Method for hydrothermal acid control degradation of mulberry leaf extracted polysaccharide and antioxidant application thereof

Abstract

The application relates to a method for hydrothermal acid-controlled degradation of mulberry leaf polysaccharide and antioxidant application thereof, and belongs to the technical field of extraction of active ingredients in natural products. The method comprises the following steps: pretreating raw materials, hydrothermally reacting and acid-catalytically treating mulberry leaves, and alcohol-sedimenting, separating and purifying mulberry leaf polysaccharide, so as to obtain mulberry leaf polysaccharide, finally, the antioxidant application of the mulberry leaf polysaccharide is explored, and a theoretical basis is provided for development of mulberry leaf resources. The method can not only obtain mulberry leaf polysaccharide, but also obtain high-value natural active ingredients such as rutin, astragalin, scopoletin and isoquercitrin in the mulberry leaves, is beneficial to development and utilization of the mulberry leaf resources, meets the needs of people's health, and has practical significance and economic value.

Description

A method for hydrothermal acid-controlled degradation of polysaccharides extracted from mulberry leaves and its antioxidant application. Technical Field

[0001] This invention belongs to the field of polysaccharide extraction technology from mulberry leaves, specifically relating to a method for extracting mulberry leaf polysaccharides by hydrothermal reaction synergistic acid catalytic degradation of mulberry leaves. Background Technology

[0002] Mulberry leaf polysaccharides are natural high-molecular polymers with various physicochemical properties. Their molecular structures are linear or branched; for example, mulberry melanin polysaccharide is α-glucan, and mulberry bark polysaccharide is β-glucan. Mulberry leaf polysaccharides exhibit optical rotation and good thermal stability, maintaining their chemical structure and biological activity at high temperatures. They possess multiple effects, including lowering blood sugar and lipids, antioxidation, lowering blood pressure, immune regulation, and reducing cardiovascular disease, gradually becoming a research hotspot. Currently, the main extraction methods for mulberry leaf polysaccharides include decoction extraction, water extraction, ultrasound-assisted extraction, bio-enzyme extraction, and supercritical fluid extraction. These methods, to varying degrees, suffer from problems such as low polysaccharide yield, low polysaccharide content, complex processes, long extraction times, low raw material utilization, and excessive use of organic solvents, impacting the environment. Therefore, finding an environmentally friendly, efficient, and convenient method for extracting mulberry leaf polysaccharides is of significant practical importance for improving the utilization of plant resources and meeting the needs of public health.

[0003] Existing polysaccharide extraction methods, such as the "Extraction Process of Selenium-Containing Phellinus linteus Polysaccharide" published on August 4, 2023 (CN116535533A), involve: first, pulverizing Phellinus linteus and mixing it with water at a ratio of 1:30-50 g / mL, stirring for 10-15 minutes to ensure thorough soaking; then, applying ultrasonic vibration at 200-300 W power at 80-90℃ for assisted extraction for 2-3 hours; filtering to obtain Phellinus linteus residue and extract; and then, decolorizing at a temperature of 40-50℃ for 25 minutes using a solid decolorizing agent (diatomaceous earth, adsorption resin) at a ratio of 3-5 g / L to the extract. After 40 min, the solid decolorizing agent was removed by filtration. Then, the extract was deproteinized using the Sevage method (chloroform: n-butanol = 5:1 (V / V)) to obtain a crude extract of Phellinus linteus polysaccharide. The extract was then concentrated at 70–80℃. Selenized carrageenan was added to the crude extract at a ratio of 1:0.5–0.72 L / g, and the mixture was stirred. Finally, the mixture was stirred and allowed to stand at a volume ratio of 1:3–4.5 mL / mL to ethanol (≥95%). The mixture was then separated into solid and liquid components and dried to obtain selenium-containing Phellinus linteus polysaccharide with a content of 37.58–41.67% and a selenium content of 14.62–16.03 μg / g. The method of this patent has the following shortcomings: (1) In addition to raw material Sanghuang, the extraction of selenium-containing Sanghuang polysaccharide also requires solid decolorizing agent (diatomaceous earth, adsorption resin) and Sevage reagent (chloroform: n-butanol = 5:1 (V / V)). Among them, chloroform and n-butanol are toxic solvents and are used in large quantities, which is not good for personnel health and increases management costs. The use of solid decolorizing agent and Sevage reagent increases the difficulty of equipment processing, causes secondary pollution to the environment, increases treatment costs, and thus increases production costs; (2) The extraction of selenium-containing Sanghuang polysaccharide requires pretreatment, ultrasonic-assisted hydrothermal extraction, filtration, decolorization, deproteinization, concentration, addition of selenized carrageenan, stirring, alcohol precipitation and static separation, drying, etc., which requires many steps, many reagents, complex operation, long equipment occupation time, increases the number of production equipment and production sites, and thus increases production costs, which is not conducive to promotion; (3) The content of selenium-containing Sanghuang polysaccharide obtained is 37.58-41.67%, and the raw material Sanghuang is limited by processing and source, resulting in high production costs and affecting the application and promotion of the technology. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing polysaccharide extraction methods by providing a method for hydrothermal acid-controlled degradation of mulberry leaf polysaccharides and their antioxidant applications. This method features readily available and low-cost raw materials, fewer steps, simple operation, low and recyclable organic solvent usage, rich extraction of natural components, thorough and high content of mulberry leaf polysaccharides, high utilization rate of mulberry leaves, and environmental friendliness.

[0005] The mechanism of this invention is as follows: A hydrothermal reaction uses water as the reaction medium, creating a high-temperature (pressure) extraction environment in a closed reaction vessel. Heating forms a subcritical region, reducing solvent viscosity, disrupting the interaction between the dissolved substance and the matrix, and intensifying intermolecular or intramolecular group vibrations, thus promoting water dissociation to generate H₂. + and OH - This facilitates the breakdown of plant cell walls and disrupts the dense network structure of mulberry leaf tissue; the organic acids in the solution medium provide H+. + It can increase the H in the reaction system + Ion concentration, hydrothermal reaction and citric acid synergistically catalyze the degradation of mulberry leaves, accelerate the change of redox potential, and promote the breakage of glycosidic bonds in hemicellulose and cellulose under acidic or alkaline conditions; the reaction medium water in the subcritical state is a solvent, a mineralizer and a pressure transmission medium, which is conducive to the dissolution and mass transfer of sparingly soluble or insoluble substances. The hydrothermal reaction coupling enhances acid catalytic oxidation and thermochemical conversion, strengthens biomass pyrolysis or hydrolysis, reduces the mass transfer barrier between cell wall and cytoplasm, and improves the dissolution efficiency of active ingredients such as mulberry leaf polysaccharides.

[0006] The technical solution to achieve the purpose of the invention is: a method for hydrothermal acid-controlled degradation of mulberry leaf extract polysaccharides and its antioxidant application. Using mulberry leaves as raw material, the method involves a simple three-step process: raw material pretreatment, hydrothermal reaction combined with acid catalysis to treat the mulberry leaves, and alcohol precipitation and purification of mulberry leaf polysaccharides to obtain the mulberry leaf polysaccharide product. The specific steps of the method are as follows:

[0007] (1) Raw material pretreatment

[0008] Using fresh mulberry leaves as raw material, the raw material is first washed with water and then dried naturally. Then it is crushed by a crusher and passed through a 20-200 mesh sieve. The raw material that does not pass through the sieve is returned to the crusher for further crushing. The sieved raw material is collected, which is the pre-treated mulberry leaf powder.

[0009] (2) Hydrothermal reaction synergistic acid catalysis treatment of mulberry leaves

[0010] After step (1) is completed, weigh 1-100g of mulberry leaf powder into a reaction vessel; then, according to the ratio of raw material mass to solution volume of 1:5-100g / mL, add a mixed solution of citric acid and ethanol and stir evenly; then, after hydrothermal reaction at a hydrothermal temperature of 60-180℃ for 20-90min, circulate cooling water to cool to room temperature; then filter, collect the filtrate and filter residue separately, the filtrate being the extract containing mulberry leaf polysaccharides; finally, transfer the extract to a rotary evaporator to obtain a concentrated mulberry leaf polysaccharide solution, and use the filter residue as raw material for preparing adsorbent materials. The mixed solution is a citric acid aqueous solution with a mass concentration of 0-2g / L and an ethanol solution with a volume fraction of 1-70%.

[0011] (3) Purification of mulberry leaf polysaccharide by alcohol precipitation

[0012] After step (2) is completed, first add 70-100% ethanol solution at a volume ratio of 1:2-5 (1:2-5) to concentrated mulberry leaf polysaccharide solution (mL), and mix well; then let it stand at room temperature (pressure) for ethanol precipitation for 6-24 hours, and pour off the supernatant; then set the centrifugation speed to 3000-6000 r / min and centrifuge the suspension for 10-30 min; then collect the supernatant and the centrifuged precipitate separately. The centrifuged precipitate is the precipitate containing mulberry leaf polysaccharide, and the supernatant contains the solution of active ingredients from mulberry leaves; finally, dry the centrifuged precipitate to obtain mulberry leaf polysaccharide with a yield of 31.15-55.85 mg / g. The active ingredients in the mulberry leaves may be rutin, astragaloside, scopolamine lactone, isoquercitrin, etc.

[0013] After adopting the above technical solution, the present invention mainly has the following effects:

[0014] (1) This invention uses citric acid instead of traditional acids and bases, and the solvent used is only ethanol which can be recycled. The extraction conditions are at room temperature (pressure). The process is simple, easy to operate, and has low energy consumption. It can not only obtain mulberry leaf polysaccharides, but also high-value natural active ingredients such as rutin, astragaloside, scopolamine lactone, and isoquercitrin in mulberry leaves, which is beneficial to the development and utilization of mulberry leaf resources.

[0015] (2) The mulberry leaf polysaccharide extracted by the present invention has a yield of 31.15 to 55.85 mg / g. The mulberry leaf polysaccharide has poor water solubility at room temperature (pressure), is difficult to dissolve in water, has good alcohol solubility, and is slightly soluble in alcohol solvents. It has a good scavenging effect on OH- free radicals, DPPH free radicals, NO free radicals and total antioxidant capacity.

[0016] (3) The raw material of the present invention is mulberry leaves. Mulberry leaves are widely planted in various regions of my country, with large yields, are not affected by the seasons, and have a wide range of raw material sources. Other plants can also be used directly as raw materials, which are convenient for storage, transportation and management, and have low production costs, thus meeting the needs of polysaccharide extraction. Detailed Implementation Methods

[0017] The present invention will be further described in detail below with reference to specific embodiments: Embodiment 1

[0018] The specific steps of a method for hydrothermal acid-controlled degradation of polysaccharides extracted from mulberry leaves and its antioxidant application are as follows:

[0019] (1) Raw material pretreatment

[0020] Using fresh mulberry leaves as raw material, the raw material is first washed with water and then dried naturally. Then it is crushed by a crusher and passed through an 80-mesh sieve. The raw material that does not pass through the sieve is returned to the crusher for further crushing. The sieved raw material is collected, which is the pre-treated mulberry leaf powder.

[0021] (2) Hydrothermal reaction synergistic acid catalysis treatment of mulberry leaves

[0022] After step (1) is completed, 30g of mulberry leaf powder is weighed into the reaction vessel; then, according to the ratio of raw material mass to solution volume of 1:25g / mL, a mixed solution of citric acid and ethanol is added and stirred evenly; then, after hydrothermal reaction at a hydrothermal temperature of 110℃ for 50min, circulating cooling water is introduced to cool to room temperature; then, the mixture is filtered, and the filtrate and filter residue are collected separately. The filtrate is the extract containing mulberry leaf polysaccharides; finally, the extract is transferred to a rotary evaporator to obtain a concentrated mulberry leaf polysaccharide solution, and the filter residue is used as the raw material for preparing the adsorbent material. The mixed solution is a 0.5g / L citric acid aqueous solution and a 45% ethanol solution.

[0023] (3) Purification of mulberry leaf polysaccharide by alcohol precipitation

[0024] After step (2) is completed, add 90% ethanol solution at a volume ratio of 1:4 (mulberry leaf polysaccharide concentrate, mL) to ethanol solution, mix and stir well; then let it stand at room temperature (pressure) for 12 hours for alcohol precipitation, and pour off the supernatant; then set the centrifugation speed to 4000 r / min and centrifuge the suspension for 20 min; then collect the supernatant and the centrifuged precipitate separately. The centrifuged precipitate is the precipitate containing mulberry leaf polysaccharide, and the supernatant is the solution containing the active ingredient raw material from mulberry leaves; finally, dry the centrifuged precipitate to obtain mulberry leaf polysaccharide with a yield of 39.67 mg / g and a purity of 39.67%. The active ingredients in the mulberry leaves may be rutin, astragaloside, scopolamine lactone, isoquercitrin, etc. Example 2

[0025] A method for hydrothermal acid-controlled degradation of polysaccharides extracted from mulberry leaves and its antioxidant application, the same as in Example 1, wherein:

[0026] In step (1), the mulberry leaf powder has a mesh size of 80.

[0027] In step (2), 30g of mulberry leaf powder is used, the ratio of raw material mass to solution volume is 1:20g / mL, the concentration of citric acid is 0.5g / L, the volume fraction of ethanol is 30%, the hydrothermal temperature is 80℃, and the hydrothermal time is 40min.

[0028] In step (3), the ratio of mulberry leaf polysaccharide concentrate (mL) to ethanol solution (mL) was 3:11, the ethanol solution concentration was 80%, the alcohol precipitation time at room temperature (pressure) was 14h, the centrifugation speed was 4000r / min, the centrifugation time was 20min, the yield of mulberry leaf polysaccharide was 50.26mg / g, and the purity was 11.27%. Example 3

[0029] A method for hydrothermal acid-controlled degradation of polysaccharides extracted from mulberry leaves and its antioxidant application, the same as in Example 1, wherein:

[0030] In step (1), the mulberry leaf powder has a mesh size of 80.

[0031] In step (2), 30g of mulberry leaf powder was used, the ratio of raw material mass to solution volume was 1:25g / mL, the lemon concentration was 0.5g / L, the ethanol volume fraction was 45%, the hydrothermal temperature was 110℃, and the hydrothermal time was 50min.

[0032] In step (3), the ratio of mulberry leaf polysaccharide concentrate (mL) to ethanol solution (mL) was 1:5, the concentration of ethanol solution was 90%, the alcohol precipitation time at room temperature (pressure) was 8h, the centrifugation speed was 4000r / min, the centrifugation time was 20min, the yield of mulberry leaf polysaccharide was 31.15mg / g, and the purity was 34.28%.

[0033] Experimental results

[0034] 1. Effect of different ethanol volume fractions on the extraction of mulberry leaf polysaccharides

[0035] 2. Effect of different material-to-liquid ratios on the extraction of mulberry leaf polysaccharides

[0036] 3. Effects of different hydrothermal temperatures on the extraction of mulberry leaf polysaccharides

[0037] 4. Effects of different hydrothermal times on the extraction of mulberry leaf polysaccharides

[0038] 5. Effects of different ethanol concentrations on the extraction of polysaccharides from mulberry leaves

[0039] 6. Effect of different alcohol-to-ethanol volume ratios on the extraction of mulberry leaf polysaccharides

[0040] 7. Effects of different alcohol precipitation times on the extraction of mulberry leaf polysaccharides

[0041] 8. Table of pure polysaccharide and protein content in mulberry leaf polysaccharides

[0042] 9. Analysis of the deliquescent and water-absorbing properties of mulberry leaf polysaccharides under ambient temperature and pressure

[0043] 10. Effects of mulberry leaf polysaccharide solution on antioxidant properties

[0044] 11. Solubility analysis of mulberry leaf polysaccharide in aqueous / alcoholic solutions

[0045] 12. High-performance liquid chromatography analysis of active ingredients in the supernatant of mulberry leaf polysaccharide extracted by hydrothermal acid-controlled extraction

[0046] From the above experiments, it is known that this invention uses hydrothermal acid-controlled degradation of mulberry leaves to extract mulberry leaf polysaccharides. When the mulberry leaf mesh size is 80 mesh, the raw material is 30g, the ratio of raw material mass to solution volume is 1:25g / mL, the citric acid concentration is 0.5g / L, the ethanol volume fraction is 45%, the hydrothermal temperature is 110℃, and the hydrothermal time is 50min, the mulberry leaf polysaccharide extract is obtained. The extract is concentrated, and the ratio of concentrated mulberry leaf polysaccharide solution (mL) to 90% ethanol solution (mL) is 1:4. The extract is then allowed to stand at room temperature (pressure) for 12h for alcohol precipitation, followed by centrifugation at 4000r / min for 20min, and drying to obtain mulberry leaf polysaccharides with a yield of 49.92mg / g and a purity of 39.67%. The mulberry leaf polysaccharide solution, within a concentration gradient of 0.25, 0.50, 1.00, 2.00, and 4.00mg / mL, has a significant effect on OH-. - The highest free radical scavenging rate was 96.71%, the highest DPPH free radical scavenging rate was 38.65%, the highest NO free radical scavenging rate was 5.83%, and the highest total antioxidant capacity was 0.27, demonstrating good antioxidant properties. Mulberry leaf polysaccharides exhibit poor water solubility at room temperature (pressure), are difficult to dissolve in water, but have good alcohol solubility and are slightly soluble in alcohol solvents. The production process uses citric acid instead of traditional acids and alkalis, and the only solvent used is ethanol, which can be recycled. The extraction conditions are at room temperature (pressure), making the process simple, easy to operate, and energy-efficient. In addition to mulberry leaf polysaccharides, it also yields high-value natural active ingredients from mulberry leaves such as rutin, astragaloside, scopolamine lactone, and isoquercitrin. This is beneficial for the development and utilization of mulberry leaf resources, reducing production costs, and meeting the health needs of the people, possessing practical significance and economic value.

Claims

1. A method for hydrothermal acid-controlled degradation of polysaccharides extracted from mulberry leaves, characterized in that, Includes the following steps: (1) Raw material pretreatment: Fresh mulberry leaves are used as raw materials. First, the raw materials are washed with water and dried naturally. Then, they are crushed by a pulverizer and passed through an 80-mesh sieve. The raw materials that are not sieved are returned to the pulverizer for further crushing. The sieved raw materials are collected, which are the pretreated mulberry leaf powder. (2) Hydrothermal reaction and acid catalytic treatment of mulberry leaves: After the completion of step (1), 1-100g of mulberry leaf powder is weighed into the reaction vessel. Then, according to the ratio of raw material mass to solution volume of 1:25g / mL, 0.5g / L citric acid aqueous solution and 45% ethanol solution are added. After stirring evenly, the mixture is hydrothermally reacted at a hydrothermal temperature of 110℃ for 50min. Then, circulating cooling water is introduced to cool to room temperature. Then, the mixture is filtered and the filtrate and residue are collected separately. The filtrate is the extract containing mulberry leaf polysaccharides. Liquid; Finally, the extract was transferred to a rotary evaporator to obtain a concentrated mulberry leaf polysaccharide solution, and the filter residue was used as a raw material for preparing adsorbent materials; (3) Mulberry leaf polysaccharide alcohol precipitation separation and purification After step (2) was completed, the concentrated mulberry leaf polysaccharide solution was mixed with a 90% ethanol solution at a volume ratio of 1:4 and stirred until homogeneous; then, it was allowed to stand at room temperature and pressure for 12 hours for alcohol precipitation, and the supernatant was poured off; then, the centrifugation speed was set to 4000 r / min, and the suspension was centrifuged for 20 min; then, the supernatant and the centrifuged precipitate were collected separately. The centrifuged precipitate was the precipitate containing mulberry leaf polysaccharide, and the supernatant contained the active ingredient raw material in mulberry leaves; finally, the centrifuged precipitate was dried to obtain mulberry leaf polysaccharide with a yield of 49.92 mg / g; in step (3), the purity of mulberry leaf polysaccharide was 39.67%.