A compound isatis root preparation and a preparation method thereof

Abstract

This invention discloses a compound Isatis root preparation and its preparation method, belonging to the field of traditional Chinese medicine preparation technology. It solves the problems of low extraction rate and high energy consumption of active ingredients in existing compound Isatis root preparations. The compound Isatis root preparation uses Isatis root and Isatis leaf as active ingredient raw materials, and includes steps such as swelling and cell wall disruption pretreatment, low-melting-point solvent-complex enzyme synergistic extraction, physical field cell wall disruption extraction, solid-liquid separation and concentration, and preparation molding. This invention can maximize the retention of heat-sensitive active ingredients, improve the dissolution of active ingredients in the compound Isatis root preparation, and greatly increase the extraction rate of active ingredients.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine preparation technology, and specifically to a compound Isatis indigotica preparation and its preparation method. Background Technology

[0002] Isatis root, the dried root of the cruciferous plant Isatis indigotica, is a classic traditional Chinese medicine with a long history of application in China. Compound Isatis root preparations (such as compound Isatis root granules), made by combining Isatis root with other medicinal herbs, are commonly used clinical Chinese patent medicines for clearing heat and detoxifying, possessing effects such as clearing heat and detoxifying, cooling the blood and relieving sore throat. Modern pharmacological studies have shown that Isatis root also has antiviral, antibacterial, anti-inflammatory, and immunomodulatory effects, and is widely used in the prevention and treatment of wind-heat colds, sore throats, mumps, and viral diseases.

[0003] The pharmacological basis of Compound Isatis Root Granules is complex, mainly containing alkaloids (such as quercetin), organic acids, nucleosides (such as adenosine), flavonoids, amino acids, and polysaccharides, among other active ingredients. It has a sweet and slightly bitter taste. It can be used for wind-heat colds and sore throats. Taking Isatis root during a cold helps enhance immunity, kill viruses and pathogens in the body, and aids recovery. Taking Isatis root during influenza and viral diseases helps enhance resistance and prevent infection. It can also be used for sore throat, dry mouth and throat, and acute tonsillitis caused by excessive heat in the lungs and stomach.

[0004] Currently, the conventional preparation methods for compound Isatis root preparations typically include steps such as extraction, concentration, addition of excipients, and granulation of Isatis root and other medicinal materials. Among these, the extraction process is the key step that determines the quality and efficacy of the preparation. Extraction is often carried out using water decoction or ethanol reflux extraction, followed by purification processes such as concentration and alcohol precipitation. Then, excipients such as sucrose and dextrin are added to form granules or other dosage forms.

[0005] However, the aforementioned conventional extraction methods have significant technical limitations in practical applications: On the one hand, due to the dense tissue structure of Isatis indigotica and the different dissolution rates of its active ingredients (such as water-soluble components like cypermethrin and some fat-soluble components), conventional static or simple dynamic extraction often fails to achieve intracellular and extracellular concentration equilibrium within a short time, resulting in incomplete dissolution of active ingredients. On the other hand, while prolonged heating extraction can increase the dissolution of some components, it easily causes the degradation or volatilization of heat-sensitive active ingredients (such as some glycosides and sulfur-containing compounds), which not only reduces the extraction rate of active ingredients but may also lead to an increase in the dissolution of ineffective components, making subsequent separation and purification difficult. These technical limitations ultimately result in a waste of medicinal resources and unstable efficacy of the preparations.

[0006] Therefore, how to improve the extraction rate of effective components of compound Isatis indigotica preparations while preserving their biological activity to the greatest extent and shortening the extraction time has become a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] To address the above shortcomings, this invention provides a compound Isatis root preparation and its preparation method, which solves the problems of low extraction rate and high energy consumption of effective components in existing compound Isatis root preparations. It maximizes the retention of heat-sensitive effective components, improves the dissolution of active ingredients in the compound Isatis root preparation, and significantly increases the extraction rate of effective components. The specific technical solution is as follows: A method for preparing a compound Isatis root preparation, wherein the compound Isatis root preparation uses Isatis root and Isatis leaf as active ingredients, comprising the following steps: (1) Swelling and cell wall breaking pretreatment: Wash and dry the Isatis root and Isatis leaf medicinal materials, then crush and vibrate them into medicinal powder. Add swelling agent to the obtained medicinal powder, stir and carry out swelling treatment to obtain medicinal suspension. (2) Eutectic solvent-complex enzyme synergistic extraction: Eutectic solvent, complex enzyme A and complex enzyme B are added to the medicinal material suspension obtained in step (1) in sequence, and enzymatic extraction is performed to obtain enzymatic extract; (3) Physical field cell wall disruption extraction: The enzymatic hydrolysate obtained in step (2) is placed in a pulsed electric field and an ultrasonic field for cell wall disruption extraction and enzyme inactivation to obtain the cell wall disruption extract; (4) Solid-liquid separation and concentration: The cell wall-breaking extract obtained in step (3) is subjected to solid-liquid separation, and the supernatant is concentrated under reduced pressure to a clear extract with a relative density of 1.10 to 1.30; (5) Formulation: The extract obtained in step (4) is mixed with pharmaceutically acceptable excipients to form granules, tablets or capsules to obtain the compound Isatis root preparation.

[0008] Preferably, in step (1), the mass ratio of Isatis root to Isatis leaf in the compound Isatis root preparation is (1.8-2.2):(2.8-3.3).

[0009] Preferably, in step (1), the pulverization involves pulverizing the medicinal materials to pass through a 50-80 mesh sieve.

[0010] Preferably, in step (1), the amplitude of the vibratory mill is 3-8 mm, the frequency is 20-30 Hz, and the processing time is 60-80 min.

[0011] Preferably, in step (1), the swelling agent is one of a glycerol aqueous solution, a propylene glycol aqueous solution, or a polyethylene glycol aqueous solution with a mass fraction of 20% to 50%.

[0012] Preferably, in step (1), the stirring is carried out at 40-60°C for 20-30 minutes.

[0013] Preferably, in step (2), the eutectic solvent is a mixture of hydrogen bond acceptor and hydrogen bond donor in a molar ratio of 1:(2-4), and its final concentration in the extraction system is 30%-50% (v / v).

[0014] Preferably, in step (2), the hydrogen bond acceptor is one of choline chloride and betaine.

[0015] Preferably, in step (2), the hydrogen bond donor is one of ethylene glycol, 1,2-propanediol, and citric acid.

[0016] Preferably, in step (2), the eutectic solvent is a choline chloride-ethylene glycol eutectic solvent with a molar ratio of 1:3.

[0017] Preferably, in step (2), the complex enzyme A contains cellulase and xylanase in a mass ratio of (2-4):1, and the amount added is 0.1% to 0.3% of the mass of the medicinal material suspension.

[0018] Preferably, in step (2), the complex enzyme B comprises β-glucanase and acidic protease in a mass ratio of 1:(1-3), and the amount added is 0.1% to 0.2% of the mass of the medicinal material suspension.

[0019] Preferably, in step (2), the enzymatic extraction is performed at a temperature of 45–55°C and a pH of 4.5–6.0 for 30–40 min.

[0020] Preferably, in step (2), during the enzymatic extraction process, intermittent stirring at 20 to 50 rpm is performed every 25 to 35 minutes.

[0021] Preferably, in step (3), the electric field strength of the pulsed electric field is 8-12 kV / cm, the pulse frequency is 200-400 Hz, and the processing time is 60-80 s.

[0022] Preferably, in step (3), the ultrasonic power of the ultrasonic field is 300-600W, the ultrasonic frequency is 20-40kHz, and the processing time is 180-200s.

[0023] Preferably, in step (4), the solid-liquid separation is centrifugal separation and membrane separation, wherein the centrifugal separation speed is 300-500 r / min and the centrifugation time is 10-20 minutes.

[0024] Preferably, in step (4), the membrane separation uses an ultrafiltration membrane with a molecular weight cutoff of 500 to 800 Da.

[0025] Preferably, step (4) further includes a eutectic solvent recovery step, specifically: collecting the condensate generated during the vacuum concentration process, removing the water by vacuum evaporation, recovering the eutectic solvent, and reusing the recovered eutectic solvent for the next batch of extraction.

[0026] Preferably, in step (5), the excipients include sucrose powder and starch, and the mass ratio of sucrose powder to Isatis root is (4.5-5):(1.8-2.2).

[0027] Preferably, in step (5), the mass ratio of starch to Isatis root is (0.08-0.12):(1.8-2.2).

[0028] Preferably, in step (5), the granules are granulated by fluidized bed granulation or spray drying.

[0029] The preparation method of the granules is as follows: the obtained extract is mixed with pharmaceutically acceptable excipients (sucrose powder or starch), and granulated by fluidized bed granulation or spray drying to obtain compound Isatis root granules.

[0030] The tablet preparation method is as follows: the obtained extract is mixed with pharmaceutically acceptable excipients (sucrose powder or starch), compressed into tablets, dried, and coated with a film to obtain compound Banlangen tablets.

[0031] The preparation method of the capsule is as follows: the obtained extract is mixed with pharmaceutically acceptable excipients (sucrose powder or starch), dried, and filled into capsules to obtain compound Banlangen capsules.

[0032] Preferably, a compound Isatis root preparation is prepared using the preparation method described above.

[0033] The present invention achieves at least the following beneficial effects: 1. This invention utilizes grinding to rupture cell walls, followed by the addition of a swelling agent to swell the ruptured cell walls, thereby maximizing cell wall rupture and increasing the dissolution rate of effective components within the cells, particularly the release rate of glycosides located in the cytoplasm.

[0034] 2. This invention utilizes a low-eutectic solvent-complex enzyme synergistic extraction method for cell wall-broken Chinese medicinal materials. The low-eutectic solvent can form a hydrogen bond network, selectively dissolving the target components; while the complex enzyme specifically degrades cell wall polysaccharides. The two work synergistically to improve the extraction rate. Moreover, the low-eutectic solvent can be recycled, making it green and environmentally friendly, and in line with the green trend of modern pharmaceutical industry.

[0035] 3. The present invention further treats the enzymatic extract with pulsed electric field and ultrasound, using the electric field pulse to break down the cell membrane and the ultrasonic cavitation effect to further destroy the incompletely ruptured cell structure, accelerate the mass transfer process, shorten the extraction time, reduce the extraction temperature, and protect the heat-sensitive active ingredients (such as quercetin).

[0036] 4. The enzymatic extraction method of this invention adopts the method of first breaking the cell wall and then releasing it. First, cell wall decomposing enzymes (cellulase + xylanase) are added to destroy the plant cell wall, and then target component-directed release enzymes (β-glucanase + acidic protease) are added to promote the dissolution of specific components, which can precisely control the dissolution of effective components.

[0037] 5. The compound Isatis root preparation obtained by the method of the present invention has a high polysaccharide content, the extracted extract has a high purity, and the extraction amount and extraction rate of epigallocatechin in the extracted extract are both higher. Detailed Implementation

[0038] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Unless otherwise defined, all technical terms used below have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of protection of the present invention. Unless otherwise specifically stated, all raw materials, reagents, instruments, and equipment used in the present invention are commercially available or can be prepared by existing methods.

[0039] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0040] Example 1 A method for preparing a compound Isatis root preparation, wherein the compound Isatis root preparation uses Isatis root and Isatis leaf as active ingredients, comprising the following steps: (1) Swelling and cell wall breaking pretreatment: Wash and dry the Isatis root and Isatis leaf medicinal materials and pulverize them until they pass through a 50-mesh sieve. Then, they are subjected to vibration milling at an amplitude of 3 mm and a frequency of 20 Hz for 60 min to obtain medicinal material powder. Add one of the following to the obtained medicinal material powder: 20% by mass of glycerol aqueous solution, propylene glycol aqueous solution, or polyethylene glycol aqueous solution. Stir at 40℃ for 20 min to obtain medicinal material suspension. The mass ratio of Isatis root and Isatis leaf in the compound Isatis root preparation is 1.8:2.8. (2) Eutectic solvent-complex enzyme synergistic extraction: Eutectic solvent, complex enzyme A, and complex enzyme B were added sequentially to the medicinal material suspension obtained in step (1). Complex enzyme A contained cellulase and xylanase in a mass ratio of 2:1, and its addition amount was 0.1% of the mass of the medicinal material suspension. Complex enzyme B contained β-glucanase and acidic protease in a mass ratio of 1:1, and its addition amount was 0.1% of the mass of the medicinal material suspension. Enzymatic extraction was carried out at a temperature of 45℃ and a pH of 4.5 for 30 min. During the enzymatic extraction process, intermittent stirring at 20 rpm was performed every 25 min to obtain the enzymatic extract. The eutectic solvent was a mixture of choline chloride and ethylene glycol in a molar ratio of 1:2, and its final concentration in the extraction system was 30% (v / v). (3) Physical field cell wall disruption extraction: The enzymatic hydrolysate obtained in step (2) was placed in a pulsed electric field and an ultrasonic field for cell wall disruption extraction and enzyme inactivation to obtain a cell wall disruption extract; the electric field strength of the pulsed electric field was 8kV / cm, the pulse frequency was 200Hz, and the treatment time was 60s; the ultrasonic power of the ultrasonic field was 300W, the ultrasonic frequency was 20 kHz, and the treatment time was 180s; (4) Solid-liquid separation and concentration: The cell wall-breaking extract obtained in step (3) is centrifuged and membrane separated. The centrifugation speed is 300 r / min and the centrifugation time is 10 minutes. The membrane separation uses an ultrafiltration membrane with a molecular weight cutoff of 500 Da. The supernatant is concentrated under reduced pressure to a clear paste with a relative density of 1.10. The eutectic solvent is recovered. Specifically, the condensate generated during the reduced pressure concentration process is collected, and the water is removed by reduced pressure evaporation. The eutectic solvent is recovered and can be reused for the next batch of extraction. (5) Formulation: The extract obtained in step (4) is mixed with pharmaceutically acceptable excipients, including sucrose powder and starch. The mass ratio of sucrose powder to Isatis root is 4.5:1.8, and the mass ratio of starch to Isatis root is 0.08:1.8. Granules, tablets or capsules are prepared to obtain the compound Isatis root preparation. The granules are granulated by fluidized bed granulation or spray drying granulation.

[0041] A compound Isatis root preparation was prepared using the above-mentioned method.

[0042] Example 2 A method for preparing a compound Isatis root preparation, wherein the compound Isatis root preparation uses Isatis root and Isatis leaf as active ingredients, comprising the following steps: (1) Swelling and cell wall breaking pretreatment: Wash and dry the Isatis root and Isatis leaf medicinal materials and pulverize them to pass through an 80-mesh sieve. Then, perform vibration milling at an amplitude of 8 mm and a frequency of 30 Hz for 80 min to obtain medicinal material powder. Add one of the following to the obtained medicinal material powder: 50% by mass of glycerol aqueous solution, propylene glycol aqueous solution, or polyethylene glycol aqueous solution. Stir at 60℃ for 30 min to obtain medicinal material suspension. The mass ratio of Isatis root and Isatis leaf in the compound Isatis root preparation is 2.2:3.3. (2) Eutectic solvent-complex enzyme synergistic extraction: Eutectic solvent, complex enzyme A, and complex enzyme B were added sequentially to the medicinal material suspension obtained in step (1). Complex enzyme A contained cellulase and xylanase in a mass ratio of 4:1, and its addition amount was 0.3% of the mass of the medicinal material suspension. Complex enzyme B contained β-glucanase and acidic protease in a mass ratio of 1:3, and its addition amount was 0.2% of the mass of the medicinal material suspension. Enzymatic extraction was carried out at a temperature of 55℃ and a pH of 6.0 for 40 min. During the enzymatic extraction process, intermittent stirring at 50 rpm was performed every 35 min to obtain the enzymatic extract. The eutectic solvent was a mixture of betaine and 1,2-propanediol in a molar ratio of 1:4, and its final concentration in the extraction system was 50% (v / v). (3) Physical field cell wall disruption extraction: The enzymatic hydrolysate obtained in step (2) was placed in a pulsed electric field and an ultrasonic field for cell wall disruption extraction and enzyme inactivation to obtain a cell wall disruption extract; the electric field strength of the pulsed electric field was 12kV / cm, the pulse frequency was 400Hz, and the treatment time was 80s; the ultrasonic power of the ultrasonic field was 600W, the ultrasonic frequency was 40kHz, and the treatment time was 200s. (4) Solid-liquid separation and concentration: The cell wall-breaking extract obtained in step (3) is centrifuged and membrane separated. The centrifugation speed is 500 r / min and the centrifugation time is 20 minutes. The membrane separation uses an ultrafiltration membrane with a molecular weight cutoff of 800 Da. The supernatant is concentrated under reduced pressure to a clear paste with a relative density of 1.30. The eutectic solvent is recovered. Specifically, the condensate generated during the reduced pressure concentration process is collected, and the water is removed by reduced pressure evaporation. The eutectic solvent is recovered and can be reused for the next batch of extraction. (5) Formulation: The extract obtained in step (4) is mixed with pharmaceutically acceptable excipients, including sucrose powder and starch. The mass ratio of sucrose powder to Isatis root is 5:2.2, and the mass ratio of starch to Isatis root is 0.12:2.2. Granules, tablets or capsules are prepared to obtain the compound Isatis root preparation. The granules are granulated by fluidized bed granulation or spray drying granulation.

[0043] A compound Isatis root preparation was prepared using the above-mentioned method.

[0044] Example 3 A method for preparing a compound Isatis root preparation, wherein the compound Isatis root preparation uses Isatis root and Isatis leaf as active ingredients, comprising the following steps: (1) Swelling and cell wall breaking pretreatment: Wash and dry the Isatis root and Isatis leaf medicinal materials and pulverize them to pass through a 60-mesh sieve. Then, perform vibration milling at an amplitude of 4 mm and a frequency of 22 Hz for 65 min to obtain medicinal material powder. Add one of the following to the obtained medicinal material powder: 30% by mass of glycerol aqueous solution, propylene glycol aqueous solution, or polyethylene glycol aqueous solution. Stir at 45℃ for 22 min to obtain medicinal material suspension. The mass ratio of Isatis root and Isatis leaf in the compound Isatis root preparation is 1.9:2.9. (2) Eutectic solvent-complex enzyme synergistic extraction: Eutectic solvent, complex enzyme A, and complex enzyme B were added sequentially to the medicinal material suspension obtained in step (1). Complex enzyme A contained cellulase and xylanase in a mass ratio of 2.5:1, and its addition amount was 0.15% of the mass of the medicinal material suspension. Complex enzyme B contained β-glucanase and acidic protease in a mass ratio of 1:1.5, and its addition amount was 0.12% of the mass of the medicinal material suspension. Enzymatic extraction was carried out at a temperature of 47℃ and a pH of 5.0 for 32 min. During the enzymatic extraction process, intermittent stirring at 30 rpm was performed every 28 min to obtain the enzymatic extract. The eutectic solvent was a mixture of choline chloride and 1,2-propanediol in a molar ratio of 1:2.5, and its final concentration in the extraction system was 35% (v / v). (3) Physical field cell wall disruption extraction: The enzymatic hydrolysate obtained in step (2) was placed in a pulsed electric field and an ultrasonic field for cell wall disruption extraction and enzyme inactivation to obtain a cell wall disruption extract; the electric field strength of the pulsed electric field was 9kV / cm, the pulse frequency was 250Hz, and the treatment time was 65s; the ultrasonic power of the ultrasonic field was 400W, the ultrasonic frequency was 25kHz, and the treatment time was 185s. (4) Solid-liquid separation and concentration: The cell wall-breaking extract obtained in step (3) is centrifuged and membrane separated. The centrifugation speed is 350 r / min and the centrifugation time is 12 minutes. The membrane separation uses an ultrafiltration membrane with a molecular weight cutoff of 600 Da. The supernatant is concentrated under reduced pressure to a clear paste with a relative density of 1.15. The eutectic solvent is recovered. Specifically, the condensate generated during the reduced pressure concentration process is collected, and the water is removed by reduced pressure evaporation. The eutectic solvent is recovered and can be reused for the next batch of extraction. (5) Formulation: The extract obtained in step (4) is mixed with pharmaceutically acceptable excipients, including sucrose powder and starch. The mass ratio of sucrose powder to Isatis root is 4.6:1.9, and the mass ratio of starch to Isatis root is 0.09:1.9. The mixture is then made into granules, tablets or capsules to obtain the compound Isatis root preparation. The granules are prepared by fluidized bed granulation or spray drying granulation.

[0045] A compound Isatis root preparation was prepared using the above-mentioned method.

[0046] Example 4 A method for preparing a compound Isatis root preparation, wherein the compound Isatis root preparation uses Isatis root and Isatis leaf as active ingredients, comprising the following steps: (1) Swelling and cell wall breaking pretreatment: Wash and dry the Isatis root and Isatis leaf medicinal materials and pulverize them to pass through a 70-mesh sieve. Then, perform vibration milling at an amplitude of 7 mm and a frequency of 28 Hz for 75 min to obtain medicinal material powder. Add one of the following to the obtained medicinal material powder: 40% by mass of glycerol aqueous solution, propylene glycol aqueous solution, or polyethylene glycol aqueous solution. Stir at 55℃ for 28 min to obtain medicinal material suspension. The mass ratio of Isatis root and Isatis leaf in the compound Isatis root preparation is 2.1:3.2. (2) Eutectic solvent-complex enzyme synergistic extraction: Eutectic solvent, complex enzyme A, and complex enzyme B were added sequentially to the medicinal material suspension obtained in step (1). Complex enzyme A contained cellulase and xylanase in a mass ratio of 3.5:1, and its addition amount was 0.25% of the mass of the medicinal material suspension. Complex enzyme B contained β-glucanase and acidic protease in a mass ratio of 1:2.5, and its addition amount was 0.18% of the mass of the medicinal material suspension. Enzymatic extraction was carried out at a temperature of 53℃ and a pH of 5.5 for 38 min. During the enzymatic extraction process, intermittent stirring at 40 rpm was performed every 33 min to obtain the enzymatic extract. The eutectic solvent was a mixture of betaine and citric acid in a molar ratio of 1:3.5, and its final concentration in the extraction system was 45% (v / v). (3) Physical field cell wall disruption extraction: The enzymatic hydrolysate obtained in step (2) was placed in a pulsed electric field and an ultrasonic field for cell wall disruption extraction and enzyme inactivation to obtain a cell wall disruption extract; the electric field strength of the pulsed electric field was 11kV / cm, the pulse frequency was 350Hz, and the treatment time was 75s; the ultrasonic power of the ultrasonic field was 500W, the ultrasonic frequency was 35kHz, and the treatment time was 195s. (4) Solid-liquid separation and concentration: The cell wall-breaking extract obtained in step (3) is centrifuged and membrane separated. The centrifugation speed is 450 r / min and the centrifugation time is 18 minutes. The membrane separation uses an ultrafiltration membrane with a molecular weight cutoff of 700 Da. The supernatant is concentrated under reduced pressure to a clear paste with a relative density of 1.25. The eutectic solvent is recovered. Specifically, the condensate generated during the reduced pressure concentration process is collected, and the water is removed by reduced pressure evaporation. The eutectic solvent is recovered and can be reused for the next batch of extraction. (5) Formulation: The extract obtained in step (4) is mixed with pharmaceutically acceptable excipients, including sucrose powder and starch. The mass ratio of sucrose powder to Isatis root is 4.9:2.1, and the mass ratio of starch to Isatis root is 0.11:2.1. Granules, tablets or capsules are prepared to obtain the compound Isatis root preparation. The granules are granulated by fluidized bed granulation or spray drying granulation.

[0047] A compound Isatis root preparation was prepared using the above-mentioned method.

[0048] Example 5 A method for preparing a compound Isatis root preparation, wherein the compound Isatis root preparation uses Isatis root and Isatis leaf as active ingredients, comprising the following steps: (1) Swelling and cell wall breaking pretreatment: Wash and dry the Isatis root and Isatis leaf medicinal materials and pulverize them to pass through a 65-mesh sieve. Then, perform vibration milling at an amplitude of 6 mm and a frequency of 25 Hz for 70 min to obtain medicinal material powder. Add one of the following to the obtained medicinal material powder: 35% glycerol aqueous solution, propylene glycol aqueous solution, or polyethylene glycol aqueous solution. Stir at 50℃ for 25 min to obtain medicinal material suspension. The mass ratio of Isatis root and Isatis leaf in the compound Isatis root preparation is 2:3. (2) Eutectic solvent-complex enzyme synergistic extraction: Eutectic solvent, complex enzyme A, and complex enzyme B were added sequentially to the medicinal material suspension obtained in step (1). Complex enzyme A contained cellulase and xylanase in a mass ratio of 3:1, and its addition amount was 0.2% of the mass of the medicinal material suspension. Complex enzyme B contained β-glucanase and acidic protease in a mass ratio of 1:2, and its addition amount was 0.15% of the mass of the medicinal material suspension. Enzymatic extraction was carried out at a temperature of 50℃ and a pH of 5.2 for 35 min. During the enzymatic extraction process, intermittent stirring at 35 rpm was performed every 30 min to obtain the enzymatic extract. The eutectic solvent was a mixture of choline chloride and ethylene glycol in a molar ratio of 1:3, and its final concentration in the extraction system was 40% (v / v). (3) Physical field cell wall disruption extraction: The enzymatic hydrolysate obtained in step (2) was placed in a pulsed electric field and an ultrasonic field for cell wall disruption extraction and enzyme inactivation to obtain a cell wall disruption extract; the electric field strength of the pulsed electric field was 10kV / cm, the pulse frequency was 300Hz, and the treatment time was 70s; the ultrasonic power of the ultrasonic field was 450W, the ultrasonic frequency was 30 kHz, and the treatment time was 190s. (4) Solid-liquid separation and concentration: The cell wall-breaking extract obtained in step (3) is centrifuged and membrane separated. The centrifugation speed is 400 r / min and the centrifugation time is 15 minutes. The membrane separation uses an ultrafiltration membrane with a molecular weight cutoff of 650 Da. The supernatant is concentrated under reduced pressure to a clear paste with a relative density of 1.20. The eutectic solvent is recovered. Specifically, the condensate generated during the reduced pressure concentration process is collected, and the water is removed by reduced pressure evaporation. The eutectic solvent is recovered and can be reused for the next batch of extraction. (5) Formulation: The extract obtained in step (4) is mixed with pharmaceutically acceptable excipients, including sucrose powder and starch. The mass ratio of sucrose powder to Isatis root is 4.75:2 and the mass ratio of starch to Isatis root is 0.1:2. Granules, tablets or capsules are prepared to obtain the compound Isatis root preparation. The granules are granulated by fluidized bed granulation or spray drying granulation.

[0049] A compound Isatis root preparation was prepared using the above-mentioned method.

[0050] Comparative Example 1 The difference from Example 5 is that in step (1), the medicinal powder is not subjected to vibration milling, while other conditions remain unchanged.

[0051] Comparative Example 2 The difference from Example 5 is that in step (1), no swelling agent is added to the medicinal powder for swelling treatment, and other conditions remain unchanged.

[0052] Comparative Example 3 The difference from Example 5 is that in step (1), the freezing temperature is -10°C, and other conditions remain unchanged.

[0053] Comparative Example 4 The difference from Example 5 is that in step (2), no eutectic solvent is added to the medicinal material suspension, and other conditions remain unchanged.

[0054] Comparative Example 5 The difference from Example 5 is that in step (2), the eutectic solvent is only choline chloride, and other conditions remain unchanged.

[0055] Comparative Example 6 The difference from Example 5 is that in step (2), the complex enzyme A and complex enzyme B are not added separately, but are added together at once, while other conditions remain unchanged.

[0056] Comparative Example 7 The difference from Example 5 is that in step (2), only one type of compound enzyme is used and added at once, namely, eutectic solvent-compound enzyme synergistic extraction: eutectic solvent and compound enzyme are added to the medicinal material suspension obtained in step (1) in sequence. The compound enzyme used is pectinase, cellulase and pectin lyase with a mass ratio of 1:3:1. The amount added is 0.35% of the mass of the medicinal material suspension, and other conditions remain unchanged.

[0057] Comparative Example 8 The difference from Example 5 is that in step (2), the compound enzyme B is pectinase and cellulase in a mass ratio of 1:3, and other conditions remain unchanged.

[0058] Comparative Example 9 The difference from Example 5 is that in step (3), pulsed electric field and ultrasonic field treatment are not performed, while other conditions remain unchanged.

[0059] Comparative Example 10 The difference from Example 5 is that in step (3), no pulsed electric field processing is performed, and other conditions remain unchanged.

[0060] Comparative Example 11 The difference from Example 5 is that in step (3), the cells were placed in an ultrasonic field and a pulsed electric field for cell wall breaking and extraction, while other conditions remained unchanged.

[0061] Test case 1. Measurement Method The method for determining the content of erythromycin in compound Isatis indigotica preparations or extracts of this invention is as follows: Chromatographic conditions: ZORBAX SB-C18 column (4.6 mm × 150 mm, 5 μm), mobile phase acetonitrile-(4.0 mL phosphoric acid + 0.3 mL triethylamine + 500 mL water) = 8.5:91.5; flow rate 0.7 mL / min, detection wavelength 245 nm, injection volume 20 μL, column temperature 30 °C.

[0062] Sample preparation method: Accurately weigh three portions of the compound Isatis root preparation or the extracted extract, and place each portion in a 50mL stoppered conical flask. Add 20mL of water, weigh, and ultrasonically extract at room temperature (power 250W, frequency 20kHz) for 60min. Remove, cool, and weigh again. Make up the weight loss with water, filter through a 0.45μm microporous membrane, discard the initial filtrate, and use the subsequent filtrate as the test sample. Determination results: Detect by HPLC and calculate the content of epigallocatechin gallate in the medicinal material.

[0063] 2. Experimental Methods Compound Isatis root preparations were prepared using the methods of Examples 1-5 and Comparative Examples 1-11. The raw material amounts for each preparation method were: 600g Isatis root, 900g Isatis leaf, 1425g sucrose powder, and 30g starch. The weight of the extract obtained from each preparation method was recorded. The polysaccharide content and purity of the extract obtained from each method were determined using the phenol-sulfuric acid method. The concentration of epigallocatechin in the medicinal materials and the extract obtained by HPLC was determined, and the content of epigallocatechin in the medicinal materials and the extract obtained by HPLC was calculated. The extraction rate was then calculated using the following formula: The extraction rate of phytoestrogen (%) = the amount of phytoestrogen in the extracted extract / the amount of phytoestrogen in the medicinal material × 100%.

[0064] The experimental results are shown in Table 1 below.

[0065] Table 1 As can be seen from the experimental data in Table 1, the compound Isatis root preparations prepared by the methods in Examples 1 to 5 of this invention have a high polysaccharide content, the purity of the extracted extract is high, and the extraction amount and extraction rate of isatis root extract are both higher.

[0066] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for preparing a compound Isatis root preparation, wherein the compound Isatis root preparation uses Isatis root and Isatis leaf as active ingredients, characterized in that, Includes the following steps: (1) Swelling and cell wall breaking pretreatment: Wash and dry the Isatis root and Isatis leaf medicinal materials, then crush and vibrate them into medicinal powder. Add swelling agent to the obtained medicinal powder and stir to obtain medicinal suspension. (2) Eutectic solvent-complex enzyme synergistic extraction: Eutectic solvent, complex enzyme A and complex enzyme B are added to the medicinal material suspension obtained in step (1) in sequence, and enzymatic extraction is performed to obtain enzymatic extract; (3) Physical field cell wall disruption extraction: The enzymatic hydrolysate obtained in step (2) is placed in a pulsed electric field and an ultrasonic field for cell wall disruption extraction and enzyme inactivation to obtain the cell wall disruption extract; (4) Solid-liquid separation and concentration: The cell wall-breaking extract obtained in step (3) is subjected to solid-liquid separation, and the supernatant is concentrated under reduced pressure to a clear extract with a relative density of 1.10 to 1.30; (5) Formulation: The extract obtained in step (4) is mixed with pharmaceutically acceptable excipients to form granules, tablets or capsules to obtain the compound Isatis root preparation.

2. The preparation method according to claim 1, characterized in that, In step (1), the mass ratio of Isatis root and Isatis leaf in the compound Isatis root preparation is (1.8-2.2):(2.8-3.3).

3. The preparation method according to claim 1, characterized in that, In step (1), the pulverization involves pulverizing the medicinal materials to pass through a 50-80 mesh sieve; the vibration mill has an amplitude of 3-8 mm, a frequency of 20-30 Hz, and a processing time of 60-80 min; the swelling agent is one of a 20%-50% (w / w) aqueous solution of glycerol, propylene glycol, or polyethylene glycol; and the stirring is performed at 40-60°C for 20-30 min.

4. The preparation method according to claim 1, characterized in that, In step (2), the eutectic solvent is a mixture of hydrogen bond acceptor and hydrogen bond donor in a molar ratio of 1:(2-4), and its final concentration in the extraction system is 30%-50% (v / v). The hydrogen bond acceptor is one of choline chloride and betaine, and the hydrogen bond donor is one of ethylene glycol, 1,2-propanediol, and citric acid.

5. The preparation method according to claim 1, characterized in that, In step (2), the compound enzyme A contains cellulase and xylanase in a mass ratio of (2-4):1, and its addition amount is 0.1%-0.3% of the mass of the medicinal material suspension; the compound enzyme B contains β-glucanase and acidic protease in a mass ratio of 1:(1-3), and its addition amount is 0.1%-0.2% of the mass of the medicinal material suspension; the enzymatic extraction is carried out at a temperature of 45-55℃ and a pH of 4.5-6.0 for 30-40 minutes; during the enzymatic extraction process, intermittent stirring at 20-50 rpm is performed every 25-35 minutes.

6. The preparation method according to claim 1, characterized in that, In step (3), the electric field strength of the pulsed electric field is 8-12 kV / cm, the pulse frequency is 200-400 Hz, and the processing time is 60-80 s; the ultrasonic power of the ultrasonic field is 300-600 W, the ultrasonic frequency is 20-40 kHz, and the processing time is 180-200 s.

7. The preparation method according to claim 1, characterized in that, In step (4), the solid-liquid separation is a combination of centrifugal separation and membrane separation. The centrifugal separation is performed at a speed of 300-500 r / min for 10-20 minutes. The membrane separation uses an ultrafiltration membrane with a molecular weight cutoff of 500-800 Da.

8. The preparation method according to claim 1, characterized in that, Step (4) also includes a eutectic solvent recovery step, which involves collecting the condensate generated during the vacuum concentration process, removing the water by vacuum evaporation, and recovering the eutectic solvent.

9. The preparation method according to claim 1, characterized in that, In step (5), the excipients include sucrose powder and starch, with the mass ratio of sucrose powder to Isatis root being (4.5-5):(1.8-2.2) and the mass ratio of starch to Isatis root being (0.08-0.12):(1.8-2.2); the granules are granulated by fluidized bed granulation or spray drying granulation.

10. A compound Isatis root preparation, characterized in that, It is prepared by any one of the preparation methods described in claims 1 to 8.

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