CN107898829A - Advanced glycation end products decomposition agent - Google Patents

Abstract

The invention discloses an application of buckwheat husk flavone extract in the preparation of a glycosylation end product cracking agent. The buckwheat husk flavone extract is crushed, sieved, added to water, and subjected to ultra-high pressure treatment; filtered , concentrated in vacuum, and freeze-dried to obtain buckwheat husk flavonoid extract; use phosphate buffer to configure the solution as a splitting agent for glycation end products; construct an in vitro non-enzymatic glycosylation reaction system, and the results show that buckwheat husk flavonoids The extract cleaves glycation end products.

Description

Glycation end product splitting agent

technical field

The invention belongs to the field of health food and medicine, and in particular relates to a splitting agent for end product of glycation.

Background technique

Glycation end products (Advanced Glycation End Products, AGEs) refer to the end products obtained by non-enzymatic glycosylation of macromolecules such as reducing sugars and amino acids or lipids. Excessive accumulation of AGEs in the human body can lead to diabetes and other diseases. Complications, Alzheimer's disease and the development and progression of a range of cardiovascular diseases. A large number of studies have confirmed that AGEs are the fundamental substances that cause diabetic complications. The sources of AGEs are divided into two types: endogenous and exogenous. They are produced by the saccharification reaction of carbohydrates and proteins in living organisms. They are called endogenous AGEs. The formation of AGEs in the body can increase the utilization rate of glucose in the body. Exacerbate insulin resistance and oxidative stress response, cause changes in intracellular and extracellular vascular endothelial cells through various channels, and interfere with the normal function of the tissue structure of important organs; exogenous sources mainly come from foods rich in carbohydrates and fats, and other It is reported that smoking can produce AGEs, and exogenous AGEs ingested from food, beverage or cigarette smoke can significantly increase its concentration in blood, thereby triggering insulin resistance and accelerating tissue-specific damage. Therefore, the AGEs cracking agent which has the function of cutting off the AGE cross-linking between human tissue proteins and decomposing the AGEs that should be formed has become a new target for the treatment of various diseases.

In 1996, Vasan et al. first proposed the concept of AGEs cracking agent, which is defined as the substance that can decompose AGEs and can cut off the AGE crosslinks that have formed between AGEs tissue proteins. The first synthetic AGEs cleavage agent, N-benzoylthiazole bromide, was shown to reverse AGE crosslinks with diabetic collagen, releasing immunoglobulin G (IgG) crosslinked to the surface of erythrocytes in diabetic rats, And reverse the aggregation of amyloid fibrils formed by AGEs modification in vitro. However, in subsequent studies, due to the instability of the compound in aqueous solution, N-benzylidene thiazolium bromide failed in clinical application. Subsequently, Vasan proposed a compound ALT-711 (4,5-dimethyl-3-phenylacetylthiazole chloride) with a more stable structure and higher activity in 2003, which has completed phase II clinical research. A series of similar cracking agents for AGEs have also been developed, such as C24, C36 and C36D2. After the cleavage agent is combined with AGEs, it can react to form a structure that is easy to cleavage spontaneously, cut off the bridge of cross-linked structure formed by collagen and other biomacromolecules, make the protein free again, and then restore its own function. ALT-711 also reversed cardiovascular stiffness induced by AGE crosslinking in animal models of diabetes and hypertension. The research characteristic of AGEs cracking agent is that stable AGEs are first formed under a certain reaction system, and then cracking agent is added to crack, and the cracking agent does not participate in the whole process. In order to ensure that the working environment of the cleavage agent is stable and uninterrupted, it has higher requirements for the non-enzymatic glycosylation reaction system. At present, in the existing research reports on AGEs splitting agents, the research on the influencing factors of the reaction system mainly focuses on the reactants, temperature, incubation time for preparing AGEs, and the antibacterial mode of the non-enzymatic glycosylation system.

Buckwheat, a classic crop in northern my country, is rich in flavonoids. Buckwheat belongs to the genus Fagopyrum Mill of the Polygonacca family and is widely distributed in Asia and Europe. A lot of research evidence shows that buckwheat contains a lot of flavonoids such as rutin and quercetin, which have various pharmacological effects, such as preventing and treating diabetes and lowering blood fat. As a by-product of buckwheat processing, buckwheat husk is seriously lacking in high-value utilization technology, resulting in a great waste of resources. Buckwheat husk contains a large amount of flavonoids, and its inhibitory effect on the formation of AGEs has been fully confirmed in previous studies, but the cracking effect on AGEs has not been reported.

Contents of the invention

The object of the present invention is to provide a natural glycosylation end product splitting agent.

Application of buckwheat husk flavonoid extract in the preparation of glycosylation end product splitting agent;

The buckwheat husk flavone extract is prepared by the following method:

1) Crush the buckwheat husk, pass through a 60-80 mesh sieve, add water according to the mass ratio of material to liquid 1:5-20, and heat under high pressure;

2) Filtration, vacuum concentration at 55-65°C, and freeze-drying for 20-30 hours to obtain buckwheat husk flavone extract.

The high-pressure heating described in step 1) is heating at 121° C. for 15-25 minutes.

The high-pressure heating described in step 1) adopts an ultra-high pressure treatment device, the pressure is 100-400MPa, the pressure is maintained for 1-5min, and the high-pressure treatment is stable.

The pressure described in step 1) is 300MPa, and the pressure is maintained for 2 minutes.

The invention provides the application of buckwheat husk flavonoid extract in the preparation of glycosylation end product splitting agent. The buckwheat husk flavonoid extract is obtained by crushing buckwheat husk, sieving, adding water, and ultra-high pressure treatment; filtering, vacuum concentration, and freeze-drying to obtain the buckwheat husk flavonoid extract; using phosphate buffer solution to prepare the solution as sugar glycosylation end-product cleavage agent; the in vitro non-enzymatic glycosylation reaction system was constructed, and the results showed that buckwheat husk flavonoid extract could cleavage the glycosylation end-product.

Description of drawings

Figure 1 Fluorescence value of non-enzymatic glycosylation system, A is AGE-G-BSA system, B is AGE-F-BSA system;

Fig.2 The fluorescence value of AGE-G-BSA system cracked by BHFs; A is the concentration of 0.05mg/mL under the condition of _NaN3 ; B is the concentration of 0.20mg/mL under the condition of _NaN3 ; C is the concentration of 0.05mg under the condition of MSF /mL; D is when the concentration is 0.05mg/mL under MSF conditions;

Fig.3 The fluorescence value of AGE-F-BSA system cracked by BHFs; A is the concentration of 0.05mg/mL under the condition of _NaN3 ; B is the concentration of 0.20mg/mL under the condition of _NaN3 ; C is the concentration of 0.05mg under the condition of MSF /mL; D is when the concentration is 0.05mg/mL under MSF conditions;

Figure 4 The average molecular weight of protein in the non-enzymatic glycosylation system; A is the NaN ₃ condition of the AGE-G-BSA system; B is the MSF condition of the AGE-G-BSA system; C is the NaN ₃ condition of the AGE-G-BSA system; D is AGE-G-BSA system MSF conditions;

Figure 5 Glucose content in AGE-G-BSA system before and after BHFs cleavage;

Figure 6 AGEs fructose content in AGE-F-BSA system before and after BHFs cracking;

Fig.7 Content of free amino groups in AGEs in AGE-G-BSA system before and after BHFs cracking;

Fig. 8 Content of free amino groups of AGEs in AGE-F-BSA system before and after BHFs cleavage.

Detailed ways

The preparation of embodiment 1 glycosylation end product splitting agent

Grind the dried buckwheat husks with a high-speed grinder, pass through a 60-mesh sieve, put them into a clean beaker, add distilled water at a mass ratio of 1:10, heat at 121°C for 20 minutes under high pressure; filter, and extract the filter residue twice ; The filtrates were combined, concentrated in vacuo at 60°C to a paste, and freeze-dried for 24 hours to form a powder to obtain Buckwheat Hull Flavonoid extracts (BHFs); the Buckwheat Hull Flavonoid extracts were washed with phosphate buffered saline (PBS , 200mM, pH=7.4) was configured into a 0.05mg/mL solution, as a splitting agent for glycation end products (BHFs).

The preparation of embodiment 2 glycosylation end product cracking agent

Grind the dried buckwheat husks with a high-speed grinder, pass through a 60-mesh sieve, put them into a clean beaker, add distilled water at a mass ratio of 1:10, heat at 121°C for 20 minutes under high pressure; filter, and extract the filter residue twice ; The filtrates were combined, concentrated in vacuo at 60°C to a paste, and freeze-dried for 24 hours to form a powder to obtain Buckwheat Hull Flavonoid extracts (BHFs); the Buckwheat Hull Flavonoid extracts were washed with phosphate buffered saline (PBS , 200mM, pH=7.4) was configured into a 0.2mg/mL solution as a splitting agent for glycation end products (BHFs).

Example 3 Preparation of glycosylation end product splitting agent

Dry buckwheat husks, pulverize them with a high-speed grinder, pass through an 80-mesh sieve, add water at a mass ratio of 1:20, and use an ultra-high pressure treatment device to perform high-pressure stabilization treatment at a pressure of 300 MPa and hold for 2 minutes; filter and vacuum at 60°C Concentrate to a paste, freeze-dry for 24 hours to form a powder, and then obtain Buckwheat Hull Flavonoid extracts (BHFs); the Buckwheat Hull Flavonoid extracts are prepared with phosphate buffer (PBS, 200mM, pH=7.4) to 0.2 mg/mL solution as a splitting agent for glycation end products (BHFs).

Example 4 Establishment of in vitro non-enzymatic glycosylation system

1. Establishment of in vitro non-enzymatic glycosylation system under the condition of sodium azide

Under ultra-clean conditions, 10.00mg/ _mL bovine serum albumin (Bovine Serum Albumin, BSA ) solution and 500mM glucose (Glycation, G) or fructose (Fructose, F), add 10mL BSA solution to 5mL sugar solution to prepare glycosylation-glucose-bovine serum albumin system (AGE-G-BSA) and Glycosylation-fructose-bovine serum albumin system (AGE-F-BSA), after mixing evenly, react in the dark at 37°C for 6 months, during this period, respectively, in the 1st, 2nd, 3rd, 4th, 5th, 6th Some samples were taken out every month and temporarily stored at -80°C.

2. Establishment of non-enzymatic glycosylation system in vitro under the condition of microporous suction filtration

On the basis of the above conditions, the reaction reagents were sterilized by means of microporous membrane suction filtration (Micro-aperture suction filter, MSF). Specifically, the reaction reagents were prepared using NaN ₃ -free phosphate buffer solution (PBS, 200 mM, pH=7.4), and then the reaction reagents were sequentially passed through microporous membranes with pore diameters of 0.45 μm and 0.22 μm. Under the condition of ℃, the reaction was protected from light for 6 months. During this period, some samples were taken out at the 1st, 2nd, 3rd, 4th, 5th, and 6th months, and temporarily stored at -80℃, which were AGEs to be cleaved.

3. Cleavage of In Vitro Non-enzymatic Glycosylation System

Add 0.05 mg/mL and 0.20 mg/mL lysing agent for end-glycosylation products to the prepared AGE-G-BSA system and AGE-F-BSA system under ultra-clean conditions, respectively. The system was used as a blank group, and the same concentration of aminoguanidine (Aminoguanidine, AG) was used as a positive control. After mixing evenly, it was lysed at 37°C in the dark for 6 days, and the samples were stored at -80°C.

Example 5 Kinetic experiment of non-enzymatic glycosylation reaction system

AGEs are a kind of complex mixture, which contains a large number of fluorescent cross-linking substances, such as pentosidine, so the fluorescence value has become an important quantitative indicator of AGEs, and is widely used by researchers. Usually, the fluorescence intensity of the sample at an excitation wavelength of 355nm and an emission wavelength of about 460nm is used to express the degree of non-enzymatic glycosylation reaction and the amount of AGEs generated in the system. Use a fluorescent microplate reader at a gain of 50, an excitation wavelength of 355nm, and an emission wavelength of 460nm to measure the fluorescence value of the system before and after lysis, and use the following formula to calculate the lysis rate:

(Formula 1)

As shown in Figure 1A, in the AGE-G-BSA system, within the reaction time of 1-4 months, the fluorescence value of the system increased significantly with the increase of the reaction time ( p<0.05 ), when the reaction At the 5th month, the fluorescence value decreased significantly compared with the 4th month ( p<0.05 ), and the reaction increased again at the 6th month, which was 135450.50±50.00, which was significantly higher than the 4th and 5th month ( p <0.05 ) , reaching more than 3 times that at 1 month. The fluorescence values in the AGE-F-BSA system are shown in Figure 1B. Under the NaN ₃ condition, the fluorescence value of the reaction increased significantly within 3 months ( p<0.05 ), but when the reaction progressed to the 4th month, the fluorescence value of the system decreased significantly to 56.08% of that in the third month ( p <0.05 ), then the fluorescence value of the system increased significantly in the reaction time of 4.5.6 months ( p<0.05 ); while under MSF conditions, the fluorescence value of the system basically showed a gradual increase trend, reaching a maximum of 205647.50±731.00. The fluorescence value of AGE-F-BSA system was significantly higher than that of AGE-G-BSA system, which may be related to the relatively violent reaction between fructose and BSA and the high content of AGEs in the system.

Comparing the two different sterilization methods, the results show that the fluorescence value of MSF group is basically higher than that of NaN ₃ group, and the difference of fluorescence value under the two conditions is not obvious in the early stage of reaction (1st and 2nd month). After 3 months, there was a significant difference ( p<0.05 ), especially in the AGE-F-BSA system, when the reaction reached the 4th month, the fluorescence value of the MSF group was about 2.11 times that of the NaN ₃ group. In the following experiments, in order to simplify the experimental steps, representative systems with reaction times of 1, 3, and 6 months were selected to study the cracking effect of BHFs.

Example 6 Characteristic experiment of glycation end product splitting agent BHFs

1. The effect of BHFs on the fluorescence value of AGEs

(1) The effect of BHFs on the fluorescence value of AGE-G-BSA system

AG was chosen as a positive control. The effect of BHFs and AG on the fluorescence value of AGE-G-BSA system is shown in Figure 2. It can be seen from the figure that under the condition of NaN ₃ , the inhibition rate of BHFs showed a significant concentration-dose dependent relationship ( p<0.05 ), and its cleavage effect was significantly stronger than that of AG ( p<0.05 ). At a concentration of 0.05 mg/mL, BHFs had the strongest cracking effect on the system with a reaction time of 3 months. The cleavage effect is equivalent, the highest reaches 30.88±0.04% (the system reaction time is 6 months), which is 2.80 times of the cleavage rate of AG (11.04±0.46%) under the same conditions.

Similar conclusions can also be obtained under MSF conditions. Under the condition of concentration of 0.20mg/mL, the cracking effect of BHFs on the system with a reaction time of 6 months was the highest, up to 27.85±0.58%. In particular, under MSF conditions, 0.05 mg/mL of BHF and AG had a weak inhibitory effect on the system with a reaction time of one month, especially the cracking effect of BHFs was only 9.74% of that under NaN ₃ conditions. At the concentration of 0.20mg/mL, the cracking effect of BHFs and AG was weaker than that of NaN ₃ group.

(2) The effect of BHFs on the fluorescence value of AGE-F-BSA system

As shown in Figure 3, the effect of BHFs on the fluorescence value of AGE-F-BSA system was different from that of AGE-G-BSA system, and the inhibition rate of BHFs showed a significant concentration-dose-dependent relationship ( p<0.05 ). It can be seen from Fig. 3A and Fig. 3B that under the condition of NaN ₃ , when the concentration is 0.05 mg/mL, the effect of BHFs and AG on the fluorescence value of the system is relatively weak, while under the condition of 0.20 mg/mL, the The lysis effect of BHFs was significantly stronger than that of AG ( p<0.05 ), and the lysis effect on the system with a reaction time of 1 month was the strongest, which was 29.69±0.67%.

Similar conclusions can also be obtained under MSF conditions. At the concentration of 0.05mg/mL, the cracking effect of BHFs on the reaction time of 6 months was significantly higher than that of AG ( p<0.05 ), and when the concentration was 0.20mg/mL , the cracking effect of AG on the system was significantly weakened with the increase of the reaction time of the system ( p<0.05 ), which was consistent with the results under the NaN ₃ condition, and the cracking effect of BHFs was significantly stronger than that of AG ( p<0.05 ), which was consistent with The difference in the results under the NaN ₃ condition is that the cracking effect of BHFs on the system with a reaction time of 6 months is the strongest, which is 28.92±0.57%, which is lower than its highest cracking rate under the NaN ₃ condition.

2. The effect of BHFs on the molecular weight of AGEs protein

The protein molecular weight of the system before and after BHFs cleavage was analyzed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF/TOF) in positive ion mode, and the original data and spectra were exported by 4000SeriesExplorer V3.5 software.

(1) Non-enzymatic glycosylation system protein molecular weight

MALDI-TOF/TOF measures the molecular weight of the sample through the main peak in the spectrum of a single system point. The increase in molecular weight is significantly related to the extension of the main peak, indicating that a series of protein modifications are produced during the non-enzymatic glycosylation reaction. It can be seen from Figure 4 , the molecular weight of BSA used in the experiment was 66993.06Da. In the blank group, with the increase of reaction time, the molecular weight of the system gradually increased. Both under MSF conditions), compared with BSA, they increased by 1765.68Da and 747.33Da respectively. In contrast, it is not difficult to find that under the same reaction conditions, the average molecular weight of the AGE-G-BSA system is greater than that of the AGE-F-BSA system, and the molecular weight of the system under the MSF condition is always greater than that under the NaN ₃ condition.

(2) The effect of BHFs on the molecular weight of AGEs protein

The average molecular weight results are shown in Table 1. After adding 0.20mg/mL cracking agent, the molecular weight of the system increased significantly compared with the control group, and compared with the blank group, the maximum increase was 209.06Da, which was about 4 carboxyl groups. Among the 24 cracked systems, the molecular weight The samples with an increase of more than 45Da accounted for more than 60% of the total. The results showed that under the NaN ₃ condition, the molecular weight of the in vitro non-enzymatic glycosylation system after AG cleavage was larger than that of the BHFs group, but this result was completely opposite under the MSF condition. And in the AGE-G-BSA system, the degree of increase in the molecular weight of the cleavage agent under the NaN ₃ condition is greater than that under the MSF condition, especially in the system with a reaction time of 3 months, the addition of AG and BHFs under the NaN ₃ condition. The molecular weights are 67804.41Da and 67740.41Da respectively, which are respectively increased by 209.06Da and 145.06Da compared with the blank group, and this increase is only 25.98Da and 39.62Da under the MSF condition, that is, the increase under the _NaN3 condition is MSF 8.04 and 3.66 times under the condition. In other words, although the blank molecular weight of the AGE-G-BSA system under MSF conditions was greater than that under NaN ₃ conditions, the increase in molecular weight was relatively small after adding the cleavage agent. But this conclusion does not hold in the AGE-G-BSA system.

Table 1 Average molecular weight of AGEs protein before and after BHFs cleavage

Note: The data in the figure are expressed in the form of mean ± standard deviation (n=3), and different lowercase letters in the same index indicate significant differences among the data ( p<0.05 ) .

3. Effect of BHFs on reducing sugar content of AGEs

The contents of glucose and fructose in the system before and after lysis were measured using Nobio glucose assay kit (glucose oxidase-peroxidase method) and Sigma Frutose Assay Kit respectively.

(1) Effect of BHFs on AGEs glucose content in AGE-G-BSA system

The effect of BHFs on the glucose content in the AGE-G-BSA system is shown in Figure 5. In the blank system with a reaction time of 1 month, the glucose content was 0.76±0.02 mg/mL and 0.55 mg/mL under NaN ₃ and MSF conditions, respectively. ±0.01mg/mL, which is only 0.10% of the initial reaction concentration (30.00mg/mL), indicating that the utilization of glucose in the Maillard reaction has basically reached saturation in one month, and the subsequent reaction time is 3, 6 In the system of 1 month, the glucose content had no significant change ( p>0.05 ), and under the MSF condition, the glucose content of the system was lower than that under the NaN ₃ condition. Under the NaN ₃ condition, neither BHFs nor AG had a significant effect on the glucose content of the system ( p>0.05 ), while in the MSF condition system, for the systems with a reaction time of 3 and 6 months, the glucose content after cleavage of BHFs was significant Increased ( p<0.05 ), respectively 0.62mg/mL±0.01 and 0.75±0.01mg/mL, reaching 1.48 times and 1.80 times of the blank group.

(2) Effect of BHFs on AGEs fructose content in AGE-F-BSA system

It can be seen from Figure 6 that the fructose content in the AGE-F-BSA system is basically the same as the glucose content in the AGE- _G -BSA system. The initial concentration was 0.02%; but different from the change trend of glucose content, under the condition of NaN ₃ , when the reaction reached the sixth month, the fructose content in the system dropped significantly to 0.44±0.02mg/mL ( p<0.05 ) , only 69.84% of the system with a reaction time of 3 months, and this result was not significant under MSF conditions ( p>0.05 ). Similar to the change of glucose content in the AGE-G-BSA system, the fructose content in the AGE-F-BSA system under MSF conditions was also lower than that of the NaN ₃ group. The difference between the fructose content and the glucose content is that AG has a significant effect on the reaction time of 3 months under the conditions of NaN ₃ and MSF ( p< 0.05 ), and the fructose content is significantly increased to 1 1.21 times and 1.14 times ( p<0.05 ), however, BHFs significantly reduced the fructose content ( p<0.05 ) in the NaN ₃ condition reaction time of 3 months, only 0.38±0.01mg/mL , while in the system with a reaction time of 6 months, with the addition of BHFs and AG, the fructose content increased significantly ( p<0.05 ), especially in the system under the condition of NaN ₃ , the fructose content after the cracking of BHFs was 1.80 times of blank group ( p<0.05 ).

4. The effect of BHFs on the content of free amino groups in AGEs

The content of free amino groups in the system was determined by OPA method. Take 4.0mL of LOPA reagent in a test tube, add 200μL of the system, add the same volume of distilled water to the control group, mix well, and react in a water bath at 35°C for 2min, then use a microplate reader to measure the absorbance A ₃₄₀ of the system at 340nm. Lysine was used instead of samples to make a standard curve using the same method, which was y=-0.1211x+3.5487 (R ² =0.998, OD value in the range of 0.18-0.90), and the content of free amino groups was calculated according to the curve. Draw the standard curve with the lysine concentration (X) (0.18～0.90mg/mL) as the abscissa and the absorbance value A ₃₄₀ (Y) at 340nm as the ordinate, and the linear regression equation is y=-0.1211x+3.5487, R ² =0.998, the content of free amino groups is expressed as a percentage.

(1) Effect of BHFs on AGEs free amino group content in AGE-G-BSA system

The free amino group in the molecular structure of BSA is an active group necessary for the generation of fluorescent AGEs, so the change of the free amino group content is often used to indicate the degree of grafting reaction between protein and sugar. Figure 7 shows that in the AGE-G-BSA system, with the increase of non-enzymatic glycosylation reaction time, the content of free amino groups after BSA was glycosylated with reducing sugars was significantly reduced ( p<0.05 ), when the reaction reached the 6th At one month, the content of free amino groups in the system under NaN ₃ and MSF conditions were 10.28±0.01% and 6.32±0.39%, respectively. The content of free amino acid in the system under MSF condition was significantly lower than that under NaN ₃ condition ( p<0.05 ).

After the cracking of BHFs and AG, the content of free amino groups in the system increased significantly ( p<0.05 ), and the cracking effects of the two were equivalent ( p< 0.05 ), and there was no significant difference between the cracking effects of the two on each system ( p>0.05 ). The content of free amino groups in the system reacted under MSF conditions for 6 months reached the highest, which was 11.56±0.04%, while the content of free amino groups after AG cleavage under the same conditions was 11.46±0.05%, which were 1.82 times and 1.83 times that of the blank group, respectively. However, this result was 11.55±0.01% and 11.50±0.03% in the NaN ₃ condition with the same reaction time.

(2) Effect of BHFs on AGEs free amino group content in AGE-F-BSA system

It can be seen from Figure 8 that the change of the free amino group content in the AGE-F-BSA system blank group is different from that in the AGE-G-BSA system, and the AGE-F-BSA system is different in the reaction time of 1, 3, and 6 months. No significant change ( p>0.05 ), fluctuating in the range of 9.76%~11.41%, this result is higher than the free amino group content in AGE-G-BSA system under the same conditions. Similar to the AGE-G-BSA system, the content of free amino groups in the AGE-F-BSA system increased after adding the cracking agent. In the NaN ₃ conditional system, although BHFs and AG were sensitive to the reaction time of 1 and 3 months The system had no significant effect, but the content of free amino groups in the system with a reaction time of 6 months was significantly increased ( p<0.05 ), and the effects of the two systems were equivalent, 11.65±0.02% and 11.63±0.02%, respectively. In the MSF condition system, the cracking effect of AG on the system with a reaction time of 1 month was significantly stronger than that of BHFs ( p< 0.05 ). The content was significantly increased, and the cracking effect of the two was equivalent ( p<0.05 ).

Claims (5)

1. Application of buckwheat husk flavonoid extract in the preparation of glycosylation end product splitting agent. 2. The application according to claim 1, characterized in that: the buckwheat husk flavone extract is prepared by the following method: 1) Crush the buckwheat husk, pass through a 60-80 mesh sieve, add water according to the mass ratio of material to liquid 1:5-20, and heat under high pressure; 2) Filtration, vacuum concentration at 55-65°C, and freeze-drying for 20-30 hours to obtain buckwheat husk flavone extract. 3. The application according to claim 2, characterized in that: the high-pressure heating described in step 1) is heating at 121° C. for 15-25 minutes. 4. The application according to claim 2, characterized in that: the high-pressure heating described in step 1) adopts an ultra-high pressure processing device, the pressure is 100-400MPa, the pressure is maintained for 1-5min, and the high-pressure stable treatment is performed. 5. The application according to claim 4, characterized in that the pressure in step 1) is 300 MPa, and the pressure is maintained for 2 minutes.