Shuanghuanglian extract, preparation method, application and related products

Abstract

The application provides a Shuanghuanglian extract, a preparation method, application and related products, and belongs to the technical field of traditional Chinese medicine preparations. The preparation method of the Shuanghuanglian extract comprises the following steps: S1, mixing honeysuckle and forsythia with a solvent, ultrasonic extraction, separation, and obtaining mixed dregs and ultrasonic extraction liquid; S2, performing carbon dioxide supercritical extraction on scutellaria, and obtaining scutellaria dregs and scutellaria extract; S3, mixing the mixed dregs obtained in the step S1, the scutellaria dregs obtained in the step S2 and an enzyme buffer, performing enzymolysis reaction, and performing molecular interception with a molecular interception membrane, and obtaining an enzymolysis extraction liquid; S4, mixing the ultrasonic extraction liquid obtained in the step S1 after solvent recovery under reduced pressure, the scutellaria extract obtained in the step S2 and the enzymolysis extraction liquid obtained in the step S3, sterilizing, and drying, and obtaining the Shuanghuanglian extract. The Shuanghuanglian extract prepared by the method has better antioxidant effect and antipyretic effect, and is suitable for the preparation of Shuanghuanglian injection.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine preparation technology, and relates to a Shuanghuanglian extract, its preparation method, its application, and related products. Background Technology

[0002] Shuanghuanglian is a classic traditional Chinese medicine formula. The traditional Shuanghuanglian formula contains three Chinese medicinal herbs: honeysuckle, scutellaria baicalensis, and forsythia. Shuanghuanglian has the effects of clearing heat and detoxifying, and clearing wind-heat. Currently, Shuanghuanglian has been developed into various dosage forms. The Chinese Pharmacopoeia (2020 edition, Part I) alone records Shuanghuanglian oral liquid, Shuanghuanglian tablets, Shuanghuanglian suppositories, Shuanghuanglian capsules, Shuanghuanglian granules, Shuanghuanglian eye drops, and Shuanghuanglian for injection (lyophilized).

[0003] Chinese invention patent CN109172643A provides a Shuanghuanglian injection and its preparation process, comprising: cooling the aqueous extract of honeysuckle and forsythia to ≤30℃, slowly adding ethanol with a concentration of 93% or higher while stirring, so that the alcohol content reaches 75%-80%, stirring thoroughly, maintaining the system temperature at ≤30℃, and letting it stand for more than 12 hours; making a concentrated paste from the supernatant; wherein the ethanol is added at a rate of 50-60 L / min; adding an appropriate amount of purified water to the concentrated paste, stirring thoroughly and heating to boiling, letting it stand for more than 48 hours, taking the supernatant and filtering; centrifuging the filtrate at high speed, taking the supernatant and making a concentrated paste; taking the scutellaria baicalensis extract and the concentrated paste, adding an appropriate amount of water for injection and activated carbon, decarbonizing and filtering, and then performing ultrafiltration using a polyethersulfone tangential flow ultrafiltration membrane with a molecular weight cutoff of 8000-12000; further preparing Shuanghuanglian injection from the obtained ultrafiltrate.

[0004] Chinese invention patent CN118453523A discloses a freeze-dried Shuanghuanglian powder for injection, its preparation method, and its application. The Shuanghuanglian extract of this technology is obtained through a process of two fermentations, ionic liquid enzymatic hydrolysis, and ethanol reflux, resulting in a Shuanghuanglian extract suitable for preparing nasal spray solutions.

[0005] However, current technology has not yet been able to provide a Shuanghuanglian extract with better antioxidant and antipyretic effects, while also being suitable for preparing injectable solutions. Summary of the Invention

[0006] In view of the above-mentioned problems with the prior art, the purpose of this invention is to provide a Shuanghuanglian extract, its preparation method, its application, and related products.

[0007] To achieve the above-mentioned objectives, the present invention provides a method for preparing Shuanghuanglian extract, comprising the following steps:

[0008] S1. Mix honeysuckle and forsythia with solvent, extract by ultrasound, and separate to obtain mixed residue and ultrasound extract;

[0009] S2. Scutellaria baicalensis was subjected to supercritical carbon dioxide extraction to obtain Scutellaria baicalensis residue and Scutellaria baicalensis extract;

[0010] S3. Mix the mixed residue obtained in step S1 and the Scutellaria baicalensis residue obtained in step S2 with enzyme buffer, perform enzymatic hydrolysis reaction, and retain the residue with molecular retention membrane to obtain enzymatic hydrolysis extract.

[0011] S4. After the ultrasonic extract obtained in step S1 is depressurized and the solvent is recovered, it is mixed with the Scutellaria baicalensis extract obtained in step S2 and the enzymatic hydrolysis extract obtained in step S3, sterilized, and dried to obtain Shuanghuanglian extract.

[0012] In step S1, the solvent is selected from at least one of acetone, methanol, ethanol and water.

[0013] In step S2, the conditions for supercritical carbon dioxide extraction are: temperature 40-50℃, pressure 25-35MPa, and entrainer is ethanol;

[0014] In step S3, the enzyme buffer comprises the following components: snail enzyme, cellulase, hemicellulase, sorbitol, lactic acid, and water, and the pH of the enzyme buffer is 5-6.5; the molecular weight cutoff range is ≤10000 Da.

[0015] Furthermore, the weight ratio of honeysuckle and forsythia in step S1 to scutellaria in step S2 is 3-13:2-9:8-11.

[0016] Furthermore, and as a specific embodiment of the present invention, the weight ratio of honeysuckle and forsythia in step S1 to scutellaria in step S2 is 8:5.5:9.5.

[0017] Further, in step S1, the ratio of the total weight of honeysuckle and forsythia to the volume of solvent is 1:8-10, in g:mL; the ultrasonic extraction time is 1-2 hours, the ultrasonic extraction temperature is 40-50℃, and the ultrasonic extraction power is 50-100W.

[0018] Furthermore, and as a specific embodiment of the present invention, in step S1, the ratio of the total weight of honeysuckle and forsythia to the volume of solvent is 1:9, in g:mL; the ultrasonic extraction time is 1.5h, the ultrasonic extraction temperature is 45℃, and the ultrasonic extraction power is 80W.

[0019] Furthermore, in step S2, the conditions for supercritical carbon dioxide extraction are: temperature 45°C, pressure 30 MPa, and entrainer is ethanol with a content of 4%-8%.

[0020] Furthermore, in step S2, the entrainer is 6% ethanol.

[0021] Further, in step S3, the ratio of the total weight of the mixed residue and the Scutellaria baicalensis residue to the volume of the enzyme buffer is 1:6-15, in g:mL; in the enzyme buffer, the concentration of the snail enzyme is 4-10 g / L, the concentration of the cellulase is 50-150 U / mL, the concentration of the hemicellulase is 10-30 U / mL, the weight concentration of the sorbitol is 20-50 g / L, and the concentration of the lactic acid is 20-40 g / mL; the conditions for the enzymatic hydrolysis reaction are: hydrolysis time 2-8 h, hydrolysis temperature 30-45℃, and shaking speed of the shaker 20-80 rpm.

[0022] Furthermore, in step S3, the ratio of the total weight of the mixed residue and the Scutellaria baicalensis residue to the volume of the enzyme buffer is 1:10, in g:mL; the concentration of snail enzyme in the enzyme buffer is 7 g / L, the concentration of cellulase is 100 U / mL, the concentration of hemicellulase is 20 U / mL, the weight concentration of sorbitol is 35 g / L, and the concentration of lactic acid is 30 g / L; the conditions for the enzymatic hydrolysis reaction are: hydrolysis time 4 h, hydrolysis temperature 40 °C, and shaking speed of the shaker 50 rpm.

[0023] Furthermore, in step S4, the sterilization is wet heat sterilization, and the drying is freeze drying.

[0024] On the other hand, the present invention provides a Shuanghuanglian extract prepared by the above-described preparation method.

[0025] Furthermore, this invention provides the application of the above-mentioned preparation method in the production of Shuanghuanglian medicine.

[0026] In another aspect, the present invention provides a medicine in which the active ingredient includes the above-mentioned Shuanghuanglian extract.

[0027] Finally, the present invention provides a Shuanghuanglian injection, comprising the above-mentioned Shuanghuanglian extract and a medically acceptable solvent.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] This invention provides a method for preparing a Shuanghuanglian extract. By optimizing the extraction process of three medicinal materials—honeysuckle, forsythia, and scutellaria—a Shuanghuanglian extract with better antioxidant and antipyretic effects was obtained. This Shuanghuanglian extract is suitable for preparing Shuanghuanglian injection, and the resulting Shuanghuanglian injection meets all the quality control indicators required by the pharmacopoeia. Detailed Implementation

[0030] Terminology and Declarations of this Invention:

[0031] 1. Articles “a,” “a kind,” and “the”: These include plural objects unless otherwise explicitly specified as a single (kind) object.

[0032] 2. Numerical Range: Unless otherwise expressly stated, all ranges or ratios disclosed herein shall be construed as including any and all subranges or subratios contained herein. For example, a stated range or ratio of 1 to 30 shall be considered to be included between the minimum value of 1 and the maximum value of 30, and includes any subranges or subratios, integers, decimals, or subranges or subratios consisting of integers or decimals, including endpoints.

[0033] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0034] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all chemical reagents used in the embodiments of the present invention were obtained through conventional commercial means. Unless otherwise specified, all contents mentioned below are mass contents. Unless otherwise specified, it is understood that the process was carried out at room temperature.

[0035] In the examples below, some of the reagents were sourced from Table 1:

[0036] Table 1

[0037]

[0038] The Chinese medicinal materials used in the following examples (honeysuckle, scutellaria, and forsythia) are all granular Chinese medicinal materials that have been crushed and passed through a 5-mesh sieve.

[0039] Example 1

[0040] A method for preparing Shuanghuanglian extract, comprising the following steps:

[0041] S1. Mix 80g of honeysuckle, 55g of forsythia, and a 50% (w / w) acetone aqueous solution (the total weight of honeysuckle and forsythia to the volume ratio of solvent is 1:9g / mL). Sonicate for 1.5 hours, controlling the ultrasonic extraction temperature at 45℃ and the ultrasonic power at 80W. After ultrasonic extraction, filter; the residue is a mixed herbal residue, and the filtrate is the ultrasonic extract.

[0042] S2. 95g of Scutellaria baicalensis was subjected to supercritical carbon dioxide extraction. The supercritical carbon dioxide extraction temperature was 45℃, the pressure was 30MPa, the entrainer was ethanol (6% by weight of Scutellaria baicalensis), and the extraction time was 3h. After the supercritical carbon dioxide extraction was completed, the liquid was distilled at atmospheric pressure to recover ethanol as Scutellaria baicalensis extract, and the solid was Scutellaria baicalensis residue.

[0043] S3. Mix the mixed residue obtained in step S1 with the Scutellaria baicalensis residue obtained in step S2, and weigh them. Mix the enzyme buffer at a ratio of (mixed residue + Scutellaria baicalensis residue): enzyme buffer = 1:10 g / mL. Incubate the enzymatic hydrolysis reaction for 4 hours at a constant temperature of 40℃ and a shaking speed of 50 rpm.

[0044] The preparation method for enzyme buffer is as follows:

[0045] Take 7g of snail enzyme, 100,000U of cellulase, 20,000U of hemicellulase, 35g of sorbitol, and 30g of lactic acid, mix them, add 600mL of water, and stir until fully dissolved. Adjust the pH of the solution to 6 using a 5% sodium hydroxide aqueous solution, and bring the volume to 1L to obtain the enzyme buffer.

[0046] After the enzymatic hydrolysis reaction is completed, the filtrate is filtered and a molecular weight cutoff membrane (10000Da, Solarbio) is used to retain components with a molecular weight ≤10000Da, which is recorded as the enzymatic hydrolysis extract.

[0047] S4. The ultrasonic extract obtained in step S1 is subjected to reduced pressure to recover the solvents acetone and water. Then, it is mixed with the Scutellaria baicalensis extract obtained in step S2 and the enzymatic hydrolysis extract obtained in step S3 at 400 rpm for 10 min. The mixture is then sterilized by 0.45 μm filter membrane and freeze-dried to obtain the Shuanghuanglian extract.

[0048] Example 2

[0049] Compared with Example 1, the difference is that in step S1, honeysuckle is replaced with 30g and forsythia with 90g, and in step S2, scutellaria is replaced with 110g. All other steps are the same.

[0050] Example 3

[0051] Compared with Example 1, the difference is that in step S1, honeysuckle is replaced with 130g and forsythia with 20g, and in step S2, scutellaria is replaced with 80g. All other steps are the same.

[0052] Example 4

[0053] Compared with Example 1, the difference lies in step S2, where the conditions for supercritical carbon dioxide extraction are 50°C, 25MPa, and 2h, and the entrainer is ethanol with a weight content of 8% of Scutellaria baicalensis. All other conditions are the same.

[0054] Example 5

[0055] Compared with Example 1, the difference lies in step S2, where the conditions for supercritical carbon dioxide extraction are 40°C, 35MPa, and 4h, and the entrainer is ethanol at 4% by weight of Scutellaria baicalensis. All other conditions are the same.

[0056] Example 6

[0057] Compared with Example 1, the difference lies in the preparation method of the enzyme buffer in step S3, which is changed as follows, while the rest are the same.

[0058] The preparation method for enzyme buffer is as follows:

[0059] Take 10g of snail enzyme, 50000U of cellulase, 30000U of hemicellulase, 20g of sorbitol, and 40g of lactic acid, mix them, add 600mL of water, and stir until fully dissolved. Adjust the pH of the solution to 5.5 using a 5% sodium hydroxide aqueous solution, and bring the volume to 1L to obtain the enzyme buffer.

[0060] Example 7

[0061] Compared with Example 1, the difference lies in the preparation method of the enzyme buffer in step S3, which is changed as follows, while the rest are the same.

[0062] The preparation method for enzyme buffer is as follows:

[0063] Take 4g of snail enzyme, 150,000U of cellulase, 10,000U of hemicellulase, 50g of sorbitol, and 20g of lactic acid, mix them, add 600mL of water, and stir until fully dissolved. Adjust the pH of the solution to 6.5 using a 5% sodium hydroxide aqueous solution, and bring the volume to 1L to obtain the enzyme buffer.

[0064] Example 8

[0065] Compared with Example 1, the difference is that in step S3, the conditions of the enzymatic hydrolysis reaction are changed to 8 hours of enzymatic hydrolysis reaction, constant temperature of 30°C, and shaking speed of 80 rpm, while the rest are the same.

[0066] Example 9

[0067] Compared with Example 1, the difference is that in step S3, the conditions of the enzymatic hydrolysis reaction are changed to 2 hours of enzymatic hydrolysis reaction, constant temperature of 45°C, and shaking speed of 20 rpm, while the rest are the same.

[0068] Comparative Example 1

[0069] Compared with Example 1, the difference is that in step S1, honeysuckle is replaced with 30g and forsythia with 20g, and in step S2, scutellaria is replaced with 180g. All other steps are the same.

[0070] Comparative Example 2

[0071] Compared with Example 1, the difference is that in step S2, acetone in an equal weight ratio is used as the entrainer, and after the supercritical carbon dioxide extraction is completed, acetone is recovered instead. All other steps are the same.

[0072] Comparative Example 3

[0073] Compared with Example 1, the difference is that in step S2, the temperature of supercritical carbon dioxide extraction is changed to 35°C and the pressure is 40MPa, while the rest are the same.

[0074] Comparative Example 4

[0075] Compared with Example 1, step S3 is omitted, and step S4 is changed to directly mixing the ultrasonic extract obtained in step S1 and the Scutellaria baicalensis extract obtained in step S2, sterilizing with a 0.45 μm filter membrane, and freeze-drying to obtain Shuanghuanglian extract.

[0076] Comparative Example 5

[0077] The freeze-dried powder of Shuanghuanglian for injection was prepared according to the preparation method under the item "Shuanghuanglian for Injection (lyophilized)" in the Chinese Pharmacopoeia (2020 edition, Part I).

[0078] Effect evaluation

[0079] 1. Evaluation of the antioxidant effect of Shuanghuanglian for injection.

[0080] The DPPH method was used as an evaluation method for antioxidant effects to compare the antioxidant effects of Shuanghuanglian freeze-dried powder provided in each example and comparative example.

[0081] The structural formula of DPPH is as follows:

[0082]

[0083] The experimental method is as follows:

[0084] (1) Accurately weigh the DPPH reagent, dissolve it in anhydrous ethanol, prepare a standard solution (50 mg / L), and store it in the dark.

[0085] (2) Take the freeze-dried Shuanghuanglian powder of each example and comparative example and mix it with deionized water to prepare a 100mg / L Shuanghuanglian solution.

[0086] (3) Three sets of experiments were conducted for each example / comparative example.

[0087] Group A: Mix 2 mL of DPPH solution with 2 mL of deionized water.

[0088] Group B: Mix 2 mL of Shuanghuanglian solution with 2 mL of anhydrous ethanol.

[0089] Group C: Mix 2 mL of DPPH solution with 2 mL of Shuanghuanglian solution.

[0090] After standing at room temperature in the dark for 30 minutes, the absorbance was measured at 517 nm using a spectrophotometer. The absorbances measured for groups a, b, and c were denoted as A0, A10, A20, A30, A40, A50, A60, A70, A80, A90, A1 ... 样品 And A2.

[0091] DPPH removal rate = [A0 - (A 样品 -A2)]÷A0×100%.

[0092] The DPPH scavenging rates of each embodiment and comparative example are shown in Table 2 below.

[0093] Table 2

[0094]

[0095] It is evident that the antioxidant effect of the Shuanghuanglian freeze-dried powder provided in the various embodiments of the present invention is significantly higher than that of the Shuanghuanglian freeze-dried powder provided in the comparative examples.

[0096] 2. Evaluation of the antipyretic effect of Shuanghuanglian injection.

[0097] Four-week-old male Wistar rats were randomly divided into groups of seven.

[0098] Blank control group and modeling control group: physiological saline solution was injected into the tail vein daily for 5 consecutive days.

[0099] Each example group: The lyophilized Shuanghuanglian powder of each example group was diluted with an appropriate amount of physiological saline to prepare Shuanghuanglian injection solution. 9g / kg of Shuanghuanglian injection solution (based on the crude drug dosage) was administered to the rat via tail vein daily for 5 consecutive days.

[0100] Comparative control groups: The lyophilized Shuanghuanglian powder of each comparative control group was diluted with an appropriate amount of physiological saline to prepare Shuanghuanglian injection solution. Each rat was given a dose of 9 g / kg of raw Shuanghuanglian injection solution via tail vein injection daily for 5 consecutive days.

[0101] Five days after drug administration, except for the blank control group, rats in each group were subcutaneously injected with 20% yeast solution (prepared by mixing Angel high-activity dry yeast with 4 times its weight of water) at a dose of 1 mL / 100g rat body weight to induce fever. The blank control group was subcutaneously injected with physiological saline. The changes in rat body temperature (rectal temperature was measured by inserting a temperature sensor probe into the rat's anus, and reading the temperature value after 30-60 seconds when the temperature stabilized) within 12 hours after modeling were measured and recorded.

[0102] The temperature measurement results at 0h, 3h, 5h, 8h, and 12h are shown in Table 3 below (n=7, mean ± standard deviation, rounded to two decimal places):

[0103] Table 3

[0104]

[0105] As can be seen from the experimental data in Table 3, the antipyretic effect of the Shuanghuanglian lyophilized powder provided in each embodiment of the present invention is significantly better than that of the Shuanghuanglian lyophilized powder provided in each comparative example, and the difference is statistically significant (in Table 3, ...). a This indicates that the data in the same column show significant differences between each embodiment and each comparative example compared to the model control group (p < 0.05). In particular, the Shuanghuanglian freeze-dried powder provided in Example 1 has the characteristics of rapid antipyretic effect (significant effect is produced in 5 hours) and better antipyretic effect, while the Shuanghuanglian freeze-dried powders provided in Comparative Examples 1-5 take up to 8 hours to produce significant effect.

[0106] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing a Shuanghuanglian extract, characterized in that, The method comprises the following steps: S1, mixing honeysuckle and forsythia with a solvent, ultrasonic extraction, separation, obtaining mixed dregs and ultrasonic extraction liquid; S2, carbon dioxide supercritical extraction of scutellaria, obtaining scutellaria dregs and scutellaria extract; S3, mixing the mixed dregs obtained in step S1 and the scutellaria dregs obtained in step S2 with an enzyme buffer, enzymatic reaction, molecular cut-off membrane cut-off, obtaining enzymatic extraction liquid; S4, mixing the ultrasonic extraction liquid obtained in step S1 after solvent recovery under reduced pressure, the scutellaria extract obtained in step S2 and the enzymatic extraction liquid obtained in step S3, sterilization, drying, obtaining Shuanghuanglian extract; In step S1, the total weight of the honeysuckle and forsythia and the volume of the solvent are in a ratio of 1:8-10, unit g:mL; the ultrasonic extraction time is 1-2h, the ultrasonic extraction temperature is 40-50℃, the ultrasonic extraction power is 50-110W, and the solvent is a 50% weight concentration acetone aqueous solution; In step S2, the carbon dioxide supercritical extraction conditions are: temperature 45℃, pressure 30MPa, entrainer is 4%-8% ethanol based on the weight of scutellaria, and the extraction time is 3h; In step S3, the enzyme buffer comprises the following components: snail enzyme, cellulase, hemicellulase, sorbitol, lactic acid and water, the pH of the enzyme buffer is 5-6.5; the cut-off molecular weight is ≤10000Da; the total weight of the mixed dregs and the scutellaria dregs and the volume of the enzyme buffer are in a ratio of 1:6-15, unit g:mL; in the enzyme buffer, the concentration of the snail enzyme is 4-10g / L, the concentration of the cellulase is 50-150U / mL, the concentration of the hemicellulase is 10-30U / mL, the weight concentration of the sorbitol is 20-50g / L, and the concentration of the lactic acid is 20-40g / mL; the enzymatic reaction conditions are: enzymatic reaction time 2-8h, enzymatic reaction temperature 30-45℃, and shaking bed shaking speed 20-80rpm. In step S4, the drying is freeze-drying.

2. The production method according to claim 1, characterized by, 3. The Shuanghuanglian extract prepared by the preparation method in any one of claims 1-2. The effective component is the Shuanghuanglian extract in claim 3.

4. A medicament, characterized by comprising: The medicine comprises the Shuanghuanglian extract in claim 3 and a medically acceptable solvent.

5. A Shuanghuanglian injection, characterized in that, ​

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