Efficient preparation method of fructus broussonetiae natural product based on ultrasonic extraction

Abstract

The invention provides a paper mulberry fruit natural product efficient preparation method based on ultrasonic extraction, which comprises the following steps: S1, raw material pretreatment: crushing dried paper mulberry fruits, and sieving with a 40-60-mesh sieve to obtain paper mulberry fruit powder; s2, composite enzymatic hydrolysis pretreatment: adding deionized water of which the mass is 0.5-1.5 times of that of the fructus broussonetiae powder into the fructus broussonetiae powder in the S1, uniformly stirring, then adding a compound enzyme of which the mass is 0.1-0.5% of that of the powder, and carrying out enzymatic hydrolysis for 30-90 minutes under the conditions that the temperature is 45-55 DEG C and the pH is 4.5-5.5; the compound enzyme is formed by compounding pectinase and cellulase according to the mass ratio of 1: 1-3: 1, the pectinase and the cellulase are subjected to compounding pretreatment, impurity structures are accurately disintegrated, dissolution of pectin and polysaccharide is reduced from the source, a single-frequency mode is abandoned in an ultrasonic link, and flavone is protected through low-frequency strong cavitation wall breaking, high-frequency mild mass transfer and pulse temperature control. The two methods are not simply superposed, enzymolysis is a foundation for ultrasonic directional extraction, ultrasonic amplification of the enzymolysis effect finally solves the traditional problems of many impurities and low efficiency, and a green and efficient flavone extraction scheme is formed.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of extraction of broussonetia papyrifera, and particularly relates to a high-efficiency preparation method of natural products of broussonetia papyrifera based on ultrasonic extraction. BACKGROUND

[0002] Broussonetia papyrifera is the dried mature fruit of a plant of the Moraceae family, and is a traditional Chinese medicinal material recorded in Chinese Pharmacopoeia. Broussonetia papyrifera is rich in active ingredients such as flavonoids, alkaloids and polysaccharides. Flavonoids are the core material basis for playing the pharmacological effects of tonifying kidney and clearing liver and improving eyesight, and are also the key target components for the current medicinal development and industrialization of broussonetia papyrifera. Traditional extraction methods such as hot reflux extraction have the problems of long time consumption and high temperature, which can easily lead to oxidative degradation of heat-sensitive flavonoids. The ultrasonic extraction technology has the advantages of high extraction efficiency, short operation time and low extraction temperature, and can effectively reduce the damage of active ingredients, so it is widely used in the preparation research of flavonoids in broussonetia papyrifera. However, the existing ultrasonic extraction technology has a core defect that cannot be broken through in the application of broussonetia papyrifera, that is, poor extraction selectivity. The core principle of ultrasonic extraction is cavitation effect. Although the strong mechanical action can quickly break the cell wall of broussonetia papyrifera and release flavonoids, it cannot distinguish target components from impurities, and will extract water-soluble impurities (such as pectin, mucilage, protein and polysaccharide) in the intercellular substance and intracellular substance at the same time. These impurities can significantly increase the viscosity of the extract, not only increase the energy consumption and operation difficulty of subsequent separation steps such as filtration and centrifugation, but also greatly reduce the efficiency of subsequent purification process (such as macroporous adsorption resin purification). The impurities will occupy the adsorption sites of the resin, resulting in a decrease in the adsorption capacity of flavonoids, and the number of elution times or additional purification steps needs to be increased, thereby prolonging the process cycle and increasing the production cost. And from the existing technology, the published patents related to the extraction of broussonetia papyrifera either do not propose a solution to the problem of impurity co-extraction, or only improve the extraction efficiency by optimizing the ultrasonic power and time, but do not fundamentally improve the purity of the extract. These defects are all caused by the lack of “directional extraction” design in the existing ultrasonic technology, which cannot reduce the dissolution of impurities from the source, and seriously restricts the quality upgrading and industrial application of flavonoids in broussonetia papyrifera. SUMMARY

[0003] Technical problems to be solved In view of the deficiencies of the prior art, the application provides a high-efficiency preparation method of natural products of broussonetia papyrifera based on ultrasonic extraction, which solves the problem of poor extraction selectivity of the existing ultrasonic extraction of broussonetia papyrifera.

[0004] Technical scheme In order to achieve the above object, the application is implemented by the following technical scheme: a high-efficiency preparation method of natural products of broussonetia papyrifera based on ultrasonic extraction, comprising the following steps: S1, raw material pretreatment: grind dry broussonetia frondosa, pass through 40-60 mesh sieve to obtain broussonetia frondosa powder; S2, composite enzymatic hydrolysis pretreatment: add deionized water with a mass of 0.5-1.5 times that of the broussonetia frondosa powder in S1, stir, then add composite enzyme with a mass of 0.1%-0.5% of the powder, and carry out enzymatic hydrolysis at 45-55°C and pH 4.5-5.5 for 30-90 minutes; the composite enzyme is prepared by compounding pectinase and cellulase at a mass ratio of 1:1-3:1; S3, multi-frequency alternating ultrasonic extraction: add 60%-80% ethanol aqueous solution to the material after enzymatic hydrolysis, and the solid-liquid ratio is 1:15-1:30; the low-frequency-high-frequency alternating + pulse mode is used for extraction, and the extraction temperature is controlled at 50-60°C; the low-frequency ultrasonic parameters are 40 kHz and 300-500 W, and the action time is 5-10 minutes; the high-frequency ultrasonic parameters are 80 kHz and 150-250 W, and the action time is 10-20 minutes; the pulse mode is working for 2 seconds and intermittent for 1 second; S4, separation post-treatment: centrifuge or filter the extraction liquid while hot, collect the filtrate, recover ethanol by concentration under reduced pressure, dry the obtained concentrate to obtain broussonetia frondosa extract.

[0005] Preferably, in step S2, the mass ratio of pectinase to cellulase in the composite enzyme is 2:1, the addition amount of the composite enzyme is 0.3% of the mass of the broussonetia frondosa powder, the enzymatic hydrolysis temperature is 40°C, the pH is 5.0, and the enzymatic hydrolysis time is 45-60 minutes.

[0006] Preferably, in S3, the concentration of the ethanol aqueous solution is 75%, and the solid-liquid ratio is 1:20; the low-frequency ultrasonic power is 400 W, the action time is 7 minutes, the high-frequency ultrasonic power is 200 W, the action time is 14 minutes, and the extraction temperature is 59°C.

[0007] Preferably, in S4, the centrifugation parameters are 3000-4000 rpm for 10 minutes; the concentration under reduced pressure is at 60°C and -0.05--0.07 MPa, and the concentrate is concentrated to a relative density of 1.2-1.36; and the drying is vacuum drying at 60°C and -0.09 MPa.

[0008] Preferably, after S4, a deep purification step is further included: dilute the filtrate or concentrate to a flavone concentration of 10-15 mg / mL, load it onto an AB-8 type macroporous adsorption resin column, sequentially elute 3 BV of deionized water, 2 BV of 70% ethanol aqueous solution, collect the ethanol eluate, and concentrate and dry.

[0009] Beneficial effects The application provides a high-efficiency preparation method of broussonetia frondosa natural product based on ultrasonic extraction, and has the following beneficial effects: The present application uses pectinase and cellulase complex pretreatment, accurately disintegrates impurity structure, reduces pectin and polysaccharide dissolution from the source, and secondly, the ultrasonic link abandons the single frequency mode, uses low frequency strong cavitation to break the wall, high frequency gentle mass transfer, and pulse temperature control to protect flavonoids. The two are not simply superimposed, enzyme hydrolysis lays the foundation for ultrasonic directional extraction, and ultrasonic amplifies the enzyme hydrolysis effect, finally solves the problems of traditional impurities and low efficiency, and forms a green and efficient flavonoid extraction scheme. DETAILED DESCRIPTION

[0010] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the scope of protection of the present application.

[0011] Embodiment one: The present application provides a kind of based on ultrasonic extraction's high-efficiency preparation method of natural products of Fructus Broussonetiae, comprising the following steps: S1, raw material pretreatment: crushing dry Fructus Broussonetiae, passing through 40-60 mesh screen, to obtain Fructus Broussonetiae powder; S2, complex enzymatic hydrolysis pretreatment: adding 0.5-1.5 times the mass of deionized water to the Fructus Broussonetiae powder in S1, stirring evenly, then adding 0.1%-0.5% of the mass of the powder of complex enzyme, and hydrolyzing at 45-55℃, pH 4.5-5.5 for 30-90 minutes; the complex enzyme is compounded by pectinase and cellulase in a mass ratio of 1:1-3:1; S3, multi-frequency alternating ultrasonic extraction: adding 60%-80% ethanol aqueous solution to the hydrolyzed material, with a solid-liquid ratio of 1:15-1:30, and using low frequency-high frequency alternating + pulse mode for extraction, with the extraction temperature controlled at 50-60℃; the low frequency ultrasonic parameters are 40kHz, 300-500W, and the action time is 5-10 minutes; the high frequency ultrasonic parameters are 80kHz, 150-250W, and the action time is 10-20 minutes; the pulse mode is working for 2s and intermittent for 1s; S4, separation post-treatment: centrifuging or filtering the extraction liquid while hot, collecting the filtrate, recovering ethanol by vacuum concentration, and drying the obtained concentrate to obtain Fructus Broussonetiae extract In step S2, the mass ratio of pectinase to cellulase in the complex enzyme is 2:1, the addition amount of the complex enzyme is 0.3% of the mass of the Fructus Broussonetiae powder, the enzyme hydrolysis temperature is 40℃, the pH is 5.0, and the enzyme hydrolysis time is 45-60 minutes In S3, the concentration of the ethanol aqueous solution is 75%, the solid-liquid ratio is 1:20; the low frequency ultrasonic power is 400W, the action time is 7 minutes, the high frequency ultrasonic power is 200W, the action time is 14 minutes, and the extraction temperature is 59℃.

[0012] In S4, the centrifugation parameters are 3000-4000 rpm for 10 minutes; the vacuum concentration conditions are 60℃ and -0.05--0.07 MPa, concentrating to an extract with a relative density of 1.2-1.36; and the drying is performed under vacuum at 60℃ and -0.09 MPa.

[0013] S4 is followed by a deep purification step: the filtrate or concentrate is diluted to a flavonoid concentration of 10-15 mg / mL, loaded onto an AB-8 macroporous adsorption resin column, and eluted sequentially with deionized water for 3 BV, 70%, and ethanol aqueous solution for 2 BV. The ethanol eluent is collected, concentrated, and dried.

[0014] Example 2: Traditional ultrasonic extraction (without enzymatic hydrolysis) The raw material pretreatment is the same as in Example 1; 20L of 75% ethanol aqueous solution was added directly, and ultrasonic extraction was performed at 40kHz and 400W for 23 minutes (the same as the total ultrasonic time in Example 1), at a temperature of 55℃, with the same pulse mode as in Example 1. The separation, concentration, and drying steps were the same as in Example 1, yielding 155g of extract; Test results: Total flavonoid purity 41.5%, flavonoid recovery rate 85.3%, impurity content 28.7%; Example 3: Enzymatic hydrolysis followed by thermal extraction (without ultrasound) The raw material pretreatment and compound enzymatic hydrolysis steps are the same as in Example 1; Add 20 L of 75% ethanol aqueous solution, place in a 100 L reflux extraction tank, and extract twice at 75℃ for 2 hours each time. Combine the extracts. The separation, concentration, and drying steps were the same as in Example 1, yielding 110g of extract; Test results showed that the total flavonoid purity was 49.8%, the flavonoid recovery rate was 88.5%, and the impurity content was 18.2%, but the total process time was 5.5 hours (1.5 times that of Example 1).

[0015] Results analysis:

[0016] In summary, this invention achieves an optimal balance among purity, recovery rate, and efficiency. Although it takes slightly longer than traditional ultrasound, it improves purity by more than 40%, significantly reduces impurities, and substantially lowers subsequent purification costs. Compared with enzymatic hydrolysis and thermal extraction, it improves efficiency by 50% while maintaining higher purity, making it advantageous for industrial applications.

[0017] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A highly efficient method for preparing natural products from Broussonetia papyrifera seeds based on ultrasonic extraction, characterized in that, Includes the following steps: S1. Raw material pretreatment: Crush the dried paper mulberry seeds and pass them through a 40-60 mesh sieve to obtain paper mulberry seed powder; S2. Pretreatment with compound enzymatic hydrolysis: Add 0.5-1.5 times the mass of deionized water to the paper mulberry fruit powder in S1, stir well, and then add 0.1%-0.5% of the powder mass of compound enzyme. Enzymatic hydrolysis is carried out at 45-55℃ and pH 4.5-5.5 for 30-90 minutes. The compound enzyme is composed of pectinase and cellulase in a mass ratio of 1:1-3:

1. S3. Multi-frequency alternating ultrasonic extraction: Add 60%-80% ethanol aqueous solution to the enzymatically hydrolyzed material, with a material-to-liquid ratio of 1:15-1:

30. Use low-frequency-high-frequency alternating + pulse mode for extraction, and control the extraction temperature at 50-60℃. The low-frequency ultrasonic parameters are 40kHz, 300-500W, and the action time is 5-10 minutes. The high-frequency ultrasonic parameters are 80kHz, 150-250W, and the action time is 10-20 minutes. The pulse mode is 2 seconds of operation followed by a 1-second interval. S4. Post-separation processing: Centrifuge or filter the extract while it is hot, collect the filtrate and concentrate it under reduced pressure to recover the ethanol. The resulting concentrate is dried to obtain the Broussonetia papyrifera fruit extract.

2. The method for efficient preparation of Broussonetia papyrifera fruit natural products based on ultrasonic extraction according to claim 1, characterized in that: In step S2, the mass ratio of pectinase to cellulase in the compound enzyme is 2:1, the amount of compound enzyme added is 0.3% of the mass of paper mulberry fruit powder, the enzymatic hydrolysis temperature is 40℃, the pH is 5.0, and the enzymatic hydrolysis time is 45-60 minutes.

3. The method for efficient preparation of Broussonetia papyrifera fruit natural products based on ultrasonic extraction according to claim 1, characterized in that: The concentration of the ethanol-water solution in S3 is 75%, and the material-to-liquid ratio is 1:20; the low-frequency ultrasonic power is 400W for 7 minutes, the high-frequency ultrasonic power is 200W for 14 minutes, and the extraction temperature is 59℃.

4. The method for efficient preparation of Broussonetia papyrifera fruit natural products based on ultrasonic extraction according to claim 1, characterized in that: The centrifugation parameters in S4 are 3000-4000 rpm for 10 minutes; the vacuum concentration conditions are 60℃ and -0.05--0.07 MPa, concentrating to an extract with a relative density of 1.2-1.36; the drying is vacuum drying at 60℃ and -0.09 MPa.

5. The method for efficient preparation of Broussonetia papyrifera fruit natural products based on ultrasonic extraction according to claim 1, characterized in that: The S4 step further includes a deep purification step: dilute the filtrate or concentrate to a flavonoid concentration of 10-15 mg / mL, load the sample onto an AB-8 macroporous adsorption resin column, and elute sequentially with deionized water for 3 BV, 70%, and ethanol aqueous solution for 2 BV. Collect the ethanol eluent, concentrate and dry it.

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