Method for extracting and preparing mangiferin from mangiferin leaves

Abstract

The invention provides a method for extracting and preparing mangiferin from Guiqiangmangifera indica leaves, which comprises the following steps: firstly, quickly freezing by liquid nitrogen to destroy cell wall structures and improve the permeability of a solvent, then treating mango leaf powder by compound enzyme to break compact cell walls, and extracting by using a deep eutectic solvent prepared from betaine, lactic acid group and ethanol to obtain mangiferin. According to the method, the mangiferin is selectively dissolved, the viscosity is reduced, the diffusion rate is increased, efficient extraction of the mangiferin is achieved, meanwhile, solvent recycling is achieved, the adopted raw materials are natural substances easy to decompose, pollution to the environment is reduced, finally, continuous purification is achieved through gradient membrane separation, use of organic solvents is reduced, and the cost is reduced. Therefore, the purity of the mangiferin product is improved.

Description

Technical Field

[0001] This invention belongs to the field of mangiferin extraction technology, and specifically relates to a method for extracting and preparing mangiferin from the leaves of the Guiqi mango tree. Background Technology

[0002] Mangiferin, also known as mangiferin or Zhimu Ning, is a natural polyphenolic compound extracted from the Liliaceae plant Anemarrhena asphodeloides. This compound can also be extracted from the leaves, fruits, and bark of mango and almond trees (Anacardiaceae), Gentiana macrophylla and Swertia japonica (Gentianaceae), and Pyrrosia lingua (Polypodiaceae). Modern pharmacological and clinical studies have demonstrated that mangiferin has various pharmacological effects on the central nervous system, respiratory system, and cardiovascular system. It also possesses anti-radiation, anti-tumor, anti-diabetic, hepatoprotective, and choleretic effects, as well as anti-inflammatory, antipyretic, and immunomodulatory effects.

[0003] Currently, research both domestically and internationally is increasingly emphasizing the development of herbal medicines. Many new drugs are first discovered from herbal medicines. Mango leaves are the leaves of the mango plant Mangifera indica L., which belongs to the Anacardiaceae family. The mangiferin contained in mango leaves is the main active ingredient for relieving cough, expectorating phlegm, and reducing inflammation. However, every year in my country, tens of millions of tons of mango leaves are burned by fruit farmers as firewood, resulting in a huge waste of resources.

[0004] Existing technologies use a 45% or higher ethanol aqueous solution to extract mango leaves, followed by concentration and extraction with a non-polar organic solvent, then extraction with a 90% or higher ethanol aqueous solution. However, the extracted product is a mixture of various glycosides, containing at least 50% mangiferin monomer, which still requires purification. Current technologies primarily use macroporous resin adsorption as the main purification step. However, this method requires pretreatment of the resin, and purification involves adsorption and elution processes, resulting in a long production cycle. During purification and elution with macroporous resin, physical adsorption can easily lead to some samples being dead-adsorbed, reducing the transfer rate. Furthermore, the large volume of eluent used in macroporous resin purification and the long solvent recovery time increase production costs and generate significant waste liquid, placing a burden on the environment. In addition, the use of macroporous resin increases the risk of introducing residual organic solvents such as benzene, toluene, xylene, styrene, alkanes, and diethylbenzene, impacting product safety. Summary of the Invention

[0005] To address the problems of low purity, long production cycle, and environmental pollution caused by residual organic solvents in existing mangiferin extraction technologies, this invention provides a method for extracting and preparing mangiferin from the leaves of the Guiqi mango tree. This method improves the purity of the finished mangiferin product while allowing the solvent used for extraction to be recycled, thus reducing the risk of organic residues.

[0006] This invention is achieved through the following technical solution: A method for extracting and preparing mangiferin from the leaves of *Mango styrax* includes the following steps: S1: Take tender leaves of Guiqi mango with a growth period of ≤90 days, freeze them in liquid nitrogen for 3-5 minutes, then freeze-dry them at -50℃~-40℃ and vacuum degree ≤10Pa for 24-36 hours, and finally pulverize them to 80-100 mesh to obtain raw material powder. S2: Add the raw material powder to an acetate-sodium acetate buffer solution with a pH of 4.0-5.0 at a material-to-liquid ratio of 1:(10-15). Then add a compound enzyme at a concentration of 450-530 U / g of raw material powder and hydrolyze at 42-48℃ for 45-75 minutes to obtain the enzymatic hydrolysate. The compound enzyme consists of cellulase, pectinase, and β-glucosidase. The oxygen concentration is maintained at ≤0.5% during the enzymatic hydrolysis process. S3: Add a eutectic solvent to the enzymatic hydrolysate, with a volume ratio of enzymatic hydrolysate to eutectic solvent of 1:(1.2-1.8). Then, perform dynamic countercurrent extraction twice at 55-65℃, each time for 25-35 minutes, maintaining the solid content of the extraction system at 3.0-4.5wt% to obtain the extract. The eutectic solvent is prepared by stirring betaine and lactic acid at a molar ratio of 1:(2.5-3.5) at 60℃ for 2 hours, and adding 18-22% ethanol by total volume. S4: Combine the extracts, first remove macromolecular impurities with a 100kDa ultrafiltration membrane, then enrich the mangiferin components with a 300kDa nanofiltration membrane, and finally concentrate the mangiferin components to 10-15% of the original volume of the juice through a reverse osmosis membrane to obtain the concentrate and the throttling liquid. S5: Adjust the pH of the concentrated solution to 1.8-2.2 with hydrochloric acid, let it stand at 2-6℃ for 10-14 hours to crystallize, wash the crystals 2-3 times with 65-75% ethanol, and then vacuum dry to obtain the mangiferin product. S6: Distill the retentate under reduced pressure at -0.08 to -0.09 MPa and 60-65℃. After dehydration by molecular sieve, the effluent is used to recover ethanol. The residue is decolorized and impurities are removed by 300 Da nanofiltration membrane to recover the eutectic solvent. Lactic acid is then added to form a new eutectic solvent, which is recycled for extraction in step S3.

[0007] This invention employs a combination of enzymatic hydrolysis, eutectic solvent extraction, and membrane separation to significantly improve the purity of mangiferin extraction while greatly reducing the amount of chemical organic matter used, thereby minimizing environmental pollution from residual chemical organic matter and improving product safety. First, liquid nitrogen flash freezing is used to disrupt cell wall structure and improve solvent permeability. Then, mango leaf powder is treated with a complex enzyme to break down its dense cell walls. Next, extraction is performed using a eutectic solvent prepared from betaine, lactic acid, and ethanol, selectively dissolving mangiferin. Ethanol reduces viscosity and increases diffusion rate, achieving efficient extraction of mangiferin while simultaneously recycling the solvent. The raw materials used are all easily decomposable natural substances, reducing environmental pollution. Finally, gradient membrane separation is used for continuous purification, reducing the use of organic solvents and thus improving the purity of the mangiferin product.

[0008] As a further improvement of the present invention, the method for preparing the eutectic solvent is as follows: (1) Add food-grade betaine and lactic acid to the reactor at a molar ratio of 1:(2.5-3.5) and react for 1.5-2.5 h at 400-600 rpm and 55-65℃ under nitrogen protection. Measure the reaction solution. Stop the reaction when the viscosity of the reaction solution at 25℃ is ≤300 cP and the conductivity is ≥0.8 S / m to obtain the mother liquor. (2) After cooling the mother liquor to 40°C, add anhydrous ethanol to the mother liquor and make the final concentration of anhydrous ethanol 15-25% v / v. After homogenization for 10-15 min, the eutectic solvent is obtained.

[0009] When the amount of lactic acid is too low, the solvent cannot form a hydrogen bond network, resulting in decreased solubility and poor dissolution of mangiferin. When the amount of lactic acid is too high, the hydrogen bond network is too dense, which can easily lead to excessive condensation of mangiferin and the generation of other byproducts, thus reducing the purity of mangiferin and increasing the difficulty of purification. After experimentation, this invention found that a molar ratio of betaine to lactic acid of 1:(2.5-3.5) achieves the best dissolution effect. When adding ethanol, the system temperature should not exceed 40°C, otherwise ethanol is prone to volatilization at high temperatures, resulting in excessive deviation in system concentration, affecting the diffusion efficiency of mangiferin, and thus leading to excessively high viscosity of the extracted solution, thereby increasing the difficulty of membrane separation.

[0010] As a further improvement of the present invention, in step S3, the amount of eutectic solvent added during the first extraction is 1.2-1.5 times the volume of the enzymatic hydrolysate; and in the second extraction, the amount of eutectic solvent added is 50-70% of the amount added during the first extraction.

[0011] As a further improvement of the present invention, the composite enzyme is composed of cellulase, pectinase and β-glucosidase in an enzyme activity ratio of (5.0-6.0):(2.5-3.0):(0.85-1.0); The amount of cellulase added is 280-320 U / g of raw material powder, the amount of pectinase added is 130-170 U / g, and the amount of β-glucosidase added is 45-55 U / g.

[0012] Cellulase primarily targets the cellulose skeleton of leaf cell walls, accounting for the highest proportion (60%±) to ensure structural disintegration; pectinase hydrolyzes pectin in the intercellular layer (approximately 30%), releasing embedded mangiferin; β-glucosidase precisely cleaves the -C glycosyl bonds of mangiferin (approximately ≤15%), avoiding excessive hydrolysis and damage to the aglycone structure. When the proportion of cellulase is <55%, leaf vein fibers are not completely disintegrated, and the yield of mangiferin decreases by ≥25%. When the total activity of β-glucosidase is >15%, it hydrolyzes the glycosyl groups of mangiferin, generating mangiferin byproducts, and the HPLC impurity peaks increase by 1.8 times. This invention has been verified through extensive experiments. Under specific activity ratios (enzyme activity ratio of (5.0-6.0):(2.5-3.0):(1.0)), the efficiency of disintegration of the leaf cell wall of *Mango guiqi* mango is improved by 40%.

[0013] As a further improvement of the present invention, in step S4, the ultrafiltration operating pressure is 0.4-0.6 MPa and the membrane flux is ≥30 L / (m²·h); the nanofiltration operating pressure is 1.0-1.5 MPa and the molecular weight cutoff is 280-320 Da.

[0014] As a further improvement of the present invention, in step S5, the crystallization process is cooled at a rate of 0.5°C / min; the solid-liquid ratio during ethanol washing is 1:3-1:5 (w / v), and the washing time is ≤5 minutes.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention employs a combination of enzymatic hydrolysis, eutectic solvent extraction, and membrane separation, which significantly improves the extraction purity of mangiferin while reducing the amount of organic solvents and reagents used. This reduces the impact of organic solvent and reagent residues on the environment and the final product, making it green and environmentally friendly.

[0016] 2. This invention uses a compound enzyme and an extraction solvent that are both natural and biodegradable substances. The compound enzyme works synergistically to efficiently degrade the cellulose skeleton of the cell wall and the pectin in the intercellular layer to release the embedded mangiferin. Then, the mangiferin is dissolved in ethanol using a betaine-lactic acid solvent to facilitate subsequent membrane separation, thereby achieving the effect of high-purity extraction of mangiferin.

[0017] 3. The eutectic solvent prepared by this invention can be recycled more than 5 times, which greatly reduces the extraction cost. At the same time, the solvent is composed of natural substances, which is greener and healthier than chemical agents. Detailed Implementation

[0018] The present invention will be further described below with reference to the embodiments. Unless otherwise specified, the technical means used in the embodiments are all conventional technical means in the art.

[0019] Example 1: A method for extracting and preparing mangiferin from the leaves of *Mango styrax* includes the following steps: S1: Take tender leaves of Guiqi mango with a growth period of ≤90 days, freeze them in liquid nitrogen for 3 minutes, then freeze-dry them in vacuum at -50℃ and vacuum degree ≤10Pa for 24 hours, and finally pulverize them to 80 mesh to obtain raw material powder.

[0020] S2: Add the raw material powder to an acetate-sodium acetate buffer solution with a pH of 4.0 at a material-to-liquid ratio of 1:10, then add a compound enzyme at a ratio of 450 U / g of raw material powder, and hydrolyze at 42°C for 45 minutes to obtain the enzymatic hydrolysate; the compound enzyme is composed of cellulase, pectinase, and β-glucosidase with an enzyme activity ratio of 5.0:2.5:0.85; the amount of cellulase added is 320 U / g of raw material powder, the amount of pectinase added is 160 U / g, and the amount of β-glucosidase added is 55 U / g; the oxygen concentration is maintained at ≤0.5% during the enzymatic hydrolysis process.

[0021] S3: Add a eutectic solvent to the enzymatic hydrolysate, with a volume ratio of 1:1.2. Then, perform dynamic countercurrent extraction twice at 55°C, 35 minutes each time, maintaining the solid content of the extraction system at 3.0 wt% to obtain the extract. The eutectic solvent is prepared by stirring betaine and lactic acid at a molar ratio of 1:2.5 at 60°C for 2 hours, and adding 18% ethanol by volume. The amount of eutectic solvent added during the first extraction is 1.2 times the volume of the enzymatic hydrolysate; the amount of eutectic solvent added during the second extraction is 50% of the amount added during the first extraction.

[0022] S4: Combine the extracts, first remove macromolecular impurities using a 100kDa ultrafiltration membrane, then enrich the mangiferin components using a 300kDa nanofiltration membrane, and finally concentrate the mangiferin components to 10% of the original volume of the juice using a reverse osmosis membrane to obtain a concentrate and a cut-off liquid; the ultrafiltration operating pressure is 0.4MPa, the membrane flux is ≥30 L / (m²·h), the nanofiltration operating pressure is 1.0MPa, and the molecular weight cut-off is 280Da.

[0023] S5: Adjust the pH of the concentrate to 1.8 with hydrochloric acid, let it stand at 2℃ for 10h to crystallize, wash the crystals twice with 65% ethanol, and dry them under vacuum to obtain the mangiferin product; the crystallization process is cooled at a rate of 0.5℃ / min; the solid-liquid ratio is 1:3 (w / v) when washing with ethanol, and the washing time is ≤5 minutes.

[0024] S6: The retentate is distilled under reduced pressure at -0.08 MPa and 60 °C. The effluent is dehydrated by molecular sieve and ethanol is recovered. The residue is decolorized and impurities are removed by 300 Da nanofiltration membrane to recover the eutectic solvent. Lactic acid is then added to form a new eutectic solvent, which is recycled for extraction in step S3.

[0025] The method for preparing the eutectic solvent is as follows: (1) Food-grade betaine and lactic acid were added to the reactor at a molar ratio of 1:2.5. The reaction was carried out at 400 rpm and 55°C for 1.5 h under nitrogen protection. The reaction solution was measured. When the viscosity of the reaction solution at 25°C was ≤300 cP and the conductivity was ≥0.8 S / m, the reaction was stopped, and the mother liquor was obtained. (2) After cooling the mother liquor to 40°C, add anhydrous ethanol to the mother liquor and make the final concentration of anhydrous ethanol 15% v / v. After homogenization for 10 min, the eutectic solvent is obtained.

[0026] Example 2: A method for extracting and preparing mangiferin from the leaves of *Mango styrax*, comprising the following steps: S1: Take tender leaves of Guiqi mango with a growth period of ≤90 days, freeze them in liquid nitrogen for 5 minutes, then freeze-dry them in vacuum at -40℃ and vacuum degree ≤10Pa for 36 hours, and finally pulverize them to 100 mesh to obtain raw material powder.

[0027] S2: Add the raw material powder to an acetate-sodium acetate buffer solution with a pH of 5.0 at a material-to-liquid ratio of 1:15, then add a compound enzyme at a ratio of 530 U / g of raw material powder, and enzymatically hydrolyze at 48°C for 75 minutes to obtain the enzymatic hydrolysate; the compound enzyme is composed of cellulase, pectinase, and β-glucosidase in an enzyme activity ratio of 6.0:3.0:1.0; the amount of cellulase added is 282 U / g of raw material powder, the amount of pectinase added is 141 U / g, and the amount of β-glucosidase added is 47 U / g; the oxygen concentration is maintained at ≤0.5% during the enzymatic hydrolysis process.

[0028] S3: Add a eutectic solvent to the enzymatic hydrolysate, with a volume ratio of 1:1.8. Then, perform dynamic countercurrent extraction twice at 65°C, 35 minutes each time, maintaining the solid content of the extraction system at 4.5 wt% to obtain the extract. The eutectic solvent is prepared by stirring betaine and lactic acid at a molar ratio of 1:3.5 at 60°C for 2 hours, and adding 22% ethanol by volume. The amount of eutectic solvent added during the first extraction is 1.5 times the volume of the enzymatic hydrolysate; the amount of eutectic solvent added during the second extraction is 70% of the amount added during the first extraction.

[0029] S4: Combine the extracts, first remove macromolecular impurities using a 100kDa ultrafiltration membrane, then enrich the mangiferin components using a 300kDa nanofiltration membrane, and finally concentrate the mangiferin components to 15% of the original juice volume using a reverse osmosis membrane to obtain a concentrate and a cut-off liquid; the ultrafiltration operating pressure is 0.6 MPa, the membrane flux is ≥30 L / (m²·h), the nanofiltration operating pressure is 1.5 MPa, and the molecular weight cut-off is 320 Da.

[0030] S5: Adjust the pH of the concentrate to 2.2 with hydrochloric acid, let it stand at 6℃ for 14 hours to crystallize, wash the crystals three times with 75% ethanol, and dry them under vacuum to obtain the mangiferin product; the crystallization process is cooled at a rate of 0.5℃ / min; the solid-liquid ratio is 1:5 (w / v) when washing with ethanol, and the washing time is ≤5 minutes.

[0031] S6: The retentate is distilled under reduced pressure at -0.09 MPa and 65 °C. The effluent is dehydrated by molecular sieve and ethanol is recovered. The residue is decolorized and impurities are removed by 300 Da nanofiltration membrane to recover the eutectic solvent. Lactic acid is then added to form a new eutectic solvent, which is recycled for extraction in step S3.

[0032] The method for preparing the eutectic solvent is as follows: (1) Food-grade betaine and lactic acid were added to the reactor at a molar ratio of 1:3.5. The reaction was carried out at 600 rpm and 55-65℃ for 2.5 h under nitrogen protection. The reaction solution was measured. When the viscosity of the reaction solution at 25℃ was ≤300 cP and the conductivity was ≥0.8 S / m, the reaction was stopped and the mother liquor was obtained. (2) After cooling the mother liquor to 40°C, add anhydrous ethanol to the mother liquor and make the final concentration of anhydrous ethanol 25% v / v. After homogenization for 15 min, the eutectic solvent is obtained.

[0033] Example 3: A method for extracting and preparing mangiferin from the leaves of *Mango styrax* includes the following steps: S1: Take tender leaves of Guiqi mango with a growth period of ≤90 days, freeze them in liquid nitrogen for 4 minutes, then freeze-dry them in vacuum at -45℃ and vacuum degree ≤10Pa for 30 hours, and finally pulverize them to 90 mesh to obtain raw material powder.

[0034] S2: Add the raw material powder to an acetate-sodium acetate buffer solution at pH 4.5 at a material-to-liquid ratio of 1:12, then add a compound enzyme at a ratio of 500 U / g of raw material powder, and hydrolyze at 45°C for 60 minutes to obtain the enzymatic hydrolysate; the compound enzyme is composed of cellulase, pectinase, and β-glucosidase in an enzyme activity ratio of 5.6:2.8:1.0; the amount of cellulase added is 300 U / g of raw material powder, the amount of pectinase added is 150 U / g, and the amount of β-glucosidase added is 53.6 U / g; maintain the oxygen concentration ≤0.5% during the enzymatic hydrolysis process.

[0035] S3: Add a eutectic solvent to the enzymatic hydrolysate, with a volume ratio of 1:1.5. Then, perform dynamic countercurrent extraction twice at 60°C, 30 minutes each time, maintaining the solid content of the extraction system at 4.0 wt% to obtain the extract. The eutectic solvent is prepared by stirring betaine and lactic acid at a molar ratio of 1:3.0 at 60°C for 2 hours, and adding 20% ​​ethanol by volume. The amount of eutectic solvent added during the first extraction is 1.3 times the volume of the enzymatic hydrolysate; the amount of eutectic solvent added during the second extraction is 65% of the amount added during the first extraction.

[0036] S4: Combine the extracts, first remove macromolecular impurities using a 100kDa ultrafiltration membrane, then enrich the mangiferin components using a 300kDa nanofiltration membrane, and finally concentrate the mangiferin components to 12% of the original juice volume using a reverse osmosis membrane to obtain a concentrate and a cut-off liquid; the ultrafiltration operating pressure is 0.5 MPa, the membrane flux is ≥30 L / (m²·h), the nanofiltration operating pressure is 1.2 MPa, and the molecular weight cut-off is 300 Da.

[0037] S5: Adjust the pH of the concentrate to 2.0 with hydrochloric acid, let it stand at 4℃ for 12 hours to crystallize, wash the crystals three times with 70% ethanol, and dry them under vacuum to obtain the mangiferin product; the crystallization process is cooled at a rate of 0.5℃ / min; the solid-liquid ratio is 1:4 (w / v) when washing with ethanol, and the washing time is ≤5 minutes.

[0038] S6: The retentate is distilled under reduced pressure at -0.085MPa and 62℃. The effluent is dehydrated by molecular sieve and ethanol is recovered. The residue is decolorized and impurities are removed by 300Da nanofiltration membrane to recover the eutectic solvent. Lactic acid is then added to form a new eutectic solvent, which is recycled for extraction in step S3.

[0039] The method for preparing the eutectic solvent is as follows: (1) Food-grade betaine and lactic acid were added to the reactor at a molar ratio of 1:3.0. The reaction was carried out at 500 rpm and 60°C for 2 hours under nitrogen protection. The reaction solution was measured. When the viscosity of the reaction solution at 25°C was ≤300 cP and the conductivity was ≥0.8 S / m, the reaction was stopped, and the mother liquor was obtained. (2) After cooling the mother liquor to 40°C, add anhydrous ethanol to the mother liquor and make the final concentration of anhydrous ethanol 20% v / v. After homogenization for 12 min, the eutectic solvent is obtained.

[0040] Example 4: The content of mangiferin prepared in Examples 1-3 was determined by chromatographic column chromatography, and the results are shown in Table 1.

[0041] Column: Supelco LC-18-DB 15cm × 4.6mm 5μm; Mobile phase: 10% acetonitrile: phosphoric acid (100:0.2, V / V, adjusted to pH 3-4 with triethylamine); Flow rate: 1 mL / min; Detection wavelength: 260nm; Column temperature: room temperature; Injection volume: 5 μL or 10 μL.

[0042] Table 1: Test sample Initial extraction content (%) Extraction content (%) after 5 cycles of eutectic solvent. Example 1 99.70 98.90 Example 2 99.65 98.85 Example 3 99.72 98.86 As shown in Table 1-2, the purity of mangiferin extracted by this invention reached a maximum of 99.72%, and the purity of mangiferin extracted after 5 cycles in the eutectic solvent was still as high as 98.90%.

[0043] The above embodiments are merely exemplary embodiments of the present invention and are not intended to limit the present invention. The scope of protection of the present invention is defined by the claims. Those skilled in the art can make various modifications or equivalent substitutions to the present invention within its spirit and scope of protection, and such modifications or equivalent substitutions should also be considered to fall within the scope of protection of the present invention.

Claims

1. A method for extracting and preparing mangiferin from the leaves of *Mango styrax*, characterized in that, Includes the following steps: S1: Take tender leaves of Guiqi mango with a growth period of ≤90 days, freeze them in liquid nitrogen for 3-5 minutes, then freeze-dry them at -50℃~-40℃ and vacuum degree ≤10Pa for 24-36 hours, and finally pulverize them to 80-100 mesh to obtain raw material powder. S2: Add the raw material powder to an acetate-sodium acetate buffer solution with a pH of 4.0-5.0 at a material-to-liquid ratio of 1:(10-15). Then add a compound enzyme at a concentration of 450-530 U / g of raw material powder and hydrolyze at 42-48℃ for 45-75 minutes to obtain the enzymatic hydrolysate. The compound enzyme consists of cellulase, pectinase, and β-glucosidase. The oxygen concentration is maintained at ≤0.5% during the enzymatic hydrolysis process. S3: Add a eutectic solvent to the enzymatic hydrolysate, with a volume ratio of enzymatic hydrolysate to eutectic solvent of 1:(1.2-1.8). Then, perform dynamic countercurrent extraction twice at 55-65℃, each time for 25-35 minutes, maintaining the solid content of the extraction system at 3.0-4.5wt% to obtain the extract. The eutectic solvent is prepared by stirring betaine and lactic acid at a molar ratio of 1:(2.5-3.5) at 60℃ for 2 hours, and adding 18-22% ethanol by total volume. S4: Combine the extracts, first remove macromolecular impurities with a 100kDa ultrafiltration membrane, then enrich the mangiferin components with a 300kDa nanofiltration membrane, and finally concentrate the mangiferin components to 10-15% of the original volume of the juice through a reverse osmosis membrane to obtain the concentrate and the throttling liquid. S5: Adjust the pH of the concentrated solution to 1.8-2.2 with hydrochloric acid, let it stand at 2-6℃ for 10-14 hours to crystallize, wash the crystals 2-3 times with 65-75% ethanol, and then vacuum dry to obtain the mangiferin product. S6: Distill the retentate under reduced pressure at -0.08 to -0.09 MPa and 60-65℃. After dehydration by molecular sieve, the effluent is used to recover ethanol. The residue is decolorized and impurities are removed by 300 Da nanofiltration membrane to recover the eutectic solvent. Lactic acid is then added to form a new eutectic solvent, which is recycled for extraction in step S3.

2. The method for extracting and preparing mangiferin from the leaves of *Mango styrax* according to claim 1, characterized in that: The method for preparing the eutectic solvent is as follows: (1) Add food-grade betaine and lactic acid to the reactor at a molar ratio of 1:(2.5-3.5) and react for 1.5-2.5 h at 400-600 rpm and 55-65℃ under nitrogen protection. Measure the reaction solution. Stop the reaction when the viscosity of the reaction solution at 25℃ is ≤300 cP and the conductivity is ≥0.8 S / m to obtain the mother liquor. (2) After cooling the mother liquor to 40°C, add anhydrous ethanol to the mother liquor and make the final concentration of anhydrous ethanol 15-25% v / v. After homogenization for 10-15 min, the eutectic solvent is obtained.

3. The method for extracting and preparing mangiferin from the leaves of *Mango styrax* according to claim 1, characterized in that: In step S3, the amount of eutectic solvent added during the first extraction is 1.2-1.5 times the volume of the enzymatic hydrolysate; during the second extraction, the amount of eutectic solvent added is 50-70% of the amount added during the first extraction.

4. The method for extracting and preparing mangiferin from the leaves of *Mango styrax* according to claim 1, characterized in that: The aforementioned complex enzyme is composed of cellulase, pectinase, and β-glucosidase in an enzyme activity ratio of (5.0-6.0):(2.5-3.0):(0.85-1.0); The amount of cellulase added is 280-320 U / g of raw material powder, the amount of pectinase added is 130-170 U / g, and the amount of β-glucosidase added is 45-55 U / g.

5. The method for extracting and preparing mangiferin from the leaves of *Mango styrax* according to claim 1, characterized in that: In step S4, the ultrafiltration operating pressure is 0.4-0.6 MPa, and the membrane flux is ≥30 L / (m²·h); the nanofiltration operating pressure is 1.0-1.5 MPa, and the molecular weight cutoff is 280-320 Da.

6. The method for extracting and preparing mangiferin from the leaves of *Mango styrax* according to claim 1, characterized in that: In step S5, the crystallization process involves cooling at a rate of 0.5℃ / min; the solid-liquid ratio during ethanol washing is 1:3-1:5 (w / v), and the washing time is ≤5 minutes.