to obtain the radix scutellariae medicinal material powder.

[0011] Furthermore, the temperature of the oven is 45°C to 55°C, for example, about 50°C.

[0012] Furthermore, the sieve is a 50-mesh sieve. Furthermore, the water extraction time is 35 to 45 minutes.

[0013] Furthermore, the first liquid-to-solid ratio is 16-18.

[0014] Furthermore, the alcohol is ethanol.

[0015] Furthermore, the concentration of the ethanol solution is 35% to 45%.

[0016] Furthermore, the extraction temperature of the alcohol extraction is 78°C to 82°C.

[0017] Furthermore, the alcohol extraction time is 40 min to 50 min, for example, about 45 min.

[0018] Furthermore, the second liquid-to-solid ratio is 14-16.

[0019] Further, in step (2), the macroporous resin is HPD100 resin, AB-8 resin, D101 resin or D140 resin. Further, the macroporous resin is HPD100 resin or AB-8 resin. Further, the pretreatment comprises soaking the macroporous resin in an ethanol solution, removing the floating debris and impurities, wet-packing the column, and eluting with an ethanol solution and water in sequence. Further, the concentration of the ethanol solution is about 95%, and the soaking time of the ethanol solution is about 24 hours. Further, the water is deionized water. Further, the concentration of the loading solution is 20 mg / ml to 100 mg / ml, for example, about 40 mg / ml. Further, the flow rate of the loading solution is 1 BV / h to 3 BV / h, for example, about 2 BV / h.

[0020] Further, in step (3), the volume of the water elution is 1BV to 4BV. Further, the flow rate of the water elution is 1BV / h to 3BV / h, for example, about 2BV / h (1.5mL / min). Further, the volume of the 50% to 70% ethanol solution elution is 3BV to 4BV, for example, about 3.5BV. Further, the flow rate of the 50% to 70% ethanol solution elution is 1BV / h to 3BV / h. Further, step (3) further includes volatilizing the ethanol in the total eluate until there is no alcohol taste, and then drying it in an oven to constant weight to obtain the Scutellaria baicalensis extract. Further, the method does not include the following steps: after the total eluate is configured into a solution of a certain mass concentration, adding acid to adjust the pH, storing it at an appropriate temperature, and then placing it in a refrigerator to stand, filtering and separating the precipitate, and washing the precipitate with water and ethanol until the pH is neutral.

[0021] Further, in step (4), the mass concentration of the drug solution is 40mg / ml to 100mg / ml, further 55mg / ml to 85mg / ml, further 60mg / ml to 80mg / ml, for example, about 60mg / ml or about 80mg / ml. Further, the acid is hydrochloric acid. Further, the acid addition is the acid addition under constant stirring. Further, the pH is 1 to 2, for example, 1.4 to 1.6. Further, the temperature is 75°C to 85°C, for example, about 80°C. Further, the storage time is 0.5 hours to 1.5 hours, for example, about 1 hour. Further, the temperature of the refrigerator is 4°C to 10°C, for example, about 6°C. Further, the standing time is 10 hours to 15 hours, for example, about 12 hours. Further, the concentration of the ethanol is 45% to 55%, for example, about 50%. Furthermore, the temperature of the oven is 45°C to 55°C, for example, about 50°C.

[0022] According to another aspect of the present invention, a Scutellaria baicalensis extract prepared by the above method is provided.

[0023] According to another aspect of the present invention, a pharmaceutical composition comprising the Scutellaria baicalensis extract is provided.

[0024] Furthermore, the pharmaceutical composition further comprises pharmaceutically acceptable excipients. Further, the excipients are selected from one or more of the following: dispersants, wetting agents, adhesives, diluents, retention aids, lubricants, sustained-release agents, plasticizers, disintegrants, inclusion agents, flavoring agents, opacifiers and antioxidants. Further, the pharmaceutical composition is in the form of tablets, pills, capsules, powders, injections, films, lozenges, granules or oral solutions.

[0025] Beneficial effects of the present invention:

[0026] The method of the present invention uses a macroporous adsorption resin and an optional acid precipitation method to purify successively, which can effectively and significantly improve the yield and content of wogonin while ensuring the content and yield of baicalin. The method is stable and reliable and can provide ideas for actual quality improvement and process development. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without exceeding the scope of protection required by the present invention.

[0028] Figure 1 This is a schematic diagram of the extraction rates of four indicator substances in the extract in the experiment on the effect of extraction solvent on the extraction effect of Scutellaria baicalensis.

[0029] Figure 2 Schematic diagram of the relationship curve between the predicted value and the actual value of the process evaluation index.

[0030] Figure 3 Schematic diagram of the design space established using the probability of achievement method.

[0031] Figure 4 Schematic diagram of the dynamic leakage curve for the maximum sample loading.

[0032] Figure 5 Schematic diagram of the elution curve of HPD100 macroporous resin when eluted with 60% ethanol.

[0033] Figure 6 This is a schematic diagram of the elution curve of HPD100 macroporous resin when eluted with 90% ethanol.

[0034] Figure 7 Schematic diagram of acid precipitation results of four indicators at different drug solution concentrations. DETAILED DESCRIPTION

[0035] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0036] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those of ordinary skill in the field of the invention or the field in which the terms are used. Although any methods, conditions, substances or materials similar to or equivalent to those disclosed herein may be used in the practice of the present invention, preferred methods, conditions, substances or materials are described herein.

[0037] The present invention is intended to encompass all alternatives, variations and equivalents that may be included in the present invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0038] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0039] In the present invention, the term "comprising" is synonymous with "including". As used herein, the terms "comprising", "including", "having", "containing" or any other variation thereof are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus comprising the listed elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0040] As described in the background technology section, the existing method for preparing the scutellaria baicalensis extract has the problems of cumbersome and complicated steps, long production cycle, large product loss, high cost, easy to cause pollution, and the need to consume a large amount of organic solvents. More importantly, the content of effective ingredients such as baicalin, wogonin and baicalein is not high. In order to solve the above problems, the present invention provides a method for increasing the content of baicalin and wogonin in the scutellaria baicalensis extract, the method comprising the following steps:

[0041] (1) Preparing a scutellaria baicalensis extract: weighing an appropriate amount of scutellaria baicalensis powder, performing reflux extraction with water at a first liquid-to-solid ratio, centrifuging and obtaining a supernatant to obtain a first extract; performing reflux extraction with alcohol at a second liquid-to-solid ratio on the obtained medicinal residue, centrifuging and obtaining a supernatant, and recovering ethanol until there is no alcohol taste to obtain a second extract; combining the first extract and the second extract to obtain the scutellaria baicalensis extract;

[0042] (2) Loading: Accurately weigh an appropriate volume of Scutellaria baicalensis extract, dilute with water to obtain a macroporous resin loading solution, and pass the macroporous resin loading solution through a pretreated macroporous resin adsorption column at a certain flow rate so that the macroporous resin loading solution is adsorbed on the macroporous resin adsorption column;

[0043] (3) collecting the eluate: using water and 50% to 70% ethanol solution to elute at a certain flow rate, collecting the eluate and combining them to obtain a total eluate; and

[0044] Optionally, (4) acid precipitation purification: after the total eluate is prepared into a solution of a certain mass concentration, acid is added to adjust the pH, the solution is stored at an appropriate temperature, and then placed in a refrigerator to stand, the precipitate is filtered and separated, and the precipitate is washed with water and ethanol until the pH is neutral, and after the ethanol is evaporated until there is no alcohol smell, the precipitate is placed in an oven and dried to constant weight to obtain the Scutellaria baicalensis extract;

[0045] Among them, the active ingredients include baicalin and wogonin.

[0046] In the present invention, when concentration, temperature, time, flow rate, volume, pH or other values ​​or parameters are expressed as a range, a preferred range, or a range defined by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether the range is disclosed separately. For example, when a range of "50% to 70%" is disclosed, the described range should be interpreted as including the range of "50% to 70%", "51% to 70%", "52% to 70%", "53% to 70%", "54% to 70%", "55% to 70%", "56% to 70%", "57% to 70%", "58% to 70%", "59% to 70%", "60% to 70%", "61% to 70%", "62% to 70%", "63% to 70%", "64% to 70%", "65% to 70%", "66% to 70%", "67% to 70%", "68% to 70%", "69% to 70%", etc. When a numerical range is described herein, unless otherwise specified, the range is intended to include its end values ​​and all integers and fractions within the range, and the technical effects of the present invention can be achieved within the above range.

[0047] In a preferred embodiment, in step (1), the method for preparing the radix scutellariae medicinal material powder comprises the following steps: weighing an appropriate amount of radix scutellariae decoction pieces, drying them in an oven to constant weight, and then crushing them with a traditional Chinese medicine grinder, collecting them, and sieving them to obtain the radix scutellariae medicinal material powder.

[0048] In a preferred embodiment, the temperature of the oven is 45°C to 55°C, such as about 50°C.

[0049] In a preferred embodiment, the sieve is a 50 mesh sieve.

[0050] In a preferred embodiment, the water extraction time is 35 to 45 minutes.

[0051] In a preferred embodiment, the first liquid-to-solid ratio is 16-18.

[0052] In a preferred embodiment, the alcohol is ethanol.

[0053] In a preferred embodiment, the concentration of the ethanol solution is 35% to 45%.

[0054] In a preferred embodiment, the extraction temperature of the alcohol extraction is 78°C to 82°C.

[0055] In a preferred embodiment, the alcohol extraction time is 40 min to 50 min, such as about 45 min.

[0056] In a preferred embodiment, the second liquid-to-solid ratio is 14-16.

[0057] In a preferred embodiment, in step (2), the macroporous resin is HPD100 resin, AB-8 resin, D101 resin or D140 resin. In a preferred embodiment, the macroporous resin is HPD100 resin or AB-8 resin. In a preferred embodiment, the pretreatment includes soaking the macroporous resin in an ethanol solution, removing the floating debris and debris, wet-packing the column, and eluting with an ethanol solution and water in sequence. In a preferred embodiment, the concentration of the ethanol solution is about 95%, and the soaking time of the ethanol solution is about 24 hours. In a preferred embodiment, the water is deionized water. In a preferred embodiment, the concentration of the loading solution is 20 mg / ml to 100 mg / ml, for example, about 40 mg / ml. In a preferred embodiment, the flow rate of the loading solution is 1 BV / h to 3 BV / h, for example, about 2 BV / h.

[0058] In a preferred embodiment, in step (3), the volume of the water elution is 1BV to 4BV. In a preferred embodiment, the flow rate of the water elution is 1BV / h to 3BV / h, for example, about 2BV / h (1.5mL / min). In a preferred embodiment, the volume of the 50% to 70% ethanol solution elution is 3BV to 4BV, for example, about 3.5BV. In a preferred embodiment, the flow rate of the 50% to 70% ethanol solution elution is 1BV / h to 3BV / h. In a preferred embodiment, step (3) further includes volatilizing the ethanol in the total eluate until there is no alcohol taste, and then drying it in an oven to constant weight to obtain the Scutellaria baicalensis extract. In a preferred embodiment, the method does not include the following steps: after the total eluate is configured into a solution of a certain mass concentration, acid is added to adjust the pH, stored at an appropriate temperature, and then placed in a refrigerator to stand, filtration and separation of the precipitate, and the precipitate is washed with water and ethanol until the pH is neutral.

[0059] In a preferred embodiment, in step (4), the mass concentration of the medicinal solution is 40 mg / ml to 100 mg / ml, further 55 mg / ml to 85 mg / ml, further 60 mg / ml to 80 mg / ml, for example, about 60 mg / ml or about 80 mg / ml. In a preferred embodiment, the acid is hydrochloric acid. In a preferred embodiment, the acid addition is the addition of acid dropwise under constant stirring. In a preferred embodiment, the pH is 1 to 2, for example, 1.4 to 1.6. In a preferred embodiment, the temperature is 75°C to 85°C, for example, about 80°C. In a preferred embodiment, the storage time is 0.5 hours to 1.5 hours, for example, about 1 hour. In a preferred embodiment, the temperature of the refrigerator is 4°C to 10°C, for example, about 6°C. In a preferred embodiment, the standing time is 10 hours to 15 hours, for example, about 12 hours. In a preferred embodiment, the concentration of the ethanol is 45% to 55%, for example, about 50%. In a preferred embodiment, the temperature of the oven is 45°C to 55°C, such as about 50°C.

[0060] According to another aspect of the present invention, a Scutellaria baicalensis extract prepared by the above method is provided.

[0061] According to another aspect of the present invention, a pharmaceutical composition comprising the Scutellaria baicalensis extract is provided.

[0062] In a preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0063] In a preferred embodiment, the pharmaceutical preparation of the present invention contains at least one pharmaceutically acceptable excipient in an amount of 0.00001 to 50 wt.%, or 0.0001 to 10 wt.%, or 0.0001 to 5 wt.%, or 0.005 to 1 wt.%, or 0.1 to 20 wt.%, or 0.5 to 15 wt.%, or 1 to 5 wt.%, based on the weight of the pharmaceutical preparation.

[0064] In the present invention, the term "pharmaceutically acceptable" refers to a substance, such as a carrier or diluent, that does not abrogate the biological activity or properties of the compound and is relatively non-toxic, i.e., a substance that does not cause undesirable biological effects or interact in a deleterious manner with any of its components when administered to an individual.

[0065] In the present invention, the term "pharmaceutically acceptable excipient" refers to carriers and / or excipients that are pharmacologically and / or physiologically compatible with subjects and active ingredients (i.e., capable of inducing the desired therapeutic effect without causing any undesirable local or systemic effects), which are well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995).

[0066] In a preferred embodiment, the auxiliary material is selected from one or more of the following: dispersants, wetting agents, binders, diluents, retention aids, lubricants, sustained-release agents, plasticizers, disintegrants, inclusion agents, flavoring agents, opacifiers and antioxidants.

[0067] These excipients are preferably pharmaceutically inert, or may have a synergistic or additive effect to enhance the therapeutic activity of the pharmaceutical composition, and the above-mentioned excipients are only listed. The excipients actually used in the present invention are not limited to the above-mentioned excipients and can be adjusted according to actual conditions to achieve the effects of the present invention.

[0068] In a preferred embodiment, the pharmaceutical composition is in the form of tablets, pills, capsules, powders, injections, films, lozenges, granules or oral solutions.

[0069] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 50" includes ±5% of 50, or from 47.5 to 52.5; "about 45" includes ±5% of 45, or from 42.75 to 47.25; "about 95" includes ±5% of 95, or from 90.25 to 99.75; "about 24" includes ±5% of 24, or from 22.8 to 25.2; "about 40" includes ±5% of 40, or from 38 to 42; "about 2" includes ±5% of 2, or from 1.9 to 2.1; "about 60" includes ±5% of 60, or from 57 to 63; “About 3.5” includes ±5% of 3.5, or from 3.325 to 3.675; “about 90” includes ±5% of 90, or from 85.5 to 94.5; “about 2.2” includes ±5% of 2.2, or from 2.09 to 2.31; “about 80” includes ±5% of 80, or from 76 to 84; “about 1” includes ±5% of 1, or from 0.95 to 1.05; “about 6” includes ±5% of 6, or from 5.7 to 6.3; “about 12” includes ±5% of 12, or from 11.4 to 12.6.

[0070] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions or conditions recommended by the manufacturer.

[0071] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0072] The above features mentioned in the present invention or the features mentioned in the embodiments can be combined in any way. All the features disclosed in this patent specification can be used in combination with any combination form, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0073] Example

[0074] 1. Analysis method of Scutellaria baicalensis extract

[0075] High performance liquid chromatography was used to qualitatively and quantitatively analyze baicalin, scutellaria baicalensis extract, wogonin and baicalein in the scutellaria baicalensis extract and the purified extract. The chromatographic method was based on the HPLC method established in the literature (Zhao Liang, Wang Xinxia, ​​Lv Lei, et al. HPLC determination of baicalin, wogonin, scutellaria baicalensis, baicalein and wogonin in Qinggan Sanjie granules [J]. Chinese Traditional Patent Medicine, 2014, 36(02): 313-318.).

[0076] 1.1 Chromatographic conditions

[0077] The chromatographic column is Waters Xterra Rp-C 18 (3.0mm×100mm,3.5μm), mobile phase A was acetonitrile solution, phase B was 0.1% formic acid water, gradient elution (0-10min, 10%-20%A; 10-30min, 20%-25%A; 30-60min, 25%-45%A), volume flow rate was 0.4mL / min, column temperature was room temperature, UV detection wavelength was 275nm, injection volume was 10μL, and running time was 60min.

[0078] 1.2. Preparation of reference solution

[0079] Accurately weigh 10 mg of baicalin reference substance and place it in a 10 mL volumetric flask, add methanol to ultrasonically dissolve it, and dilute to the scale to obtain a baicalin reference substance solution with a mass concentration of 1.0 mg / mL; accurately weigh 5 mg of scutellaria baicalin reference substance and place it in a 10 mL volumetric flask, add methanol to ultrasonically dissolve it, and dilute to the scale to obtain a scutellaria baicalin reference substance solution with a mass concentration of 0.5 mg / mL; accurately weigh 5 mg of wogonin reference substance and place it in a 25 mL volumetric flask, add methanol to ultrasonically dissolve it, and dilute to the scale to obtain a wogonin reference substance solution with a mass concentration of 0.2 mg / mL; accurately weigh 5 mg of baicalin reference substance and place it in a 10 mL volumetric flask, add methanol to ultrasonically dissolve it, and dilute to the scale to obtain a baicalin reference substance solution with a mass concentration of 0.5 mg / mL.

[0080] 1.3. Preparation of mixed reference solution

[0081] From the reference solution of baicalin, scutellarin, wogonin and baicalein under item 1.2, accurately measure 3.4mL, 0.7mL, 3.2mL and 0.4mL respectively, put them into a 10mL volumetric flask, add methanol and ultrasonically dissolve them, make up to the scale, and prepare a mixed reference solution with mass concentrations of baicalin, scutellarin, wogonin and baicalein of 340, 35, 64 and 20μg / mL respectively.

[0082] 1.4. Linear relationship investigation

[0083] Accurately pipette the mixed reference solution under 1.3, and inject 15, 10, 6, 3, 1, and 0.5 μL of the solution according to the chromatographic conditions under 1.1, respectively, and record the peak area value. Perform linear regression with the peak area as the ordinate (Y) and the mass concentration (μg / mL) as the abscissa, and obtain the regression equation as shown in Table 1.

[0084] Table 1 Regression equations and linear ranges of four compounds

[0085] Element Regression equation <![CDATA[r 2 ]]> Linear range / μg / mL Scutellarin Y=43.233X-15.626 0.9994 1.8～52.5 Baicalin Y=62.805X+55.99 0.9998 17.0～510.0 Wogonin Y=78.634X+3.8078 0.9998 3.2～96.0 Wogonin Y=117.2X-5.3247 0.9999 1.0～30.0

[0086] 2. Optimization of Scutellaria baicalensis extraction process

[0087] 2.1 Experimental medicinal materials, reagents and equipment

[0088] 2.1.1 Experimental herbs and their pretreatment

[0089] Scutellaria baicalensis slices (provided by Shanghai Kaibao Pharmaceutical Co., Ltd., batch number: 2107204) were dried in an oven at 50°C to constant weight, and then crushed with a traditional Chinese medicine grinder. The powder was collected after passing through a 50-mesh sieve and put into a plastic bag for later use.

[0090] 2.1.2 Experimental Reagents

[0091] Table 2 Experimental reagents list

[0092]

[0093]

[0094] 2.1.3 Experimental instruments and equipment

[0095] Table 3 List of instruments and equipment

[0096]

[0097] 2.2. Study on the influence of extraction solvent on the extraction effect of Scutellaria baicalensis

[0098] In order to explore the influence of water or ethanol as extraction solvent on the extraction effect of Scutellaria baicalensis, reflux extraction was considered to extract twice, and the combination of four extraction solvents, including two water extractions, two alcohol extractions, water extraction followed by alcohol extraction, and alcohol extraction followed by water extraction, was compared. The extraction effects of baicalin, scutellaria baicalin, wogonin and baicalein were used as indicators.

[0099] 2.2.1 Extraction method

[0100] Accurately weigh 5.0 g of Scutellaria baicalensis powder, extract twice with 10 times the solvent (water or 70% ethanol) by reflux, the water extraction temperature is 100°C, the alcohol extraction temperature is 80°C, each time for 45 minutes, centrifuge to obtain the two extracts, combine them, make up to 100 mL, and obtain the Scutellaria baicalensis extract, which is refrigerated for later use. The alcohol-containing extract needs to be concentrated and evaporated to remove the ethanol before combining and making up the volume, otherwise it will be unfavorable for subsequent acid precipitation.

[0101] 2.2.2 Analysis methods

[0102] The contents of four index substances in the Scutellaria baicalensis extract were determined using "1. Analysis method for Scutellaria baicalensis extract". For the determination of the Scutellaria baicalensis extract, an appropriate amount of extract sample was accurately measured with a pipette, placed in a 10 mL volumetric flask, made up to volume with 50% ethanol, and filtered through a 0.22 μm microporous filter membrane before injection.

[0103] The quality evaluation indexes of Scutellaria baicalensis extract and crude extract were calculated as follows:

[0104] (1) Yield (Y): Yield of the target substance in the Scutellaria baicalensis extract = mass of the target substance in the extract / mass of the raw material Scutellaria baicalensis × 100%;

[0105] (2) Content: Content of the index substance in the crude extract of Scutellaria baicalensis = mass of the index substance in the crude extract / mass of the crude extract × 100%;

[0106] (3) Transfer rate (relative to the extract): Transfer rate of the target substance in the crude extract of Scutellaria baicalensis = mass of the target substance in the crude extract / mass of the target substance in the extract × 100%.

[0107] The experiment was repeated 3 times for each solvent combination, and the results were averaged.

[0108] 2.2.3 Effect of extraction solvent on the extraction effect of Scutellaria baicalensis

[0109] The reflux extraction method was used to compare the extraction of baicalin, scutellaria baicalin, wogonin and baicalein in the extract. Figure 1 shown.

[0110] The results showed that the yields of baicalin and wogonin were not much different under the four extraction solvent combinations, and were slightly higher when water extraction was performed first and then alcohol extraction than other methods; the yield of baicalin was the highest when water extraction was performed twice, followed by water extraction first and then alcohol extraction. However, through repeated experiments, it was found that under the same conditions, the RSD% of baicalin yield could reach up to 10% (two water extractions and two alcohol extractions), and the lowest was 4%-5% (water first and then alcohol and alcohol first and then water), while the RSD% of the yields of the other three substances were all within 3%, indicating that the extraction stability of baicalin was poor; the introduction of ethanol significantly increased the yield of wogonin, indicating that using ethanol as a solvent is more conducive to the extraction of wogonin.

[0111] In addition, the order of water extraction and alcohol extraction also has a certain influence on the four substances. The yields of the four substances when water extraction is performed first and then alcohol extraction are slightly higher than those when alcohol extraction is performed first and then water extraction. The reason may be that the temperature of the first water extraction is very high, which can extract as many flavonoid O-glycosides in Scutellaria baicalensis as possible, while the second alcohol extraction can further extract the remaining flavonoid O-glycosides while also extracting more baicalin with better alcohol solubility.

[0112] 2.3 Optimization of Scutellaria baicalensis extraction process

[0113] 2.3.1 Preparation of Scutellaria baicalensis extract

[0114] According to the results of "2.2, the effect of extraction solvent on the extraction effect of Scutellaria baicalensis", the method of water extraction followed by alcohol extraction was selected to extract the main active ingredients in Scutellaria baicalensis. 5.0g of Scutellaria baicalensis powder was accurately weighed, and refluxed and extracted twice with water of a certain liquid-to-solid ratio and ethanol of a certain concentration. The two extracts were centrifuged and combined to obtain the Scutellaria baicalensis extract. The contents of baicalin, baicalin, wogonin and baicalein in the extract were determined according to "1, analysis method of Scutellaria baicalensis extract".

[0115] 2.3.2 Definitive screening experiment design and results

[0116] Since there are many process parameters to be considered in the extraction process of Scutellaria baicalensis, a deterministic screening experimental design was used to investigate the effects of the first extraction time (X1), the first liquid-solid ratio (X2), the ethanol concentration (X3), the second extraction temperature (X4), the second extraction time (X5) and the second liquid-solid ratio (X6) on the extraction effects of the four index substances. The deterministic screening experimental design was carried out using JMP software (SAS, USA), and the center point experiment was repeated 3 times, with a total of 19 groups of experiments. The high and low levels of each process parameter were set in combination with the pre-experiment and literature. The experimental factor level table and experimental design table and the results are shown in Tables 4 and 5. The process evaluation index of Scutellaria baicalensis extraction is the yield of scutellarin (Y1), baicalin (Y2), wogonin (Y3) and wogonin (Y4) in the Scutellaria baicalensis extract, which is calculated according to the following formula:

[0117]

[0118] Table 4 Factors and levels of the definitive screening experiment design

[0119]

[0120] Table 5 Deterministic screening experiment design table and results

[0121]

[0122]

[0123] 2.3.3 Data processing and model fitting

[0124] The following formula was used to establish a quantitative model between the yields of the process evaluation indicators baicalin (Y1), baicalin (Y2), wogonin (Y3), and wogonin (Y4) and each process parameter.

[0125]

[0126] Among them, a0 is a constant; a i , a ii and a ij are the regression coefficients of the linear term, quadratic term and interaction term respectively; X i and X j are the process parameters; Y k is the evaluation index of Scutellaria baicalensis extraction process, subscript k = 1, 2, 3, 4. In JMP software, Bayesian information criterion combined with stepwise forward method was used for fitting, the key process parameters of each index were determined by P value, and then the quantitative model was established by regression coefficient. The regression coefficient and variance analysis results of the model are shown in Table 6.

[0127] Table 6 Model regression coefficients and variance analysis results

[0128]

[0129]

[0130] As can be seen from Table 6, the models of the four process evaluation indicators all have R 2 >0.90, indicating that the model has a good fitting effect and can better describe the impact of process parameters on indicators. The relationship curves between the predicted values ​​and actual values ​​of the four indicators are shown in Figure 2. Figure 2 As shown, Figure 2 The black dot is the actual value of the indicator, the oblique straight line is the fitting model, the horizontal straight line is the mean of the actual value, and the gray area is the 95% confidence interval. The P value of the process parameters and their interactions in the model is statistically significant when it is less than 0.05, and it is very significant when it is less than 0.01.

[0131] The analysis results of the regression model show that for the yield of baicalin (Y1), the first extraction time (X1), ethanol concentration (X3) and the interaction between the first extraction time (X1) and the first liquid-solid ratio (X2) are more significant (P < 0.01), so X1, X2 and X3 are identified as the key process parameters affecting the yield of baicalin; for the yield of baicalin (Y2), the first extraction time (X1), the first liquid-solid ratio (X2) and the interaction between the first liquid-solid ratio (X2) and the second liquid-solid ratio (X6) are more significant (P < 0.01), so X1, X2 and X6 are identified as the key process parameters affecting the yield of baicalin. The key process parameters affecting the yield were as follows: for the yield of wogonin (Y3), the first liquid-solid ratio (X2), ethanol concentration (X3), the interaction between the first liquid-solid ratio (X2) and the second liquid-solid ratio (X6), and the interaction between ethanol concentration (X3) and the second extraction temperature (X4) had significant effects (P < 0.01), so X2, X3, X4 and X6 were identified as the key process parameters affecting the yield of wogonin; for the yield of baicalin (Y4), the first liquid-solid ratio (X2) and ethanol concentration (X3) had significant effects (P < 0.01), so X2 and X3 were identified as the key process parameters affecting the yield of wogonin.

[0132] 2.3.4. Establishment and verification of design space

[0133] The design space was established by the probability of reaching the target method, and the probability of reaching the target was calculated by the simulation experiment error method. Python was used for programming. The specific programming ideas were as follows (Shao Jingyuan, Qu Haibin, Gong Xingchu. Comparative study of two design space calculation methods - the probability of reaching the target method and the multi-index superposition method [J]. China Journal of Traditional Chinese Medicine, 2018, 43(10): 2074-2080.):

[0134] (1) Determine the relative standard deviation of the measured values ​​of each index: According to the measured results of Experiments 17 to 19 in Table 5, the relative standard deviations of the yields of baicalin (Y1), baicalin (Y2), wogonin (Y3), and wogonin (Y4) were calculated and recorded as RSD1=1.63, RSD2=2.17, RSD3=1.19, and RSD4=1.62, respectively.

[0135] (2) Generate random values ​​for each indicator measurement value: Assume that the indicator measurement value DR obtained in the experiment i,j is a sample from a normal distribution population N, where subscript i is the index number, subscript j is the experiment number, and it is assumed that the mean of the normal distribution population N is the measured value DR i,j , the variance is (DR i,j ×RSD i ) 2 , that is, assuming that the measured values ​​of the indicators under different experimental conditions follow a series of normal distributions with equal relative standard deviations. According to the normal distribution N, random simulation generates random values ​​DR for each measured value i,j,n , where the subscript n is the number of random simulations. Let n = 5000, thus generating a total of i×j×n, or 4×17×5000 random values.

[0136] (3) Establishing a mathematical model: Based on the random values ​​generated by the previous simulation, for a certain process evaluation index, the simulation values ​​corresponding to the 19 experiments in Table 5 are sorted by the number of simulations, grouped together into 5000 groups, and modeled according to the quantitative model formula. This will result in 5000 models. A total of 4×5000 models are established for the four process evaluation indexes.

[0137] (4) Using the model to predict the results: Use the exhaustive method to predict the results, set the calculation step size to 0.1, and the coding value range of the process parameters in the deterministic screening experimental design is [-1,1], so there are 21 discrete values ​​for each process parameter. Assume that there is t i process parameters affect the process evaluation index Yi (i = 1, 2, 3 or 4), then for each Y i Can form 21 ti By taking each process parameter combination into the 20,000 models established by the process evaluation indicators, 20,000 prediction results of the four process evaluation indicators can be obtained, and the total prediction results are

[0138] indivual.

[0139] (5) Statistical probability values: According to the results of the deterministic screening test design, the yield of scutellaria baicalin was between 0.1964% and 0.2659%, the yield of baicalin was between 14.5736% and 15.7377%, the yield of wogonin was between 2.9006% and 3.1035%, and the yield of wogonin was between 0.1862% and 0.2311%. The ranges of the evaluation indicators of the Scutellaria baicalensis extraction process were determined as shown in Table 7.

[0140] Table 7 Process evaluation index range setting

[0141]

[0142]

[0143] For each process parameter combination, the number of times that the statistical prediction results can meet the limits of all four process evaluation indicators is m, and there must be m≤5000. The following formula is used to calculate the probability Pr (0≤Pr≤1) that the process parameter combination can meet the standards for all process evaluation indicators.

[0144]

[0145] (6) Calculation of design space: The probability of reaching the target is set to 0.80. When Pr ≥ 0.80 for a process parameter combination, it means that the process parameter combination is within the design space. By counting all process parameter combinations that satisfy Pr ≥ 0.80, the design space based on the probability of reaching the target is obtained. The design space is displayed using Origin 2018 software.

[0146] The design space established by the probability of reaching the target is as follows: Figure 3 As shown by Figure 3 It can be seen that the shape of the design space is irregular, and the four indicators are different. When the first liquid-to-solid ratio (X2) is at a higher level, the four indicator substances all show a good extraction effect; when the yield of baicalin is the indicator, the interaction between the liquid-to-solid ratio (X2) of the first extraction and the extraction time (X1) is more significant, and the probability of reaching the standard is high when the first extraction time is longer and the liquid-to-solid ratio is larger or the first extraction time is shorter and the liquid-to-solid ratio is relatively smaller. When the ethanol concentration (X3) is higher, it is mainly beneficial to the extraction of wogonin, but has no obvious benefit to the extraction effect of other substances. When the second liquid-to-solid ratio (X6) is at a lower level, the yield of baicalin is not easy to reach the standard; when the yield of baicalin is the indicator, the interaction between the second liquid-to-solid ratio (X6) and the first liquid-to-solid ratio (X2) is more significant, and the probability of reaching the standard is higher when the liquid-to-solid ratios of the first and second extractions are both smaller or larger.

[0147] Taking into account the convenience and economy of the four indicators and the operation, the recommended operating range is finally determined as follows: the first extraction time is 35-45 min, the first liquid-solid ratio is 16-18, the ethanol concentration is 35%-45%, the alcohol extraction temperature is 78-82 ° C, the second extraction time is 45 min, and the second liquid-solid ratio is 14-16. Within this operating range, the four indicator substances all meet the process evaluation indicator range in Table 7, and the probability of reaching the standard is above 0.95.

[0148] To further verify the accuracy of the design space, three method parameter combination points were selected in the design space for experimental verification, and the verification method conditions and experimental results are shown in Table 8. As can be seen from Table 8, the yields of baicalin, baicalin, wogonin and baicalein all met the standards, and the actual values ​​were close to the predicted values, indicating that the design space established in this study is relatively reliable.

[0149] Table 8 Conditions and results of verification experiment

[0150]

[0151] 3. Study on the macroporous adsorption resin and acid precipitation purification process of Scutellaria baicalensis

[0152] 3.1 Experimental medicinal materials, reagents and equipment

[0153] 3.1.1. Preparation of experimental medicinal materials and extracts

[0154] Scutellariae Radix Pieces (provided by Shanghai Kaibao Pharmaceutical Co., Ltd., batch number: 2107204) were dried in an oven at 50°C to constant weight, and then crushed with a Chinese medicine grinder, and the powder was collected after passing through a 50-mesh sieve. 15.0 g of Scutellariae Radix Powder was extracted twice according to the optimal process parameter range in "2. Optimization of Scutellariae Radix Extraction Process", and the alcohol extraction solution was recovered to ethanol until there was no alcohol taste, and the two extracts were combined to obtain a certain volume of Scutellariae Radix Extract for use.

[0155] 3.1.2 Experimental Reagents

[0156] The experimental reagents are shown in Table 2.

[0157] The specific models, characteristics and manufacturers of macroporous resins are shown in Table 9.

[0158] Table 9 Macroporous resin properties

[0159]

[0160] 3.1.3 Instruments and equipment

[0161] The instruments and equipment are shown in Table 3.

[0162] 3.2 Research on the purification process of macroporous adsorption resin

[0163] 3.2.1 Pretreatment and regeneration of macroporous adsorption resin

[0164] 3.2.1.1 Preprocessing method

[0165] Take an appropriate amount of macroporous resin in a beaker, soak it in 95% ethanol for 24 hours to fully swell, remove the floating debris and debris, and then wet-pack it into a column. Wash it with 95% ethanol until the effluent is clear, and then wash the resin with deionized water until there is no ethanol smell. Seal the treated macroporous resin with deionized water and store it in a refrigerator for later use.

[0166] 3.2.1.2 Regeneration method

[0167] After use, the macroporous adsorption resin is contaminated and its adsorption capacity decreases. Its adsorption performance can be restored through certain regeneration treatment. The specific regeneration treatment method is as follows: elute the used resin with anhydrous ethanol until the eluent is colorless, rinse with 3BV of 1% NaOH, and then wash with water until neutral. Seal the treated macroporous resin with deionized water and store it in a refrigerator for future use.

[0168] 3.2.2. Screening of macroporous adsorption resin

[0169] Through literature research, six types of resins commonly used for separation and purification of flavonoid components were identified, with different polarities and average pore sizes, and relatively good effects. They are non-polar D101, HPD100, X-5, D140, weakly polar AB-8 and medium polar HPD400. The static adsorption and desorption method was used to screen the most suitable resin based on the adsorption capacity and desorption rate of different macroporous adsorption resins. Weigh 2g of the macroporous adsorption resin pretreated in 3.2.1 and place it in a 150mL conical flask. Two parallel portions were added with 60mL of Scutellaria baicalensis extract, sealed with a lid, and adsorbed at room temperature for 24h to ensure that the adsorption equilibrium was reached. Filter and determine the contents of the remaining four components of scutellaria baicalin, baicalin, wogonin and baicalin in the remaining liquid after adsorption. The adsorption capacity of the macroporous resin was calculated according to the following formula. After adsorption, the resin was rinsed with deionized water until the washing liquid was colorless. Then 1.2 g of the resin was added with 50 mL of anhydrous ethanol with 70% ethanol for desorption. The desorption was carried out at room temperature for 24 h to ensure complete desorption. The desorption was filtered and the contents of the four components in the desorption liquid were determined using "1. Analysis method for Scutellaria baicalensis extract". The desorption rate of the macroporous resin was calculated according to the following formula.

[0170]

[0171]

[0172] Among them, the unit of adsorption amount is mg / mL; C0 is the concentration of the compound in the Scutellaria baicalensis extract (mg / g); C1 is the concentration of the compound in the solution after reaching adsorption equilibrium (mg / g); C2 is the concentration of the compound in the desorption solution (mg / g); V1 is the initial solution volume (mL); V2 is the desorption solution volume (mL); W is the mass of the added macroporous adsorption resin (g).

[0173] The static adsorption and desorption effects of different macroporous resins are shown in Table 10. From the adsorption amount, the adsorption effects of HPD-400 and X-5 on the four components are not ideal, indicating that non-polar macroporous resins with relatively small pore size are more suitable for the adsorption of the four components in Scutellaria baicalensis. The other four macroporous resins HPD100, AB-8, D101 and D140 all show good adsorption effects on the four components; from the resolution rate, HPD100 has a good desorption effect on the four components, and the desorption rate is above 80%, while AB-8 with the best adsorption capacity has poor desorption effects on baicalin and baicalin. Considering the adsorption and desorption capacity comprehensively, HPD100 macroporous adsorption resin was selected in this experiment to investigate the purification process of Scutellaria baicalensis extract.

[0174] Table 10 Performance of macroporous resin adsorption / desorption of four index substances in Scutellaria baicalensis

[0175]

[0176] 3.2.3 Investigation of process parameters for purification by macroporous adsorption resin

[0177] 3.2.3.1. Investigation of drug solution concentration

[0178] Take a certain amount of pretreated HPD100 macroporous resin wet column (inner diameter 1.8cm), diameter-to-height ratio 1:10, accurately measure 5 portions of 80mL of 100 (crude drug) mg / mL extract, dilute to 100, 80, 60, 40, 20mg / mL loading solution for loading, the loading flow rate is 1.5BV / h (i.e. 1.2mL / min), after loading, wash with 3BV water, the washing speed is 2.0mL / min. Collect all the eluents, determine the content, calculate the adsorption amount of the four components by the HPD100 macroporous resin, the results are shown in Table 11.

[0179] Table 11 Adsorption amount of 4 indicator substances at different loading concentrations

[0180] Loading solution concentration Scutellarin Baicalin Wogonin Wogonin <![CDATA[ / mg·mL -1 ]]> <![CDATA[Adsorption capacity / mg·mL -1 > <![CDATA[Adsorption capacity / mg·mL -1 > <![CDATA[Adsorption capacity / mg·mL -1 > <![CDATA[Adsorption capacity / mg·mL -1 > 100 0.42 34.77 6.63 0.43 80 0.42 34.91 6.64 0.43 60 0.43 35.20 6.66 0.43 40 0.43 35.28 6.64 0.43 20 0.43 35.46 6.67 0.43

[0181] From the results, it can be seen that the change of loading concentration has a significant effect on baicalin, which has the highest content in the drug solution. The lower the loading concentration of the drug solution, the better the adsorption effect of baicalin. This may be because when the sample concentration is high, the concentration of impurities is also high. Excessive impurities compete with the target substance for the active sites on the resin, resulting in a decrease in the free active sites that can adsorb the target substance and a decrease in the adsorption amount. Considering the concentration of the extract and the loading time, 40 mg / mL was selected as the loading concentration.

[0182] 3.2.3.2. Sample flow rate investigation

[0183] Take a certain amount of pretreated HPD100 macroporous resin wet column (inner diameter 1.8cm), diameter-to-height ratio 1:10, accurately absorb 240mL of 40mg / mL sample solution, and load the sample at a speed of 1.0BV / h (i.e. 0.8mL / min), 1.5BV / h (i.e. 1.2mL / min), 2.0BV / h (i.e. 1.5mL / min), 2.5BV / h (i.e. 1.9mL / min) and 3.0BV / h (i.e. 2.3mL / min), respectively. After loading, wash with 3BV of water, and the washing speed is 2.0mL / min. Collect all the eluents, determine the content, and calculate the adsorption amount of the four components by the HPD100 macroporous resin. The results are shown in Table 12.

[0184] Table 12 Adsorption amount of 4 indicator substances at different loading flow rates

[0185] Sample flow rate Scutellarin Baicalin Wogonin Wogonin <![CDATA[ / BV·h -1 ]]> <![CDATA[Adsorption capacity / mg·mL -1 > <![CDATA[Adsorption capacity / mg·mL -1 > <![CDATA[Adsorption capacity / mg·mL -1 > <![CDATA[Adsorption capacity / mg·mL -1 > 1.0 0.55 36.01 7.57 0.46 1.5 0.57 36.20 7.43 0.46 2.0 0.60 36.88 7.55 0.46 2.5 0.58 36.28 7.54 0.46 3.0 0.55 36.18 7.54 0.46

[0186] From the results in Table 12, it can be seen that the effect of the sample flow rate on the adsorption of the four components is not significant. When the flow rate is too slow or too fast, the adsorption amount of the four components is slightly reduced. When the flow rate is 2.0BV / h (i.e., 1.5mL / min), the adsorption effect of the four components is good. When the sample flow rate is too fast, the contact time between the component and the resin is shortened, and the component is washed away by the solution before being adsorbed by the resin, which reduces the adsorption amount of the resin; when the sample flow rate is too slow, in the local space, the flavonoid component may have been fully absorbed by the resin, and the new liquid has not entered this space, resulting in a large difference between the concentration of the component adsorbed by the resin and the concentration of the component in the liquid in the space, and the substance adsorbed in the resin returns to the liquid again. In addition, the too slow flow rate also gives other impurities in the liquid time to compete for adsorption sites, resulting in a decrease in the adsorption amount of the four components as a whole. Therefore, a relatively moderate flow rate is more conducive to creating an appropriate concentration difference and adsorption time, and improving the adsorption effect, so 2.0BV / h (i.e., 1.5mL / min) is selected as the sample flow rate.

[0187] 3.2.3.3. Maximum sample loading amount

[0188] Take a certain amount of pretreated HPD100 macroporous resin wet column (inner diameter 1.8cm), with a diameter-to-height ratio of 1:10, accurately draw 400mL of 40mg / mL sample solution, load at a speed of 2.0BV / h (i.e. 1.5mL / min), collect one portion every 10mL, a total of 40 portions, determine the concentration of the four components in each portion, and draw a dynamic leakage curve, such as Figure 4 shown.

[0189] Depend on Figure 4 It can be seen that after the sample volume reached 160mL, baicalin began to leak a small amount, and the leakage increased sharply after 320mL. Baicalin and wogonin leaked significantly after the sample volume reached 360mL, and wogonin did not leak at all and could be completely adsorbed. Therefore, the maximum sample volume should not exceed 320mL. Next, the sample volume was further determined based on the loss of the four components during water washing and impurity removal.

[0190] Take a certain amount of pretreated HPD100 macroporous resin wet column (inner diameter 1.8cm), diameter-to-height ratio 1:10, accurately draw 40mg / mL sample solution 200mL, 240mL, 280mL and 320mL, respectively, at a speed of 2.0BV / h (i.e. 1.5mL / min), and wash with 2BV of water after loading, the washing speed is 1.5mL / min. Collect all the eluents, determine the content, and calculate the transfer rate of the four components adsorbed by the macroporous resin from the extract. The results are shown in Table 13.

[0191] Table 13 Transfer rate of 4 indicator substances with different loading amounts after water washing

[0192]

[0193]

[0194] Under the same washing volume, the transfer rate of baicalin decreased significantly with the increase of sample loading, indicating that baicalin was more likely to be lost during the washing process when the loading amount was high; the transfer rates of baicalin and wogonin also decreased with the increase of loading amount. Based on the leakage curve, transfer rate after washing and operation time, the loading amount was determined to be 200 mL.

[0195] 3.2.3.4. Investigation of eluent concentration

[0196] Take a certain amount of pretreated HPD100 macroporous resin wet column (inner diameter 1.8cm), diameter-to-height ratio 1:10, load the sample at a loading concentration of 40mg / mL, loading speed 2.0BV / h (i.e. 1.5mL / min), and loading volume 200mL. After loading, wash with 2BV of water at a washing speed of 1.5mL / min. Then, use 50%, 60%, 70%, 80%, 90% respectively.

[0197] (v / v) ethanol 3BV was eluted at a rate of 2.0BV / h (i.e., 1.5mL / min), the eluate was collected, the content was determined, and the elution rates of the four components were calculated; the solid mass was obtained after sampling and evaporation, and the contents of the four components were further calculated. The results are shown in Table 14.

[0198] Table 14 Elution effect of 4 indicator substances at different eluent concentrations

[0199]

[0200] From the results in Table 14, it can be seen that 50% to 70% ethanol has the best elution effect on baicalin and wogonin, while the elution effect on wogonin is significantly poor.

[0201] 3.2.3.5. Elution flow rate investigation

[0202] A certain amount of pretreated HPD100 macroporous resin was wet packed into a column (inner diameter 1.8 cm) with a diameter-to-height ratio of 1:10. The sample was loaded at a loading concentration of 40 mg / mL, a loading speed of 2.0 BV / h (i.e., 1.5 mL / min), and a loading volume of 200 mL. After loading, 2 BV of water was used for washing at a washing speed of 1.5 mL / min. Subsequently, 3BV of 60% (v / v) ethanol was used as eluent, and elution was performed at a rate of 1.0BV / h (i.e. 0.8mL / min), 1.5BV / h (i.e. 1.2mL / min), 2.0BV / h (i.e. 1.5mL / min), 2.5BV / h (i.e. 1.9mL / min) and 3.0BV / h (i.e. 2.3mL / min), respectively. The eluate was collected, the content was determined, and the elution rate of the four components was calculated; after sampling and evaporation, the solid mass was obtained, and the content of the four components was calculated. The results are shown in Table 15.

[0203] Table 15 Elution effect of 4 index substances at different elution flow rates

[0204]

[0205] It can be seen from the results in Table 15 that an elution flow rate that is too fast or too slow is not conducive to the elution of scutellaria baicalin, baicalin and wogonin. When the elution flow rate is too fast, the contact time between the eluent and the macroporous resin becomes shorter, and the substances adsorbed by the resin cannot be fully replaced, resulting in incomplete desorption; when the elution flow rate is too slow, it is particularly affected on scutellaria baicalin with greater polarity, and the elution rate and content are significantly reduced. It is possible that in the process of slow elution, other impurity liquids with relatively greater polarity are also desorbed in full contact with the eluent, reducing the selectivity of desorption and reducing the overall effect of the desorption index components. For wogonin, the elution rate and content both increased with the increase of elution flow rate. This is because wogonin is easily adsorbed by HPD100 macroporous resin, and the eluent with 60% ethanol concentration is not suitable for the desorption of wogonin. When the elution flow rate is fast, wogonin can be taken away by the solvent before being re-adsorbed, so the elution effect is improved. In summary, for baicalin and wogonin, 1BV / h~3BV / h is selected as the elution flow rate.

[0206] 3.2.3.6. Water washing volume investigation

[0207] A certain amount of pretreated HPD100 macroporous resin was wet packed into a column (inner diameter 1.8 cm) with a diameter-to-height ratio of 1:10. The sample was loaded at a loading concentration of 40 mg / mL, a loading speed of 2.0 BV / h (i.e., 1.5 mL / min), and a loading volume of 200 mL. After loading, 1 BV, 2 BV, 3 BV, and 4 BV of water were used to wash the column, respectively, at a washing speed of 1.5 mL / min, and then 3 BV of 60% (v / v) ethanol was used as an eluent at a speed of 2.0 BV / h (i.e., 1.5 mL / min). The eluent was collected, the content was determined, and the elution rates of the four components were calculated. The solid mass was obtained after sampling and evaporation, and the contents of the four components were calculated. The results are shown in Table 16.

[0208] Table 16 Elution effect of 4 indicator substances at different washing volumes

[0209]

[0210] From the results in Table 16, it can be seen that the increase in the washing volume cannot further increase the content of the four components in the eluent, and even the content decreases due to the decrease in the elution rate; the reason why the increase in the washing volume reduces the elution rate is that excessive washing will cause the loss of the four components. In summary, for baicalin and wogonin, the washing volume after loading is selected to be 1BV to 4BV.

[0211] 3.2.3.7. Elution curve drawing

[0212] Take a certain amount of pretreated HPD100 macroporous resin wet column (inner diameter 1.8cm), diameter-to-height ratio 1:10, load the sample at a loading concentration of 40mg / mL, a loading speed of 2.0BV / h (i.e., 1.5mL / min), and a loading volume of 200mL. After loading, wash with 2BV of water at a washing speed of 1.5mL / min, then elute with 275mL (about 6BV) of 60% (v / v) ethanol eluent at a speed of 2.0BV / h (i.e., 1.5mL / min). Collect one portion of the eluent every 10mL, for a total of 28 portions, determine the concentrations of the four components in each portion, and draw an elution curve when eluting with 60% ethanol, as shown in the figure. Figure 5 shown.

[0213] Depend on Figure 5 It can be seen that almost no components were eluted in the first 40mL; the 5th to 15th tubes, i.e., the 50th to 150mL, are the main elution intervals of scutellarin and baicalin. After reaching the peak in the 6th tube, the concentration slowly decreases. After the eluent is collected in the 16th tube, scutellarin is basically eluted completely. After the 18th tube, baicalin is basically eluted completely; the 5th to 17th tubes, i.e., 50th to 170mL, are the main elution intervals of baicalin. After reaching the peak in the 6th tube, the concentration slowly decreases. After the eluent is collected in the 19th tube, baicalin is basically eluted completely; the elution curve of baicalin has no peak, but the concentration slowly increases from 0 to 0.036 mg / mL in the 28th tube. Therefore, the volume of 60% ethanol eluent is set to 160mL, which can basically completely elute scutellarin, baicalin and baicalin.

[0214] The experiment was carried out according to the same loading and washing conditions as above, and then eluted with 160 mL of 60% (v / v) ethanol eluent at a rate of 2.0 BV / h (i.e., 1.5 mL / min), and then eluted with 140 mL (about 3 BV) of 90% (v / v) ethanol eluent. One portion of 10 mL was collected, for a total of 14 portions, and the concentrations of the four components in each portion were determined. The elution curve when eluted with 90% ethanol was plotted, as shown in FIG. Figure 6 shown.

[0215] Depend on Figure 6It can be seen that after elution with 160mL of 60% ethanol, baicalin has been completely eluted, and the remaining small amount of baicalin and wogonin can be further eluted by 90% ethanol; the 3rd to 11th tubes, i.e. 30-110mL, are the main elution intervals of baicalin, and when the eluent is collected in the 13th tube, baicalin is basically eluted completely. Considering the elution curve, operation time and cost, the volume of 90% ethanol eluent is set to 100mL, which can basically completely elute baicalin. In summary, using 160mL of 60% ethanol and 100mL of 90% ethanol to elute the main components of Scutellaria baicalensis adsorbed by macroporous resin can achieve a better elution effect.

[0216] 3.2.4. Macroporous adsorption resin purification process verification

[0217] A certain amount of pretreated HPD100 macroporous resin was wet packed into a column (inner diameter 1.8 cm) with a diameter-to-height ratio of 1:10. The sample was loaded at a loading concentration of 40 mg / mL, a loading speed of 2.0 BV / h (i.e., 1.5 mL / min), and a loading volume of 200 mL. After loading, 2 BV of water was used for washing at a washing speed of 1.5 mL / min. Subsequently, 160 mL (about 3.5 BV) of 60% (v / v) ethanol eluent was used at a speed of 2.0 BV / h (i.e., 1.5 mL / min), and then 100 mL (about 2.2 BV) of 90% (v / v) ethanol eluent was used at a speed of 2.0 BV / h (i.e., 1.5 mL / min). The eluent was collected, the content was determined, and the elution rates of the four components were calculated. The solid mass was obtained after sampling and evaporation, and the contents of the four components were further calculated.

[0218] According to this process, the experiment was carried out in parallel for 3 times. The transfer rates of scutellarin, baicalin and wogonin compared with the extract were all above 95%, and the transfer rate of wogonin was above 85%. The contents of scutellarin, baicalin, wogonin and baicalin in the eluent were 0.68%, 55%, 8.5% and 0.48%, respectively, and the process stability was good. It can be seen that compared with the acid precipitation method, the use of macroporous adsorption resin for preliminary purification of Scutellaria baicalensis extract can better recover the index components from the extract while effectively removing impurities. Considering that the acid precipitation method has a higher purification selectivity for flavonoid components in Scutellaria baicalensis, it is planned to use the acid precipitation method to further purify the eluent after purification by macroporous adsorption resin to investigate its purification effect.

[0219] 3.3. Study on acid precipitation purification process

[0220] 3.3.1. Investigation of acid precipitation solution concentration

[0221] The macroporous adsorption resin purified eluate obtained with the optimal process parameters was concentrated into a 100 (crude drug) mg / mL drug solution, which was diluted to 100, 80, 60, and 40 mg / mL for acid precipitation. Under constant stirring, hydrochloric acid was slowly added to adjust the pH of the drug solution to 1.4-1.6, and the solution was kept at 80°C for 1 hour, placed in a refrigerator at 6°C for 12 hours, filtered to separate the precipitate, and the precipitate was washed with 30 mL of water and 50% ethanol respectively until the pH value was neutral, the ethanol was evaporated, and then dried in an oven at 50°C to constant weight, and the mass was weighed to obtain the scutellaria extract. The scutellaria extract, baicalin, wogonin, and baicalin were quantitatively analyzed by HPLC. The quality evaluation indexes of the scutellaria extract were measured according to "1. Analysis method of scutellaria extract".

[0222] According to the experimental results, the quality of the solid product obtained after acid precipitation purification increases with the increase of the concentration of the drug solution. The changes in the yield, content and transfer rate of each component are as follows: Figure 7 shown.

[0223] For baicalin, as the concentration of the drug solution increased, the yield and transfer increased first, reaching the maximum when the drug solution concentration was 80 mg / mL, with the yield and transfer rate being 0.1537% and 69.59% respectively, and then decreasing; the change in content was more complicated, with no obvious regularity. When the drug solution concentration was 100 mg / mL, the content of baicalin in the Scutellaria baicalensis extract was the lowest, at 0.9075%, and the content was the highest, at 80 mg / mL, at 0.9808%.

[0224] For baicalin, when the concentration of the drug solution increased from 40mg / mL to 60mg / mL, the yield and transfer rate increased significantly, and then slowly increased with the increase of the drug solution concentration. When the drug solution concentration was 100mg / mL, the yield and transfer rate were 13.94% and 79.53%, respectively; the content of baicalin in the Scutellaria baicalensis extract first decreased with the increase of the drug solution concentration, reaching the highest level of 93.48% when the drug solution concentration was 40mg / mL, and the lowest level of 88.64% when the drug solution concentration was 80mg / mL, and then rebounded. Referring to the changes in the contents of the other three components, it can be seen that the reason why the content of baicalin is low at 80mg / mL may be that the contents of the other three components are high at this drug solution concentration.

[0225] For baicalin, the yield, content and transfer rate showed basically the same trend of change. They all increased significantly when the concentration of the drug solution increased from 40 mg / mL to 60 mg / mL, and then reached the maximum value when the concentration of the drug solution increased to 80 mg / mL. The yield, content and transfer rate were 1.39%, 8.90% and 42.24%, respectively. They all decreased when the concentration of the drug solution reached 10 mg / mL.

[0226] The yield, content and transfer rate of wogonin showed the same change pattern as baicalin, reaching the maximum value when the concentration of the drug solution was 80 mg / mL, and the yield, content and transfer rate were 0.0927%, 0.5921% and 38.98% respectively.

[0227] Compared with baicalin and baicalin, wogonin and baicalein have greater losses after two-step purification by macroporous resin and acid precipitation. The transfer rate from the extract to the final extract is only about 40%, and the loss is mainly caused by acid precipitation. Therefore, acid precipitation is not very conducive to the purification of these two components.

[0228] 3.3.2. Acid precipitation purification process verification

[0229] When the concentration of the drug solution is 80 (crude drug) mg / mL, except for baicalin, the other three components can reach the relatively optimal yield, content and transfer rate. This process was carried out in parallel for three times. The experimental results are shown in Table 17. The process stability is good, and the contents of baicalin, wogonin and baicalein are effectively increased. The baicalin content can reach more than 88%.

[0230] Table 17 Process verification when the acid precipitation solution concentration is 80 mg / mL

[0231]

[0232] If the baicalin content is to be above 90%, and taking into account the concentration of the eluent, the acid precipitation solution concentration of 60 (raw drug) mg / mL can be selected. This process was used for three parallel experiments. The experimental results are shown in Table 18. The process can basically meet the requirement of a baicalin content of more than 90%, but the yields and contents of the other three components were reduced.

[0233] Table 18 Process verification when the acid precipitation solution concentration is 60 mg / mL

[0234]

[0235] Although baicalin has the greatest impact on the efficacy of Scutellaria baicalensis extract, the higher the content, the better. Increasing the content of other flavonoid components is beneficial to improving the overall efficacy. The present invention uses a method of purifying by macroporous adsorption resin and acid precipitation, wherein the concentration of the acid precipitation liquid is 55mg / mL to 85mg / mL, preferably 60mg / mL to 80mg / mL, which can effectively and significantly increase the yield and content of wogonin while ensuring the content and yield of baicalin.

[0236] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, changes or deformations made by those skilled in the art based on the ideas of the present invention, the specific implementation methods and application scope of the present invention, all belong to the scope of protection of the present invention. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A method for increasing the content of baicalin and wogonin in a scutellaria baicalensis extract, characterized in that: The method comprises the following steps: (1) preparing a scutellaria baicalensis extract: weighing an appropriate amount of scutellaria baicalensis powder, performing reflux extraction with water at a first liquid-to-solid ratio, centrifuging and obtaining a supernatant to obtain a first extract; performing reflux extraction with alcohol at a second liquid-to-solid ratio on the obtained medicinal residue, centrifuging and obtaining a supernatant, and recovering ethanol until there is no alcohol taste to obtain a second extract; combining the first extract and the second extract to obtain the scutellaria baicalensis extract; (2) Loading: Accurately weigh an appropriate volume of Scutellaria baicalensis extract, dilute with water to obtain a macroporous resin loading solution, and pass the macroporous resin loading solution through a pretreated macroporous resin adsorption column at a certain flow rate so that the macroporous resin loading solution is adsorbed on the macroporous resin adsorption column; (3) Collecting the eluate: using water and 50% to 70% ethanol solution to elute at a certain flow rate, collecting the eluate and combining them to obtain a total eluate; as well as Optionally, (4) acid precipitation purification: after the total eluate is prepared into a solution of a certain mass concentration, acid is added to adjust the pH, the solution is stored at an appropriate temperature, and then placed in a refrigerator to stand, and the precipitate is filtered and separated, and the precipitate is washed with water and ethanol until the pH is neutral, and after the ethanol is evaporated until there is no alcohol smell, the precipitate is placed in an oven and dried to constant weight to obtain the Scutellaria baicalensis extract; Wherein, the effective ingredients include baicalin and wogonin.

2. The method according to claim 1, characterized in that: In step (1), the method for preparing the radix scutellariae medicinal material powder comprises the following steps: weighing an appropriate amount of radix scutellariae medicinal pieces, drying them in an oven to a constant weight, and then crushing them with a traditional Chinese medicine grinder, collecting them, and sieving them to obtain the radix scutellariae medicinal material powder; Preferably, the temperature of the oven is 45°C to 55°C, for example, about 50°C; More preferably, the sieve is a 50 mesh sieve; Preferably, the water extraction time is 35 to 45 minutes; Particularly preferably, the first liquid-to-solid ratio is 16 to 18; Particularly preferably, the alcohol is ethanol; Particularly preferably, the concentration of the ethanol solution is 35% to 45%; Particularly preferably, the extraction temperature of the alcohol extraction is 78°C to 82°C; Particularly preferably, the alcohol extraction time is 40 min to 50 min, for example, about 45 min; Most preferably, the second liquid-to-solid ratio is 14-16.

3. The method according to claim 1, characterized in that In step (2), the macroporous resin is HPD100 resin, AB-8 resin, D101 resin or D140 resin; Preferably, the macroporous resin is HPD100 resin or AB-8 resin; Preferably, the pretreatment comprises soaking the macroporous resin in an ethanol solution, removing floating debris and impurities, wet-packing the column, and eluting with an ethanol solution and water in sequence; More preferably, the concentration of the ethanol solution is about 95%, and the immersion time of the ethanol solution is about 24 hours; More preferably, the water is deionized water; Particularly preferably, the concentration of the loading solution is 20 mg / ml to 100 mg / ml, for example about 40 mg / ml; Particularly preferably, the flow rate of the loading liquid is 1 BV / h to 3 BV / h, for example, about 2 BV / h.

4. The method according to claim 1, characterized in that: In step (3), the volume of the water elution is 1 BV to 4 BV; Preferably, the flow rate of the water elution is 1 BV / h to 3 BV / h, for example, about 2 BV / h (1.5 mL / min); More preferably, the volume of the 50% to 70% ethanol solution elution is 3 BV to 4 BV, such as about 3.5 BV; More preferably, the flow rate of the 50% to 70% ethanol solution elution is 1 BV / h to 3 BV / h; Optionally, step (3) further comprises volatilizing the ethanol in the total eluate until there is no alcohol smell, and then drying it in an oven to constant weight to obtain the Scutellaria baicalensis extract.

5. The method according to claim 1, characterized in that The method does not include the following steps: after the total eluate is configured into a solution of a certain mass concentration, acid is added to adjust the pH, the solution is stored at an appropriate temperature, the solution is placed in a refrigerator to stand, filtration is performed to separate the precipitate, and the precipitate is washed with water and ethanol until the pH is neutral.

6. The method according to claim 1, characterized in that In step (4), the mass concentration of the drug solution is 40 mg / ml to 100 mg / ml, preferably 55 mg / ml to 85 mg / ml, preferably 60 mg / ml to 80 mg / ml, for example about 60 mg / ml or about 80 mg / ml; Preferably, the acid is hydrochloric acid; Preferably, the acid addition is dropwise addition of acid under continuous stirring; Preferably, the pH is 1 to 2, such as 1.4 to 1.6; More preferably, the temperature is 75°C to 85°C, such as about 80°C; Preferably, the storage time is 0.5 hours to 1.5 hours, for example, about 1 hour; Also preferably, the temperature of the refrigerator is 4°C to 10°C, for example, about 6°C; Particularly preferably, the standing time is 10 to 15 hours, for example, about 12 hours; Particularly preferably, the concentration of ethanol is 45% to 55%, for example about 50%; Most preferably, the temperature of the oven is 45°C to 55°C, such as about 50°C.

7. A Scutellaria baicalensis extract prepared by the method according to any one of claims 1 to 6.

8. A pharmaceutical composition comprising the Scutellaria baicalensis extract according to claim 7; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient; More preferably, the excipient is selected from one or more of the following: dispersants, wetting agents, binders, diluents, retention aids, lubricants, sustained-release agents, plasticizers, disintegrants, inclusion agents, flavoring agents, opacifiers and antioxidants; Also preferably, the pharmaceutical composition is in the form of tablets, pills, capsules, powders, injections, films, lozenges, granules or oral solutions.

Images