CN103705620B - The preparation method of indissoluble combined state polyphenol in a kind of Fructus Litchi - Google Patents

Abstract

The invention discloses a method for preparing insoluble bound polyphenols in litchi. The method improves the traditional acid hydrolysis process by adding litchi pulp residues after extracting free polyphenols into the acid hydrolyzate, and hydrolyzing under magnetic stirring. Continue the ultrasonic treatment to obtain the extract, adjust the pH of the extract, centrifuge, collect the supernatant, add ethyl acetate to the supernatant for extraction, combine the organic phases, and rotary evaporate to dryness, that is, the solvent insoluble combination in litchi is prepared. state polyphenols. The method has the advantages of simple steps, short time consumption, low hydrolysis temperature and high yield of litchi insoluble bound polyphenols.

Description

A preparation method of insoluble bound polyphenols in litchi

technical field

The invention belongs to the technical field of litchi polyphenols, and in particular relates to a preparation method of insoluble bound polyphenols in litchi.

Background technique

A large number of recent epidemiological surveys suggest that increasing the intake of fresh vegetables and fruits is of great significance for reducing the incidence of cancer and other chronic diseases. Studies have also pointed out that polyphenols rich in fresh vegetables and fruits are important active ingredients for these health-promoting effects. Polyphenols are a large class of plant secondary metabolites widely present in roots, stems, leaves, flowers, fruits and seeds of plants. Plant polyphenols exist in two forms, free and bound. Free polyphenols refer to polyphenols that can be extracted by conventional solvent extraction methods and are not combined with other macromolecular substances in plant cells. This type of polyphenol is also mainly involved in research. In addition, some polyphenols in plants are covalently bound to macromolecules such as polysaccharides and cellulose on plant cell walls through ester bonds, which are called bound polyphenols. These phenolic substances cannot be extracted by conventional solvent extraction methods, therefore, these phenolic substances are often ignored in research. However, with the deepening of people's understanding of the health protection effect of phenolic substances, in recent years, researchers have found that the health protection effect of bound polyphenols has its own advantages. Since this bound polyphenol is protected by the bound macromolecular components, it will not be absorbed and destroyed by the human body in the upper digestive tract and enter the large intestine. It will be slowly released under the decomposition of bacteria and then absorbed into the blood through the mucosa, thus becoming human Sustained source of polyphenol supply in the body. Therefore, in-depth study of the composition and content of different plant-bound polyphenols is of great significance for a comprehensive understanding of the biological activities of plant polyphenols from different sources.

There are many extraction methods for plant-bound polyphenols, the commonly used ones are acid hydrolysis extraction and alkali hydrolysis extraction. At present, there are more researches on bound polyphenols in corn, rice and other grains, and researchers usually use alkaline hydrolysis extraction method to prepare their bound phenols. However, there are few studies on the extraction methods of bound phenols in fruits and vegetables. Researchers mostly use the alkaline hydrolysis extraction method of grains, and have not evaluated the extraction effect of alkaline hydrolysis extraction methods on fruit and vegetable bound phenols. Due to the large difference in the composition of phenolic substances in grains and fruits and vegetables, the former is dominated by phenolic acids, while the latter contains more flavonoids. Due to the difference in the composition of the phenolic substances, the alkaline hydrolysis method is not necessarily suitable for the extraction of fruit and vegetable bound phenolic substances. Therefore, for different materials, it is necessary to optimize and establish a suitable preparation method for binding phenols. In addition, the methods currently used to prepare bound polyphenols by alkali or acid-catalyzed hydrolysis all have disadvantages such as cumbersome operation steps, high temperature, and long time consumption. Therefore, there is an urgent need to improve the existing methods.

Litchi is an important subtropical characteristic fruit, which is deeply loved by consumers at home and abroad. Studies have found that the content of phenolic substances in litchi pulp is high, and has strong antioxidant activity, which is an important active ingredient. At the same time, the study also found that the content of bound phenolic substances in litchi pulp is relatively high, accounting for nearly 30% of the total phenolic content in some varieties. However, the above research results are also based on the direct application of alkaline hydrolysis to prepare bound polyphenols, and the preparation methods of bound phenols have not been compared and optimized.

Contents of the invention

The object of the present invention is to provide a method for preparing insoluble bound polyphenols in litchi, which has simple steps, short time consumption, low hydrolysis temperature and high yield of litchi bound polyphenols.

The above object of the present invention is achieved through the following technical scheme: a preparation method of insoluble bound polyphenols in litchi, comprising the following steps:

(1) Prepare acid hydrolyzate, take concentrated hydrochloric acid, vitamin C aqueous solution and methanol, and prepare acid hydrolyzate;

(2) Take the lychee pulp residue after extracting free polyphenols, add it to the acid hydrolyzate prepared in step (1), adjust the temperature to 55-65°C for 1 hour under magnetic stirring, then continue ultrasonic treatment for 20-40 minutes, and repeat the magnetic force Stirring hydrolysis and ultrasonic treatment 1-2 times to obtain the extract;

(3) Adjust the pH of the extract to 2-3, and collect the supernatant after centrifugation;

(4) Ethyl acetate was added to the supernatant for extraction, the organic phases were combined, and rotary evaporated to dryness to obtain insoluble bound polyphenols in litchi.

The present invention mainly improves the traditional acid hydrolysis method, mainly including reducing the concentration of hydrochloric acid in the acid hydrolyzate, adding vitamin C in the hydrolyzate as an antioxidant to prevent the oxidative decomposition of bound polyphenols in the extraction process; The hydrolysis time is to reduce the decomposition of bound polyphenols caused by heat treatment; in addition, heat extraction and ultrasonic extraction are combined to shorten the extraction time. After the above-mentioned various improvements, the extract yield of bound polyphenols can be increased and Increased cellular antioxidant activity of conjugated polyphenols.

In step (1) of the present invention, the volume percentage of concentrated hydrochloric acid is 20～35% (the concentration of concentrated hydrochloric acid is 2.4～4.2mol/L at this moment), the volume percentage of methyl alcohol is 40～60%, surplus is Vitamin C aqueous solution.

The mass percent content of vitamin C in the vitamin C aqueous solution of the present invention is 0.8-1.2%.

The extraction process of litchi pulp residue after free polyphenols is extracted in the step (2) of the present invention is preferably as follows: fresh litchi pulp is mixed with frozen acetone aqueous solution for beating, after stirring and extraction, homogenate treatment, and the homogenate is centrifuged to take the precipitate The product is extracted again with frozen acetone aqueous solution, and the remaining precipitate after two extractions is dried to obtain the litchi pulp residue after free polyphenols are extracted.

Using this method to extract free polyphenols, the extraction degree of free polyphenols is high, and most of the free polyphenols in fresh litchi pulp can be extracted, thereby providing a basis for obtaining the extraction efficiency of bound polyphenols in the next step.

The volume percentage of acetone in the frozen acetone aqueous solution according to the present invention is preferably 70-80%, and the mass volume ratio of fresh lychee pulp to the frozen acetone aqueous solution is preferably 1:2-3 (unit: g/mL) , the stirring extraction time is preferably 5-10min, and a refrigerated centrifuge is used for centrifugation, the centrifugal speed is preferably 3000-5000rpm, the temperature is preferably 4°C, and the centrifugation time is preferably 10-15min.

The optimum volume percentage of acetone in the frozen acetone aqueous solution according to the present invention is 80%, and the optimal mass-volume ratio of fresh lychee pulp to the frozen acetone aqueous solution is 1:2 (unit: g/mL), stirring The optimum extraction time is 5 minutes, and a refrigerated centrifuge is used for centrifugation. The optimum centrifugal speed is 3000 rpm, the temperature is 4° C., and the centrifugal time is 10 minutes.

In step (2) of the present invention, the mass volume ratio of litchi pulp residue to acid hydrolyzate is preferably 1:5-15 (unit: g/mL).

In the step (2) of the present invention, take the litchi pulp residue after extracting free polyphenols, add it to the acid hydrolyzate prepared in step (1), preferably adjust the temperature to 60°C for 1 hour under magnetic stirring, and then continue the ultrasonic treatment for 30 minutes, and repeat Magnetic stirring hydrolysis and ultrasonic treatment 1-2 times to obtain the extract; the power of the ultrasonic equipment during ultrasonic treatment is 450~750W.

During centrifugation in step (3) of the present invention, the rotational speed of the centrifugal equipment is preferably 3000-5000 rpm; the centrifugation time is preferably 10-15 min.

In the step (4) of the present invention, an equal volume of ethyl acetate is added to the supernatant for extraction, a total of 4 to 6 extractions, the organic phases are combined, and rotary steamed to dryness below 40°C, that is, the insoluble combined state of litchi is prepared. phenol.

Compared with prior art, the present invention has following advantage:

(1) The traditional acid hydrolysis method is cumbersome and time-consuming, and the extraction time is as long as 18 hours, while the method of the present invention is simple and time-consuming, and the extraction time can be shortened to within 4.5 hours;

(2) The traditional acid hydrolysis method requires extraction at a higher temperature (80°C); the extraction temperature is relatively low, and the extraction temperature can be lowered to below 60°C, thereby reducing the decomposition of bound polyphenols caused by heat treatment;

(3) The concentration of hydrochloric acid in the acid hydrolyzate is high in the traditional acid hydrolysis method, but the method of the present invention reduces the concentration of hydrochloric acid in the acid hydrolyzate, which can be reduced to 2.4mol/L;

(4) The method of the present invention combines heating extraction and ultrasonic extraction, which can greatly shorten the extraction time;

(5) Using the method of the present invention to extract bound polyphenols, the yield of bound polyphenols is significantly improved compared with the traditional acid hydrolysis method, and the antioxidant activity of cells is also significantly improved.

detailed description

Since the extraction efficiency of free polyphenols in litchi pulp will affect the evaluation of the extraction effect of bound polyphenols in pulp residues, in order to extract free polyphenols as completely as possible, the present invention has the following several different solvents for free polyphenols in litchi pulp. The extraction performance was compared.

Get 150g of fresh lychee pulp, select respectively 300mL of frozen methanol, ethanol, acetone and ethyl acetate as extraction solvents with a volume percentage of 80%, stir and crush the lychee pulp for 5min, and use a homogenizer 4 for the crushed lychee pulp mixture Homogenize for 5 minutes at ℃, centrifuge the obtained homogenate at 3000 rpm for 10 minutes in a low-temperature refrigerated centrifuge, separate the supernatant, resuspend the precipitate with 300 mL of the above-mentioned extraction solvent, and homogenize again at 4 °C for 5 minutes, centrifuge at 3000 rpm in a low-temperature refrigerated centrifuge for 10 minutes, and combine two centrifuges The supernatant was evaporated to dryness at 45°C. Dilute the extract to 50mL with an aqueous methanol solution with a submission percentage of 85%, and use it for the determination of the total phenol content by the Folin phenol method (the determination method is as described above). The specific results are shown in Table 1. It can be seen from Table 1 Find out, among four kinds of extraction solvents, the free state polyphenol that 80% acetone extracts obtains is the most, namely 80% acetone can more completely extract the free state polyphenol in litchi pulp, so the litchi used in the following examples of the present invention The pulp residues were obtained after extracting free polyphenols with 80% acetone.

Table 1 Total phenolic content in litchi pulp extracted by different extraction solvents (mg gallic acid equivalent/100g fresh pulp)

Example 1

(1) Preparation of acid hydrolyzate: concentrated hydrochloric acid: 0.8% vitamin C aqueous solution: methanol = 3:2:5 (v:v:v); among them, concentrated hydrochloric acid is a commercially available product, generally referring to concentrated hydrochloric acid with a concentration of 12mol/L. hydrochloric acid;

(2) Obtaining lychee pulp residue: to extract the lychee pulp residue after free polyphenols in fresh lychee pulp, the specific process of obtaining is: mix fresh lychee pulp with frozen 80% (volume percentage) acetone aqueous solution 1:2 (w/v, g/mL) mixing and beating, stirring and extracting for 5 minutes, the mixture was homogenized at 4°C for 5 minutes with a homogenizer, and the obtained homogenate was centrifuged at 3000 rpm for 10 minutes, and 80% frozen Acetone (volume percentage) aqueous solution is extracted again, and after two extractions, the remaining precipitate is evaporated to dryness, which is litchi pulp residue;

(3) Weigh 10 g of lychee pulp residue after extracting free polyphenols into a flat-bottomed flask, add 100 mL of acid hydrolyzate, and hydrolyze at 60 ° C for 1 h under magnetic stirring, then put the flask into an ultrasonic pulverizer for 450 w ultrasonic treatment for 0.5 h, repeat The above cycle is repeated twice (referring to the process of magnetic stirring hydrolysis and ultrasonic ultrasonic treatment);

(3) Add NaOH with a concentration of 10mol/L to the extract mixture to neutralize to pH2;

(4) Centrifuge the neutralized extract mixture at 3000rpm for 10min, and collect the supernatant;

(5) adding an equal volume of ethyl acetate to the supernatant for extraction, a total of 6 extractions, combining the organic phases to obtain an ethyl acetate extract;

(6) Rotate the ethyl acetate extract to dryness at 40°C to obtain 148mg of litchi bound polyphenol extract; yield of polyphenol extract (%) = dry powder weight of polyphenol extract/weighed in step 3 pomace weight;

(7) Dissolve the evaporated extract to 25mL in phosphate buffer to obtain an extract of lychee pulp bound phenolics, which is stored at -80°C until testing.

(8) Determination of polyphenol content in the extract: The polyphenol content was determined by the folin phenol method with reference to the literature report (DewantoV, WuXZ, AdomKK, LiuRH: Thermal Processing Enhances the Nutritional Value of Tomatoes by Increasing Total Antioxidant Activity. JAgric FoodChem 2002, 50 : 3010-3014), as follows: in a 5mL test tube Add 0.5 mL of distilled water, then add 125 μL of the above extract or 125 μL of gallic acid standard solution of different concentrations, and finally add 125 μL of Folin’s phenol reagent to each tube. After 6 minutes, add 1.25mL of ₇ % _Na2CO3 solution and 1mL of distilled water to the test tube, mix well, and react in the dark at room temperature for 90min, and measure the absorbance value with a UV-visible spectrophotometer at a wavelength of 760nm. The phenolic content is calculated in gallic acid equivalent.

(9) Determination of cellular antioxidant activity: refer to the literature method (WolfeKL, KangX, HeX, DongM, ZhangQ, LiuRH: CellularAntioxidantActivityofCommonFruits . JAgricFoodChem 2008, 56 : 8418-8426). HepG2 cells in the logarithmic growth phase were inoculated into black-bottom transparent 96-well plates with a cell density of 5×10 ⁴ /well, cultured in a CO ₂ incubator at 37°C for 24 hours, the culture medium was sucked off, and injected into each well. Add serum-free medium containing different concentrations of quercetin standards or the above-mentioned litchi pulp residue-bound polyphenol extract to be tested, and add DCFH-DA with a final concentration of 25 μM to the medium at the same time to continue culturing in a CO ₂ incubator For 1 h, only DCFH-DA was added to the serum-free medium replaced by the control wells and blank wells. Take out the culture plate, discard the medium in each well, immediately add 200 μL of balanced salt solution (HBSS) containing 600 μM AAPH, and add HBSS without AAPH to the blank well. Immediately place the culture plate in a multifunctional microplate reader preheated to 37°C to measure the fluorescence intensity. The absorption wavelength is 538nm, and the emission wavelength is 485nm, measured every 5 minutes for a total of 1h. Three replicate wells were set for each same treatment. According to the fluorescence intensity (Y) at different time points of each well and each time point (X), the area under the fluorescence curve was calculated. According to the change of the area under the curve, the CAA value of the sample was calculated with quercetin as a reference. For the convenience of comparison, the CAA value of the samples is expressed in μmol quercetin equivalent/100g pomace.

Example 2

(1) Preparation of acid hydrolyzate: concentrated hydrochloric acid: 1% vitamin C aqueous solution: methanol = 2.5:3:4.5 (v:v:v); among them, concentrated hydrochloric acid is a commercially available product, generally referring to concentrated hydrochloric acid with a concentration of 12mol/L. hydrochloric acid;

(2) Weigh 10g of lychee pulp residue after extracting free polyphenols into a flat-bottomed flask, add 100mL of acid hydrolyzate, and hydrolyze at 60°C for 1h under magnetic stirring, then put the flask into an ultrasonic pulverizer for 750w ultrasonic treatment for 0.5h, repeat The above cycle is 1 time (referring to the process of magnetic stirring hydrolysis and ultrasonic ultrasonic treatment);

(3) Add NaOH with a concentration of 10mol/L to the extract mixture to neutralize to pH3;

(4) Centrifuge the neutralized extract mixture at 3000rpm for 15min, and collect the supernatant;

(5) adding an equal volume of ethyl acetate to the supernatant for extraction, a total of 5 extractions, combining the organic phases to obtain an ethyl acetate extract;

(6) The ethyl acetate extract was evaporated to dryness at 40°C to obtain 140 mg of litchi bound polyphenol extract.

Example 3

(1) Preparation of acid hydrolyzate: concentrated hydrochloric acid: 1.2% vitamin C aqueous solution: methanol = 3:1:6 (v:v:v); among them, concentrated hydrochloric acid is a commercially available product, generally referring to concentrated hydrochloric acid with a concentration of 12mol/L. hydrochloric acid;

(2) Weigh 5g of lychee pulp residue after extracting free polyphenols into a flat-bottomed flask, add 75mL of acid hydrolyzate, and hydrolyze at 60°C for 1h under magnetic stirring, then put the flask into an ultrasonic pulverizer for 600w ultrasonic treatment for 0.5h, repeat The above cycle is repeated twice (referring to the process of magnetic stirring hydrolysis and ultrasonic ultrasonic treatment);

(3) Add 10mol/L NaOH to the extract mixture to neutralize to pH2;

(4) Centrifuge the neutralized extract mixture at 4000rpm for 10min, and collect the supernatant;

(5) adding an equal volume of ethyl acetate to the supernatant for extraction, a total of 4 extractions, combining the organic phases to obtain an ethyl acetate extract;

(6) Ethyl acetate extract was evaporated to dryness at 40°C to obtain 99.5 mg of litchi bound polyphenol extract.

In order to provide a methodological basis for comprehensive evaluation of the biological activity of litchi phenols, since the alkaline hydrolysis method is currently the most commonly used method for extracting bound phenols, the polyphenols in the bound state of litchi pulp extracted by the present invention and the bound polyphenols obtained by using the alkali hydrolysis method Polyphenols were compared in terms of total phenol content and antioxidant activity.

Comparative Example 1

Extraction by traditional alkaline hydrolysis method

The specific steps are as follows: collect the litchi pulp residue after extracting free polyphenols, add 2mol/L NaOH at a weight-to-volume ratio of 1:10 (g/mL), stir and hydrolyze at room temperature for 18 hours under nitrogen protection, and dissolve the mixture in concentrated hydrochloric acid. Blend to pH 2, centrifuge at 3000rpm for 15min, collect the supernatant, extract 6 times with an equal volume of ethyl acetate, combine the organic phases, spin evaporate to dryness at 35°C, dissolve the dry powder of the extract in PBS, and freeze at -80°C for testing.

Comparative example 2

Extraction by traditional acid hydrolysis method

The specific steps are as follows: collect the litchi pulp residue after extracting free polyphenols, add 6mol/L HCl at a weight-to-volume ratio of 1:10 (g/mL), place it in a closed container, and hydrolyze the mixture at 80°C for 18 hours under magnetic stirring , neutralized to pH 2 with 10mol/L NaOH, centrifuged at 3000rpm for 15min, collected the supernatant, extracted 6 times with an equal volume of ethyl acetate, combined the organic phases, rotary evaporated to dryness at 35°C, dissolved the dry powder of the extract with PBS,- Freeze at 80°C until testing.

In order to understand the extraction effect of this method on the bound polyphenols of litchi pulp, the bound polyphenols extracted by the method in Example 1 were compared with those extracted by the traditional alkali hydrolysis method (comparative example 1) and acid hydrolysis method (comparative example 2). The combined polyphenols in litchi pulp were compared in terms of total phenol content and cellular antioxidant activity.

The total phenol content of the litchi pulp bound phenolic substances extracted by the above three different methods (Example 1, Comparative Example 1 and Comparative Example 2) was determined by the Folin phenol method, and the cellular antioxidant capacity was determined by the CAA method. The results of the assay are shown in Table 2 below.

Table 2 The content and antioxidant activity of bound phenols in litchi pomace extracted by different methods

Note: When the letters next to the numbers in the same row in the table are different, it means the difference is statistically significant, p <0.05

From Table 2 above, it can be seen that the yield of bound polyphenols extracted from litchi pulp by the traditional alkaline hydrolysis method, the polyphenol content (i.e. purity) of the extract, and the antioxidant activity of cells are significantly lower than those of the traditional acid hydrolysis method, indicating that litchi pulp Pulp-bound polyphenols are more suitable to be prepared by acid hydrolysis. However, the traditional acid hydrolysis method is cumbersome and time-consuming. The extraction time is as long as 18 hours, and it needs to be extracted at a higher temperature (80°C).

In order to simplify the process of extracting bound polyphenols by acid hydrolysis, the present invention makes the following adjustments to the traditional acid hydrolysis method: ① reduce the concentration of HCl in the hydrolyzate to 2.4mol/L; ② add a certain concentration of vitamins to the hydrolysis reaction solution C acts as an antioxidant to prevent the oxidative decomposition of polyphenols during the extraction process; ③Reduce the extraction temperature from 80°C to 60°C to reduce the decomposition of polyphenols caused by heat treatment; ④Combine heating extraction and ultrasonic extraction to shorten the extraction time to 3- 4.5h (total extraction time, including 2-3 cycles of acid hydrolysis + ultrasound). According to the results in Table 1 above, the extract yield and cell antioxidant activity obtained by the improved acid hydrolysis extraction method are significantly improved compared with the traditional acid hydrolysis method.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplification should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims (8)

1. A preparation method for insoluble bound polyphenols in litchi, characterized in that it contains the following steps: (1) Prepare acid hydrolyzate, take concentrated hydrochloric acid, vitamin C aqueous solution and methanol, and prepare acid hydrolyzate; (2) Take the lychee pulp residue after extracting free polyphenols, add it to the acid hydrolyzate prepared in step (1), adjust the temperature to 55-65°C for 1 hour under magnetic stirring, then continue ultrasonic treatment for 20-40 minutes, and repeat the magnetic force Stirring hydrolysis and ultrasonic treatment 1-2 times to obtain the extract; (3) Adjust the pH of the extract to 2~3, and collect the supernatant after centrifugation; (4) Ethyl acetate was added to the supernatant for extraction, the organic phases were combined, and rotary evaporated to dryness to obtain insoluble bound polyphenols in litchi; In step (1), the volume percentage of concentrated hydrochloric acid in the acid hydrolyzate is 20-35%, the volume percentage of methanol is 40-60%, and the balance is vitamin C aqueous solution; The extraction process of lychee pulp residue after free polyphenols are extracted in step (2) is as follows: fresh lychee pulp is mixed with frozen acetone aqueous solution for beating, stirred and extracted, homogenized, and the homogenate is centrifuged, and the precipitate is frozen The acetone aqueous solution was extracted again, and the remaining precipitate after two extractions was dried to obtain the litchi pulp residue after free polyphenols were extracted. 2. The method for preparing insoluble bound polyphenols in litchi according to claim 1, characterized in that: the mass percentage of vitamin C in the vitamin C aqueous solution is 0.8-1.2%. 3. The preparation method of insoluble bound polyphenols in litchi according to claim 1, characterized in that: the volume percentage of acetone in the frozen acetone aqueous solution described in step (2) is 70-80%, The mass-volume ratio of fresh lychee pulp to frozen acetone aqueous solution is 1:2~3, and the stirring and extraction time is 5~10 minutes. A refrigerated centrifuge is used for centrifugation. 10~15min. 4. the preparation method of insoluble bound polyphenols in litchi according to claim 3 is characterized in that: the volume percentage composition of acetone in the described frozen acetone aqueous solution is 80%, fresh litchi pulp and frozen The mass volume ratio of the acetone aqueous solution is 1:2, the stirring and extraction time is 5 min, a refrigerated centrifuge is used for centrifugation, the centrifuge speed is 3000 rpm, the temperature is 4 °C, and the centrifugation time is 10 min. 5. The method for preparing insoluble bound polyphenols in litchi according to claim 1, characterized in that: in step (2), the mass-volume ratio of litchi pulp residue to acid hydrolyzate is 1:5-15. 6. The method for preparing insoluble bound polyphenols in litchi according to claim 1, characterized in that: in step (2), take litchi pulp residue after extracting free polyphenols, add the acid hydrolysis prepared in step (1) Under magnetic stirring, adjust the temperature to 60°C for 1 hour, then continue ultrasonic treatment for 30 minutes, and repeat the magnetic stirring hydrolysis and ultrasonic treatment 1-2 times to obtain the extract; the power of the ultrasonic equipment during ultrasonic treatment is 450~750W. 7. The method for preparing insoluble bound polyphenols in litchi according to claim 1, characterized in that: in step (3), the rotational speed of the centrifugal equipment is 3000-5000 rpm, and the centrifugation time is 10-15 minutes. 8. The method for preparing insoluble bound polyphenols in litchi according to claim 1, characterized in that: in step (4), an equal volume of ethyl acetate is added to the supernatant for extraction, and a total of 4 to 6 extractions are performed. The organic phases were combined and rotated to dryness at a temperature below 40° C. to obtain litchi insoluble bound polyphenols.