Shuanghuanglian extract, preparation method, application and related products

Abstract

The invention 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, fructus forsythiae and a solvent, and performing ultrasonic extraction and separation to obtain mixed medicine residues and an ultrasonic extracting solution; s2, performing carbon dioxide supercritical extraction on scutellaria baicalensis to obtain scutellaria baicalensis dregs and a scutellaria baicalensis extract; s3, mixing the mixed decoction dregs obtained in the step S1 and the radix scutellariae decoction dregs obtained in the step S2 with an enzyme buffer solution, carrying out an enzymolysis reaction, and intercepting by a molecular interception membrane to obtain an enzymolysis extracting solution; and S4, recovering the solvent from the ultrasonic extracting solution obtained in the step S1 under reduced pressure, mixing with the scutellaria baicalensis extract obtained in the step S2 and the enzymolysis extracting solution obtained in the step S3, sterilizing, and drying to obtain the Shuanghuanglian extract. The Shuanghuanglian extract prepared by the method has better antioxidant effect and antipyretic effect, and is suitable for preparation of Shuanghuanglian injection.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine preparations and relates to a Shuanghuanglian extract, a preparation method, applications and related products. Background Art

[0002] Shuanghuanglian is a classic Chinese medicine formula. The traditional Shuanghuanglian formula contains three traditional Chinese medicinal ingredients: honeysuckle, scutellaria, and forsythia. Shuanghuanglian has the effects of clearing heat and detoxifying, and dispelling wind-heat. Currently, Shuanghuanglian has been developed into a variety of dosage forms. The Chinese Pharmacopoeia (2020 edition, Part I) alone lists 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 a preparation process thereof, comprising: cooling an aqueous extract of honeysuckle and forsythia to ≤30°C, slowly adding ethanol with a concentration of 93% or more, stirring while adding, until the alcohol content reaches 75%-80%, stirring thoroughly, maintaining the system temperature at ≤30°C, and standing for more than 12 hours; preparing the supernatant into a concentrated paste, 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, standing for more than 48 hours, collecting the supernatant, and filtering; centrifuging the filtrate at high speed, collecting the supernatant, and preparing 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 ultrafiltration using a polyethersulfone tangential flow ultrafiltration membrane with a molecular weight cutoff of 8000-12000; and further preparing the obtained ultrafiltrate into Shuanghuanglian injection.

[0004] Chinese invention patent CN118453523A provides a Shuanghuanglian freeze-dried powder for injection, its preparation method, and its application. This technology uses a double fermentation, ionic liquid enzymatic hydrolysis, and ethanol reflux process to extract the Shuanghuanglian extract, which is suitable for preparing a nasal spray solution.

[0005] However, the prior art has not yet provided a Shuanghuanglian extract that has better antioxidant and antipyretic effects and is suitable for preparing an injection solution. Summary of the Invention

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

[0007] To achieve the above object of the invention, on the one hand, the present invention provides a method for preparing a Shuanghuanglian extract, comprising the following steps: S1. Mixing honeysuckle and forsythia with a solvent, performing ultrasonic extraction, and separating to obtain a mixed medicinal residue and an ultrasonic extract; S2. performing supercritical carbon dioxide extraction on the scutellaria baicalensis to obtain scutellaria baicalensis residues and a scutellaria baicalensis extract; S3, mixing the mixed medicinal residue obtained in step S1 and the scutellaria baicalensis medicinal residue obtained in step S2 with an enzyme buffer, performing an enzymatic hydrolysis reaction, and intercepting the reaction with a molecular interception membrane to obtain an enzymatic hydrolysis extract; S4, after recovering the solvent from the ultrasonic extract obtained in step S1 by reducing the pressure, the extract was mixed with the scutellaria baicalensis extract obtained in step S2 and the enzymatic hydrolysis extract obtained in step S3, sterilized, and dried to obtain a Shuanghuanglian extract; Wherein, in step S1, the solvent is selected from at least one of acetone, methanol, ethanol and water; In step S2, the conditions for supercritical carbon dioxide extraction are: temperature 40-50°C, pressure 25-35 MPa, and ethanol as the entrainer; 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 molecular weight range of the cut-off is ≤10000Da.

[0008] Furthermore, the weight ratio of honeysuckle and forsythia in step S1 to scutellaria in step S2 is 3-13:2-9:8-11.

[0009] Furthermore, and as a specific embodiment of the present invention, the weight ratio of honeysuckle and forsythia in step S1 and scutellaria in step S2 is 8:5.5:9.5.

[0010] Furthermore, in step S1, the ratio of the total weight of the honeysuckle and forsythia to the volume of the solvent is 1:8-10, in units of g:mL; the time of the ultrasonic extraction is 1-2h, the temperature of the ultrasonic extraction is 40-50°C, and the power of the ultrasonic extraction is 50-100W.

[0011] Furthermore, and as a specific embodiment of the present invention, in step S1, the ratio of the total weight of the honeysuckle and forsythia to the volume of the solvent is 1:9, in units of g:mL; the time of the ultrasonic extraction is 1.5h, the temperature of the ultrasonic extraction is 45°C, and the power of the ultrasonic extraction is 80W.

[0012] Furthermore, in step S2, the conditions for the supercritical extraction of carbon dioxide are: temperature 45°C, pressure 30 MPa, and entrainer ethanol with a content of 4%-8%.

[0013] Furthermore, in step S2, the entrainer is 6% ethanol.

[0014] Furthermore, in step S3, the ratio of the total weight of the mixed medicinal residue and the scutellaria baicalensis medicinal residue to the volume of the enzyme buffer is 1:6-15, in g:mL; in the enzyme buffer, the concentration of the snailase 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 of the enzymatic hydrolysis reaction are: enzymatic hydrolysis time 2-8 h, enzymatic hydrolysis temperature 30-45°C, and shaking speed of the shaker is 20-80 rpm.

[0015] Furthermore, in step S3, the ratio of the total weight of the mixed medicinal residue and the Scutellaria baicalensis medicinal residue to the volume of the enzyme buffer is 1:10, and the unit is g:mL; the concentration of snailase in the enzyme buffer is 7g / L, the concentration of the cellulase is 100U / mL, the concentration of the hemicellulase is 20U / mL, the weight concentration of the sorbitol is 35g / L, and the concentration of the lactic acid is 30g / L; the conditions of the enzymatic hydrolysis reaction are: enzymatic hydrolysis time 4h, enzymatic hydrolysis temperature 40°C, and shaking speed of the shaker 50rpm.

[0016] Furthermore, in step S4, the sterilization is moist heat sterilization, and the drying is freeze drying.

[0017] On the other hand, the present invention provides a Shuanghuanglian extract prepared by the above preparation method.

[0018] In another aspect, the present invention provides application of the above-mentioned preparation method in the production of Shuanghuanglian medicine.

[0019] In another aspect, the present invention provides a medicine, the active ingredient of which includes the Shuanghuanglian extract.

[0020] In the final aspect, the present invention provides a Shuanghuanglian injection, comprising the Shuanghuanglian extract and a medically acceptable solvent.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for preparing a Shuanghuanglian extract. By optimizing the extraction process for three medicinal materials (honeysuckle, forsythia, and scutellaria), a Shuanghuanglian extract with enhanced antioxidant and antipyretic effects is obtained. This Shuanghuanglian extract is suitable for preparing Shuanghuanglian injection, and the resulting Shuanghuanglian injection meets all quality control indicators required by the pharmacopoeia. DETAILED DESCRIPTION

[0022] Terms and Claims of the Present Invention: 1. The articles "a", "an" and "the" include plural referents unless expressly limited to one or more referents otherwise.

[0023] 2. Numerical ranges: Unless expressly stated otherwise, all ranges or ratios disclosed herein are to be understood to include any and all subranges or subratios contained therein. For example, a range or ratio stated as 1 to 30 is to be considered inclusive of any and all subranges or subratios, integers, decimals, or subranges or subratios comprised therein, between a minimum of 1 and a maximum of 30, including any subranges or subratios, integers, decimals, or subranges or subratios comprised therein.

[0024] The following non-limiting examples are provided to enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way. The following are merely illustrative of the scope of the present invention, and those skilled in the art may make various changes and modifications to the present invention based on the disclosed content, which should also fall within the scope of the present invention.

[0025] The present invention will be further described below by way of specific examples. The various chemical reagents used in the examples of the present invention were obtained through conventional commercial channels unless otherwise specified. Unless otherwise specified, the contents described below are all by weight. Unless otherwise specified, it is understood that the experiments were conducted at room temperature.

[0026] In the following examples, some reagents are sourced from Table 1: Table 1

[0027] 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.

[0028] Example 1 A method for preparing a Shuanghuanglian extract comprises the following steps: S1. Mix 80 g of honeysuckle, 55 g of forsythia, and a 50% acetone aqueous solution (the volume ratio of the total weight of honeysuckle and forsythia to the solvent is 1:9 g / mL). Ultrasonicate for 1.5 hours at a temperature of 45°C and a power of 80 W. After ultrasonic extraction, filter the mixture, leaving the residue as the mixed residue and the filtrate as the ultrasonic extract.

[0029] S2. 95 g of Scutellaria baicalensis was subjected to supercritical carbon dioxide extraction. The supercritical carbon dioxide extraction temperature was 45°C and the pressure was 30 MPa. The entrainer was ethanol containing 6% of the weight of Scutellaria baicalensis. The supercritical carbon dioxide extraction time was 3 hours. After the supercritical carbon dioxide extraction was completed, the liquid was distilled at atmospheric pressure to recover the ethanol as the Scutellaria baicalensis extract, and the solid was the Scutellaria baicalensis residue.

[0030] S3. Mix the mixed medicinal residue obtained in step S1 and the scutellaria baicalensis residue obtained in step S2 and weigh them. Add enzyme buffer at a ratio of (mixed medicinal residue + scutellaria baicalensis residue): enzyme buffer = 1:10 g / mL. Incubate the enzymatic reaction for 4 hours at a constant temperature of 40°C and a shaking speed of 50 rpm.

[0031] The enzyme buffer is prepared as follows: 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 to fully dissolve. Use 5% sodium hydroxide aqueous solution to adjust the solution pH to 6 and dilute to 1L to obtain enzyme buffer.

[0032] After the enzymatic hydrolysis reaction was completed, the filtrate was filtered and a molecular cutoff membrane (10,000 Da, Solebol) was used to retain components with a molecular weight of ≤10,000 Da, which was recorded as the enzymatic extract.

[0033] S4. The ultrasonic extract obtained in step S1 is decompressed to recover the solvent acetone and water, and then the extract is mixed with the scutellaria baicalensis extract obtained in step S2 and the enzymatic hydrolysis extract obtained in step S3 under stirring at 400 rpm for 10 min, sterilized with a 0.45 μm filter membrane, and freeze-dried to obtain the Shuanghuanglian extract.

[0034] Example 2 Compared with Example 1, the difference is that in step S1, 30 g of honeysuckle and 90 g of forsythia are used instead, and in step S2, 110 g of scutellaria is used instead, and the rest are the same.

[0035] Example 3 Compared with Example 1, the difference is that in step S1, 130 g of honeysuckle and 20 g of forsythia are used instead, and in step S2, 80 g of scutellaria are used instead, and the rest are the same.

[0036] Example 4 Compared with Example 1, the difference is that in step S2, the conditions for supercritical carbon dioxide extraction are temperature 50°C, pressure 25 MPa, time 2 h, and the entrainer is ethanol with a weight content of 8% of Scutellaria baicalensis, and the rest are the same.

[0037] Example 5 Compared with Example 1, the difference is that in step S2, the conditions of carbon dioxide supercritical extraction are temperature 40°C, pressure 35 MPa, time 4 h, and the entrainer is ethanol with a weight content of 4% of Scutellaria baicalensis, and the rest are the same.

[0038] Example 6 Compared with Example 1, the difference is that in step S3, the preparation method of the enzyme buffer is changed to the following, and the rest are the same.

[0039] The enzyme buffer is prepared as follows: Take 10g of snail enzyme, 50,000U of cellulase, 30,000U of hemicellulase, 20g of sorbitol, and 40g of lactic acid, mix them, add 600mL of water, and stir to dissolve thoroughly. Use 5% sodium hydroxide aqueous solution to adjust the solution pH to 5.5 and dilute to 1L to obtain enzyme buffer.

[0040] Example 7 Compared with Example 1, the difference is that in step S3, the preparation method of the enzyme buffer is changed to the following, and the rest are the same.

[0041] The enzyme buffer is prepared as follows: 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 to dissolve thoroughly. Use 5% sodium hydroxide aqueous solution to adjust the solution pH to 6.5 and dilute to 1L to obtain enzyme buffer.

[0042] Example 8 Compared with Example 1, the difference is that in step S3, the enzymatic reaction conditions are changed to 8 hours, the constant temperature is 30° C., and the shaking speed is 80 rpm. The rest are the same.

[0043] Example 9 Compared with Example 1, the difference is that in step S3, the enzymatic reaction conditions are changed to enzymatic reaction for 2 hours, constant temperature of 45° C., and shaking speed of 20 rpm, and the rest are the same.

[0044] Comparative Example 1 Compared with Example 1, the difference is that in step S1, 30 g of honeysuckle and 20 g of forsythia are used instead, and in step S2, 180 g of scutellaria is used instead, and the rest are the same.

[0045] Comparative Example 2 Compared with Example 1, the difference is that in step S2, acetone in an equal weight ratio is used as the entrainer, and acetone is recovered after the supercritical carbon dioxide extraction is completed. The rest are the same.

[0046] Comparative Example 3 Compared with Example 1, the difference is that in step S2, the temperature of carbon dioxide supercritical extraction is changed to 35° C. and the pressure is changed to 40 MPa, and the rest are the same.

[0047] Comparative Example 4 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 the Shuanghuanglian extract.

[0048] Comparative Example 5 Shuanghuanglian lyophilized powder for injection is prepared according to the preparation method under the Shuanghuanglian for injection (lyophilized) in the "Chinese Pharmacopoeia" (Part 1, 2020 edition).

[0049] Effect evaluation 1. Evaluation of the antioxidant effect of Shuanghuanglian for injection.

[0050] The DPPH method was used as an evaluation method for the antioxidant effect, and the antioxidant effects of the Shuanghuanglian freeze-dried powder provided in each embodiment and comparative example were compared.

[0051] The structural formula of DPPH is as follows:

[0052] The experimental method is as follows: (1) Accurately weigh the DPPH reagent, dissolve it in anhydrous ethanol, prepare a standard solution (50 mg / L), and store it in a dark place.

[0053] (2) The Shuanghuanglian freeze-dried powder of each example and comparative example was mixed with deionized water to prepare a 100 mg / L Shuanghuanglian solution.

[0054] (3) Three groups of experiments were conducted for each embodiment / comparative example.

[0055] Group a. 2 mL of DPPH solution was mixed with 2 mL of deionized water.

[0056] Group b: 2 mL of Shuanghuanglian solution was mixed with 2 mL of anhydrous ethanol.

[0057] Group c: 2 mL of DPPH solution was mixed with 2 mL of Shuanghuanglian solution.

[0058] After standing at room temperature in the dark for 30 minutes, the absorbance was measured at 517 nm using a spectrophotometer. The absorbances of group a, group b, and group c were recorded as A0, A 样品 and A2.

[0059] DPPH clearance rate = [A0-(A 样品 -A2)]÷A0×100%.

[0060] The DPPH removal rates of the various embodiments and comparative examples are shown in Table 2 below.

[0061] Table 2

[0062] It can be seen that the antioxidant effect of the Shuanghuanglian freeze-dried powder provided by each embodiment of the present invention is significantly higher than the antioxidant effect of the Shuanghuanglian freeze-dried powder provided by each comparative example.

[0063] 2. Evaluation of the antipyretic effect of Shuanghuanglian injection.

[0064] Four-week-old male Wistar rats were randomly divided into two groups, with 7 rats in each group.

[0065] Blank control group and modeling control group: physiological saline solution was injected into the tail vein every day for 5 consecutive days.

[0066] Each Example Group: Shuanghuanglian freeze-dried powder of each Example Group was diluted with an appropriate amount of physiological saline to prepare Shuanghuanglian injection, which was injected into the tail vein daily at a dose of 9 g / kg rat body weight for 5 consecutive days.

[0067] Each comparative group: Shuanghuanglian freeze-dried powder of each comparative group was diluted with an appropriate amount of normal saline to prepare Shuanghuanglian injection, and the Shuanghuanglian injection was injected into the tail vein at a dose of 9 g / kg rat body weight daily for 5 consecutive days.

[0068] After five days of dosing, all rats in the blank control group, except the blank control group, were subcutaneously injected with 20% yeast solution (Angel high-activity dry yeast mixed with 4 times the weight of water) at a dose of 1 mL / 100 g body weight to induce fever. The blank control group was subcutaneously injected with normal saline. The rats' body temperatures were measured and recorded for 12 hours after modeling (rectal temperature measurement: insert a temperature sensor into the rat's anus and wait 30-60 seconds for the temperature to stabilize, then read the temperature).

[0069] The temperature measurement results at 0h, 3h, 5h, 8h, and 12h are shown in Table 3 below (n=7, mean ± standard deviation, retaining 2 decimal places): Table 3

[0070] It can be seen from the experimental data in Table 3 that the antipyretic effect of the Shuanghuanglian freeze-dried powder provided by each embodiment of the present invention is significantly better than that of the Shuanghuanglian freeze-dried powder provided by each comparative example, and there is a significant difference (Table 3, a Indicates that the data in the same column for each Example and each Comparative Example are significantly different from the model control group and p < 0.05). In particular, the Shuanghuanglian freeze-dried powder provided in Example 1 has the characteristics of rapid antipyretic effect (significant effect in 5 hours) and better antipyretic effect, while the Shuanghuanglian freeze-dried powder provided in Comparative Examples 1 to 5 only produces significant effect in as fast as 8 hours.

[0071] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate 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 following steps are involved: S1. Mixing honeysuckle and forsythia with a solvent, performing ultrasonic extraction, and separating to obtain a mixed medicinal residue and an ultrasonic extract; S2. performing supercritical carbon dioxide extraction on the scutellaria baicalensis to obtain scutellaria baicalensis residues and a scutellaria baicalensis extract; S3, mixing the mixed medicinal residue obtained in step S1 and the scutellaria baicalensis medicinal residue obtained in step S2 with an enzyme buffer, performing an enzymatic hydrolysis reaction, and intercepting the reaction with a molecular interception membrane to obtain an enzymatic hydrolysis extract; S4, after recovering the solvent from the ultrasonic extract obtained in step S1 by reducing the pressure, the extract was mixed with the scutellaria baicalensis extract obtained in step S2 and the enzymatic hydrolysis extract obtained in step S3, sterilized, and dried to obtain a Shuanghuanglian extract; Wherein, in step S2, the conditions of the supercritical carbon dioxide extraction are: temperature 40-50°C, pressure 25-35 MPa, entrainer ethanol, and extraction time 2-4 hours; 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 molecular weight range of the cut-off is ≤10000Da.

2. The preparation method according to claim 1, characterized in that The weight ratio of honeysuckle and forsythia in step S1 to scutellaria in step S2 is 3-13:2-9:8-11.

3. The preparation method according to claim 1, characterized in that In step S1, the ratio of the total weight of the honeysuckle and forsythia to the volume of the solvent is 1:8-10, in units of g:mL; the time of the ultrasonic extraction is 1-2h, the temperature of the ultrasonic extraction is 40-50°C, and the power of the ultrasonic extraction is 50-110W.

4. The preparation method according to claim 1, characterized in that In step S2, the conditions for the supercritical carbon dioxide extraction are: temperature 45° C., pressure 30 MPa, entrainer ethanol containing 4%-8% of the weight of Scutellaria baicalensis, and extraction time 3 h.

5. The preparation method according to claim 1, characterized in that In step S3, the ratio of the total weight of the mixed medicinal residues and the scutellaria baicalensis medicinal residues to the volume of the enzyme buffer is 1:6-15, in g:mL; in the enzyme buffer, the concentration of the snailase 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 of the enzymatic hydrolysis reaction are: enzymatic hydrolysis time 2-8 h, enzymatic hydrolysis temperature 30-45°C, and shaking speed of the shaker is 20-80 rpm.

6. The preparation method according to claim 1, characterized in that In step S4, the drying is freeze-drying.

7. The Shuanghuanglian extract obtained by the preparation method according to any one of claims 1 to 6.

8. Use of the preparation method according to any one of claims 1 to 6 in the production of Shuanghuanglian medicines.

9. A drug, characterized in that The active ingredient includes the Shuanghuanglian extract according to claim 7.

10. A Shuanghuanglian injection, characterized in that: The invention comprises the Shuanghuanglian extract according to claim 7 and a medically acceptable solvent.

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