CN115317532A

CN115317532A - Application of rosemary alcohol extract in inhibiting glycosylated end products AGEs induced by glyoxal GO

Info

Publication number: CN115317532A
Application number: CN202211159775.4A
Authority: CN
Inventors: 许明峰; 曹颖; 韩佳慧; 朱芹
Original assignee: Hangzhou Normal University
Current assignee: Hangzhou Normal University
Priority date: 2022-09-22
Filing date: 2022-09-22
Publication date: 2022-11-11

Abstract

The invention discloses application of a rosemary alcohol extract in inhibiting glycosylation end products AGEs induced by glyoxal GO. The invention selects the rosemary alcohol extract as the glycosylation inhibitor, specifically studies the glycosylation inhibiting effect of the rosemary alcohol extract, establishes an in-vitro reaction system of WPI-GO, and explores the structural change and the functional change of the protein in the middle and later periods of the reaction system and the influence of the rosemary alcohol extract on the middle and later periods of the reaction system.

Description

Application of rosemary alcohol extract in inhibiting glycosylated end products AGEs induced by glyoxal GO

Technical Field

The invention belongs to the technical field of AGEs (advanced glycation end products) inhibitor preparation, and relates to application of a rosemary alcohol extract in inhibiting AGEs (advanced glycation end products) induced by glyoxal GO.

Background

Non-enzymatic glycosylation (NEG) is a process in which macromolecules with amino groups, such as proteins, and reducing sugars, such as glucose, undergo a series of complex reactions under non-enzymatic conditions to finally form irreversible non-enzymatic glycosylation end products (AGEs). In the process, oxidation reaction participates in glycosylation reaction, and the glycosylation reaction promotes the oxidation reaction, so that part of the existing methods for inhibiting glycosylation is completed by oxidation resistance.

The glycosylation end products are stable compounds formed by Maillard reaction of free amino and reducing sugar carbonyl, and the specific forming process is as follows: the method comprises the following steps of carrying out nucleophilic addition reaction on free amino groups of macromolecular substances such as proteins and nucleic acids and carbonyl groups of reducing sugars such as glucose under non-enzymatic conditions to generate reversible Schiff bases, carrying out Amadori rearrangement on the Schiff bases to generate stable aldehyde amine substances, carrying out dehydrogenation-oxidation rearrangement on the aldehyde amine substances to generate high-activity carbonyl compounds, and carrying out amino/sulfhydryl reaction on the carbonyl compounds, arginine, lysine and cysteine to finally generate AGEs.

The active carbonyl compounds RCCs mainly refer to α, β -unsaturated aldehydes, ketoaldehydes, and dialdehyde carbonyl compounds, including 3-deoxyglucosone (3-DG), acrolein (acrlein, ACR), glyoxal (GO), methylglyoxal (MGO), and the like. RCCs have very strong reactivity and can rapidly perform addition reaction with nucleophilic groups (such as sulfhydryl of cysteine, amino of lysine and arginine, and the like). Wherein GO and MGO are main precursors of fluorescent and non-fluorescent AGEs, are active intermediates formed in the Maillard reaction and caramelization process, and have the reaction activity 200-50000 times higher than that of glucose. Although they play a key role in flavor development, they react with free amino groups or thiol groups of protein side chains to generate AGEs, thereby generating toxic effects on the human body.

With the development of research, some drugs that inhibit AGEs by reacting with GO precursor, such as pyridoxamine, which is a natural component of vitamin B6, have been developed to inhibit the formation of fluorescent AGEs. The principle is that pyridoxamine and GO can rapidly react to generate Schiff bases in physiological environment, the Schiff bases are cyclized into a hemiaminal adduct through intramolecular reaction, and the two hemiaminal adducts finally form the five-membered ring adduct containing the piperazine ring. In this way, pyridoxamine inhibits the reaction of ribonuclease (RNase) with GO, protecting the activity of RNase, inhibiting the modification of lysine residues. There are also studies that have demonstrated that some plant extracts have certain AGEs inhibitory effects both in vivo and in vitro. However, many potential plant extracts, such as rosemary extract, have not been reported.

Rosemary is a well-known high-efficiency natural antioxidant, is widely applied to the fields of medicines, foods, cosmetics and the like, and is a spice frequently used in western food. The rosemary water soluble extract mainly comprises rosmarinic acid, caffeic acid, ferulic acid, L-ascorbic acid, hesperidin and isohesperidin. The fat soluble extract comprises carnosic acid, carnosol, ursolic acid, sesamol, rosmanol, rosmarin, etc. Wherein, carnosic acid and carnosol contribute more than 90% to the antioxidant property of rosemary extract, and carnosic acid is the active component with the strongest antioxidant capacity and the highest content. Other antioxidant components in rosemary extract include rosmarinic acid, rosmarinic phenol, etc., and various phenols in rosemary extract can be added to exhibit the whole antioxidant property. The oxidation resistance mechanism of rosemary is mainly to inhibit the peroxidation chain reaction by capturing peroxy radicals, thereby inhibiting the oxidation process. In the current research, rosemary extract is generally used as an antioxidant in grease, and no specific report on the aspects of protein antioxidation or glycosylation is found.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides application of a rosemary alcohol extract in inhibiting AGEs (advanced glycation end products) induced by glyoxal GO, a WPI-GO system is established by taking whey protein isolate abundantly existing in dairy products as a protein model and AGEs intermediate product GO as a representative, and the inhibition effect of the rosemary alcohol extract on the AGEs is proved by measuring some related indexes.

The preparation process of the rosemary alcohol extract comprises the following steps:

after being dried and crushed, the rosemary is placed in an extraction container, ethanol is added, and reflux extraction is carried out twice, wherein each time lasts for 1-2 hours; mixing the two extractive solutions, recovering ethanol, concentrating to obtain paste, drying, and pulverizing into fine powder to obtain rosemary alcohol extract; wherein the volume ratio of the air-dried and crushed rosemary to the ethanol is 1.

Preferably, the extraction time per reflux is 1 hour.

Preferably, the volume ratio of the air-dried and crushed rosemary to the ethanol is 1.

Preferably, the volume fraction of ethanol is 95%.

The second purpose of the invention is to provide an inhibitor of glycosylation end products AGEs induced by glyoxal GO, which comprises the rosemary alcohol extract.

The invention has the beneficial effects that:

the invention selects the rosemary alcohol extract as the glycosylation inhibitor, specifically studies the glycosylation inhibiting effect of the rosemary alcohol extract, establishes an in-vitro reaction system of WPI-GO, and explores the structural change and the functional change of the protein in the middle and later periods of the reaction system and the influence of the rosemary alcohol extract on the middle and later periods of the reaction system.

Drawings

FIG. 1 shows the inhibition of fluorescent AGEs by rosemary alcohol extracts of different concentrations;

FIG. 2 is the mercapto content of each treatment group; wherein C: WPI; f, WPI + GO; m0.1, WPI + GO + rosemary alcohol extract with final concentration of 0.1 mg/mL; m1: WPI + GO + rosemary alcohol extract with final concentration of 1 mg/mL; m2: WPI + GO + rosemary alcohol extract with final concentration of 2 mg/mL; significant difference (P < 0.05)

FIG. 3 shows the results of polyacrylamide gel electrophoresis of each treatment group; wherein 1: WPI;2 WPI +GO;3, WPI, GO + rosemary alcohol extract with final concentration of 0.1 mg/mL; 4: WPI + GO + rosemary alcohol extract with final concentration of 1 mg/mL; 5: WPI + GO + rosemary alcohol extract with final concentration of 2 mg/mL;

FIG. 4 shows the protein surface hydrophobicity for each treatment group; c: WPI; f, WPI + GO; m0.1, WPI + GO + rosemary alcohol extract with final concentration of 0.1 mg/mL; m1: WPI + GO + rosemary alcohol extract with final concentration of 1 mg/mL; m2: WPI + GO + rosemary alcohol extract with final concentration of 2 mg/mL; significant difference (P < 0.05)

FIG. 5 shows the solubility of whey protein isolates of each treatment group; c: WPI; f, WPI + GO; m0.1, WPI + GO + rosemary alcohol extract with final concentration of 0.1 mg/mL; m1: WPI + GO + rosemary alcohol extract with final concentration of 1 mg/mL; m2: WPI + GO + rosemary alcohol extract with final concentration of 2 mg/mL; significant difference (P < 0.05).

Detailed Description

The present invention is further analyzed with reference to the following specific examples.

The in vitro simulation reaction system is an effective method for researching AGEs inhibition mechanism, and has the advantages of short research period, easy control of reaction and the like. The WPI-GO system is adopted by taking whey protein isolate which exists in a large amount in dairy products as a protein model, incubating the whey protein isolate which participates in non-enzymatic glycosylation reaction and a glycosylation intermediate product GO for 2 hours at 75 ℃, and detecting indexes such as fluorescence AGEs content, protein structure and the like in the system during the test period to research the harmful effect of the generation of AGEs on the WPI physicochemical and functional characteristics and the inhibiting effect of rosemary alcohol extract on the AGEs. In addition, the WPI-GO model is mainly used to assess the middle-later stages of protein glycosylation, since it omits the conversion from sugar to carbonyl.

Under non-enzymatic conditions, the whey protein isolate is gradually saccharified in the environment of a high-activity carbonyl compound GO to generate glycosylated proteins, namely AGEs, and meanwhile, the concentration of the AGEs is gradually increased along with the progress of glycosylation reaction of the whey protein isolate.

Setting C, F, M _0.1 、M ₁ 、M ₂ Five experimental groups.

Group C is blank: 5mL of 7mg/mL whey protein isolate solution and the balance of PBS solution;

group F was control group: 5mL of 7mg/mL whey protein isolate solution, 2.56mM GO mother liquor and the balance of PBS solution;

M _0.1 group (2): whey protein isolate solution of 7mg/mL, GO mother liquor of 2.56mM, rosemary alcohol extract of 0.1mg/mL and PBS solution in balance, wherein the total volume is 5mL;

M ₁ group (2): 5mL of 7mg/mL whey protein isolate solution, 2.56mM GO mother liquor, 1mg/mL rosemary alcohol extract and the balance of PBS solution;

M ₂ group (2): 7mg/mL whey protein isolate solution, 2.56mM GO mother liquor, 2mg/mL rosemary alcohol extract, and the balance PBS solution, and the total volume is 5mL.

Example 1: inhibition rate of different content of rosemary alcohol extract on fluorescence AGEs

According to the fluorescence characteristic that AGEs generate an emission wavelength of 440nm under an excitation wavelength of 370nm, C, F and M are measured by a microplate reader _0.1 、M ₁ 、M ₂ Fluorescence intensities of reaction solutions of five experimental groups, total fluorescence AGEs were quantified. Calculating the inhibition rate of the rosemary alcohol extract on fluorescence AGEs according to the following formula:

inhibition ratio (%) = ((F0-F)/F0) × 100

In the formula: f0 denotes the fluorescent AGEs fluorescence of the negative control group, F denotes C, F, M _0.1 、M ₁ 、M ₂ Fluorescence AGEs fluorescence values for the five experimental groups.

TABLE 1 content of fluorescent AGEs in the simulated system

Note: a. b, c, d, e represent significance comparisons of values in the same column, with no significant difference for the same letter (P > 0.05) and significant differences for different letters (P < 0.05).

As can be seen from Table 1, whether the addition of rosemary alcohol extract has a significant effect on the content of fluorescent AGEs in the system (P < 0.05). And in a certain concentration range (0.1-2 mg/mL), the inhibition rate of fluorescent AGEs is dependent on the concentration of the rosemary alcohol extract. With the increase of the concentration of the rosemary alcohol extract, the inhibition rate of AGEs is also increased. However, the increase of the inhibition of the rosemary extract concentration from 0.1mg/mL to 1mg/mL was higher than the increase of the inhibition of the rosemary extract concentration from 1mg/mL to 2 mg/mL.

Comparative example 1: inhibition rate of rosemary aqueous extract on fluorescent AGEs

Air drying and crushing rosemary, placing the rosemary in an extraction container, adding 10 times of purified water, and performing reflux extraction twice for 1 hour each time; mixing the two extractive solutions, concentrating into paste, drying, and pulverizing into fine powder to obtain herba Rosmarini officinalis water extract.

Setting C, F, W _0.1 、W ₁ 、W ₂ Five experimental groups.

group F was control: 5mL of whey protein isolate solution of 7mg/mL, GO mother liquor of 2.56mM and PBS solution in balance;

W _0.1 group (2): 5mL of 7mg/mL whey protein isolate solution, 2.56mM GO mother liquor, 0.1mg/mL rosemary aqueous extract and the balance of PBS solution;

W ₁ group (2): whey protein isolate solution of 7mg/mL, GO mother liquor of 2.56mM, rosemary aqueous extract of 1mg/mL, and PBS solution in balance, wherein the total volume is 5mL;

W ₂ group (2): 7mg/mL whey protein isolate solution, 2.56mM GO stock solution, 2mg/mL rosemary extract, and the balance PBS solution, for a total of 5mL.

According to the fluorescence characteristic that AGEs generate an emission wavelength of 440nm under the excitation wavelength of 370nm, C, F and W are measured by a microplate reader _0.1 、W ₁ 、W ₂ Fluorescence intensity of reaction liquid of five experimental groups, total fluorescence AGEs is quantified. Calculating the inhibition rate of the rosemary aqueous extract on fluorescent AGEs according to the following formula:

inhibition ratio (%) = ((F0-F)/F0) × 100

In the formula: f0 denotes the fluorescent AGEs fluorescence of the negative control group, F denotes C, F, W _.1 、W ₁ 、W ₂ Fluorescence AGEs fluorescence values for the five experimental groups.

TABLE 2 content of fluorescent AGEs in the simulated system

As can be seen from table 2, whether the addition of rosemary aqueous extract has no significant effect on the fluorescent AGEs content in the system. And in a certain concentration range (0.1-2 mg/mL), the inhibition rate of fluorescent AGEs has no correlation with the concentration of rosemary aqueous extract. Compared with example 1, the rosemary alcohol extract has a remarkably stronger inhibition effect on AGEs than the rosemary aqueous extract.

Example 2: effect of Rosemary alcohol extract on protein thiol content variation in simulation System

AGEs undergo an oxidation process in the formation process, the protein thiol content is closely related to the protein oxidation degree, and the total thiol content of the oxidized protein is reduced. The sulfhydryl-containing compound can react with DTNB to break the disulfide bond of DTNB to produce 2-nitro-5-thiobenzoic acid (NTB-), which can be ionized in water at neutral or alkaline pH to produce NTB ^2- Divalent anions, such NTB ^2- The ions appear yellow. The thiol content of the protein was determined because 5-thiol-2 nitrobenzoic acid has a maximum absorption at 412 nm.

The non-enzymatic glycosylation and the oxidation reaction are closely related, the oxidation reaction participates in the glycosylation reaction, and the change of the sulfhydryl content of the protein is an important sign for whether the protein is oxidized or not. The glycosylation reaction can be divided into three stages, the last stage being the further reaction of the carbonyl compound with the free amino group. In the GO-WPI system, GO serving as one of the most important precursors of AGEs and WPI directly enter the third stage of the glycosylation reaction, and the oxidation condition of the direct reaction of GO and WPI is researched by measuring the change of the sulfhydryl content.

(1) Preparation of Tris-Gly:

10.4g of Tris, 6.9g of glycine and 1.2g of EDTA are accurately weighed in a test tube, and the volume is determined to 1000mL after ultrasonic dissolution.

(2) Preparation of 8M Urea-Tris:

48.048g of urea is accurately weighed, dissolved by Tris-Gly, the pH is adjusted to 8, and the volume is adjusted to 100mL.

(3) Preparation of a solution of 10mM 5,5' -dithiobis (2-nitrobenzoic acid) DTNB:

0.0198175g of DTNB was accurately weighed and dissolved in 5mL of Tris-Gly.

(4) Taking C, F and M with the concentration of 20mg/mL for 100ul _0.1 、M ₁ 、M ₂ Five groups of protein solutions to be detected are put into a 2mL centrifuge tube, 1600uL 8M urea-Tris is added, and then 200uL of DTNB solution is added. Meanwhile, as a control group, 100ul of a 20mg/mL protein solution to be detected was put into a 2mL centrifuge tube, and 1600uL 8M urea-Tris was added. And setting a blank control, using Tris-Gly to replace protein solution as a reagent blank, fully shaking all samples, and standing for thirty minutes at room temperature in a dark place. After standing, 200. Mu.L of the supernatant was pipetted into an ELISA plate and the absorbance at 412nm was measured. Using molar extinction coefficient 13600M ^-1 cm ^-1 The total thiol content was calculated.

The results in fig. 2 show that the addition of rosemary alcohol extract has no significant effect on the changes in sulfhydryl content due to glycosylation. The sulfhydryl content was higher than that of the blank control group, whether or not rosemary alcohol extract was added. Thus, from the results of this example, it is clear that oxidation is not significant when GO reacts directly with WPI. The reason why the thiol content rises after the glycosylation reaction is presumed to be because hydrolysis of the protein is promoted during the reaction, resulting in hydrolysis of disulfide bonds, and the thiol content increases.

Example 3: influence of rosemary alcohol extract on change of protein crosslinking aggregation condition in simulation system

The late glycosylation phase induces protein cross-linking and aggregation, and reducing sugars readily react with lysine and arginine residues to form intermolecular cross-links and high molecular weight protein aggregates. The polyacrylamide gel electrophoresis is adopted to separate proteins with different molecular weights in the electrophoresis gel, so that the molecular weight can be analyzed: judging whether cross-linking can be inhibited by adding rosemary alcohol extract. The method comprises the following steps:

the analysis was performed by polyacrylamide gel electrophoresis using 15% separation gel and 5% concentration gel. Mixing C, F and M _0.1 、M ₁ 、M ₂ Samples of five experimental groups are diluted to 4mg/mL, equal volume of loading buffer solution is added, after centrifugation, the samples are heated for 5min at 95 ℃, after cooling, the samples are centrifuged again, and the loading amount of each well is 10 mu L. Adopting a constant voltage mode, the voltage of the sample is 80V when the gel is concentrated, and the sample is adjusted after entering the separation gelThe node voltage is 120V. After the electrophoresis, the gel was stained with a staining solution (1 mg/mL Coomassie brilliant blue, 45% ethanol, 45% distilled water and 10% glacial acetic acid) for 20min, and then destained with a destaining solution (45% ethanol, 45% distilled water and 10% glacial acetic acid) for 1h.

The preparation process of the loading buffer solution comprises the following specific steps: mixing 2mL of Tris-HCl with the concentration of 1M and the pH value of 6.8, 4mL of glycerol, 0.8g of Sodium Dodecyl Sulfate (SDS) and 0.1mL of bromophenol blue with the mass fraction of 1%, and adding water to the mixture until the volume is 10mL to prepare a loading buffer solution.

The formulations of the separation gel and the concentrated gel are shown in Table 3.

TABLE 3 Split gum at 15% and concentrated gum at 5% formulations

	15% separation gel	5% concentrated gum
			ddH ₂ O	6.90mL	6.89mL
30％Acr-Bis	15.00mL	1.25mL
			Tris-HCl	(1.5M、PH8.8)7.5mL	(0.5M、PH6.8)1.25mL
10％APS	300uL	100uL
			10％SDS	300uL	100uL
TEMED	12uL	10uL
			In all	30.012mL	9.600mL

Whey protein is a generic term for the various protein components that remain in the supernatant when casein is precipitated out of milk when the pH of milk is lowered to 4.6, including beta-lactoglobulin, alpha-lactalbumin, milk serum albumin, and the like. The content of beta-lactoglobulin in whey is higher, the beta-lactoglobulin is mainly contained in whey protein, and the relative molecular mass is about 18200; the alpha-lactalbumin accounts for about 20 percent of the lactalbumin, has the relative molecular mass of 14175, contains 8 cysteine residues, forms 4 intramolecular disulfide bonds, and has stable structure; bovine serum albumin is about 5% of whey protein, and has a relative molecular mass of 66000.

The results in FIG. 3 show that the molecular domains of greater than 45kDa appear diffuse in coating, probably because WPI is a mixed protein, the composition is complex, and there is a varying degree of hydrolysis and cross-linking during incubation at 75 ℃ resulting in a large number of large proteins of varying sizes. And as can also be seen from fig. 3, after WPI reacts with GO, the bands of β -lactoglobulin, α -lactalbumin, bovine milk serum albumin became lighter in color, while the corresponding bands of the rosemary alcohol extract group with the concentrations of 0.1mg/mL and 1mg/mL became darker in color than the rosemary alcohol extract group without the addition of rosemary alcohol extract. This result shows that certain concentration of rosemary extract has certain protective effect on protein in glycosylation reaction.

Example 4: effect of Rosemary alcohol extracts on protein hydrophobicity variation in simulated System

Hydrophobic forces are non-covalent interactions that contribute to the formation and stabilization of the quaternary structure of proteins and have a significant impact on the maintenance of protein conformation. Saccharides have more hydroxyl hydrophilic groups, and after glycosylation reaction between protein molecules and saccharides, a great amount of hydroxyl groups can be introduced into the generated glycoprotein molecules, so that the hydrophilicity is increased. And protein cross-linking can result in a change in protein structure that exposes the original hydrophobic groups or the formation of larger polymers that result in tight encapsulation of the hydrophobic residues. Generally, 8-anilino-1-naphthalenesulfonic acid sodium salt (ANS) is used as a fluorescent probe for hydrophobicity to directly measure the hydrophobicity of protein. ANS can emit green fluorescence under ultraviolet irradiation, the fluorescence can be detected after the ANS is combined with protein, and the slope obtained by plotting the fluorescence value and the protein concentration is the protein surface hydrophobicity index. The method comprises the following steps:

the surface hydrophobicity characteristics were determined using ANS as a fluorescent probe. C, F, M were mixed with PBS buffer (10mM, pH 7.4) _0.1 、M ₁ 、M ₂ The samples in the five experimental groups were diluted to concentrations of 0.1, 0.2, 0.3, 0.4, 0.5mg/mL, respectively. After 3. Mu.LANS reagent was injected into the microplate, 0.2mL of the diluted protein sample was added thereto. The fluorescence intensity of the sample was measured at an excitation wavelength of 365nm and an emission wavelength of 484 nm.

Five protein concentrations within each group and the fluorescence values measured at that concentration were plotted and the slope calculated to obtain five results, as shown in figure 4. Crosslinking reactions and glycosylation modifications have varying degrees of influence on the surface hydrophobicity of proteins. The hydrophobicity results measured by the ANS method are mainly influenced by two factors: 1) The change in the number of hydrophobic groups causes a change in the number of groups bound to the fluorochrome 2) the difference in the hydrophobic regions exposed affects the ANS fluorescence intensity. Figure 4 results show that the hydrophobicity of the protein surface is significantly reduced after glycosylation of WPI. The reason may be that the structure of the protein molecule is changed during the glycosylation reaction, and part of the hydrophobic groups are embedded or combined, so that the hydrophobicity of the protein is reduced; or because the hydrophobic groups which are masked before are exposed to cause the protein to be polymerized and the crosslinking caused by glycosylation causes the hydrophobicity of the surface of the protein to be reduced. In addition, the addition of rosemary extract does not increase the hydrophobicity of the protein back, and 2mg/mL of rosemary extract causes the surface hydrophobicity of the protein to be further significantly reduced, presumably because the rosemary extract is further combined with some hydrophobic groups of the protein, so that the hydrophobicity of the protein is continuously reduced.

Example 5: effect of Rosemary alcohol extracts on protein solubility Change in the simulated System

Solubility of a protein (solubility) refers to the amount or level of dispersion of the protein in water. In certain cases, the protein is in the form of a solution or colloidal dispersion, and does not precipitate under low centrifugal force conditions. When proteins are oxidized, they cause a series of physicochemical changes that lead to amino acid breakdown, ultimately manifesting as reduced protein solubility. The effect of rosemary alcohol extract on inhibiting protein glycosylation and inhibiting protein oxidation is analyzed by measuring the protein solubility. Therefore, the change in the solubilization behavior of the protein can be used as an index for evaluating the degree of protein denaturation.

Compounds having two or more peptide bonds are all reacted with biurets, proteins and Cu in alkaline solution ²⁺ A purple complex is formed with an absorption maximum at 540 nm. Within a certain concentration range, the protein concentration is in direct proportion to the color shade of the biuret reaction, and can be quantitatively measured by a colorimetric method. The protein concentration of the stock solution and each centrifuged sample can then be calculated from the standard curve and finally the desired protein solubility (%) is obtained by the formula 100% centrifuged protein concentration/stock solution protein concentration.

The method comprises the following steps:

(1) Taking 300 mu LC, F and M _0.1 、M ₁ 、M ₂ Centrifuging the five experimental group samples at the rotating speed of 5000rab for 15min, taking 100 mu L of supernatant, adding 400 mu L of biuret respectively, and waiting for reaction for 30min. Simultaneously taking 100 mu LC, F and M _0.1 、M ₁ 、M ₂ Adding the five experimental group samples respectively400 μ L of biuret was added and the reaction was allowed to proceed for 30min.

(2) After the reaction is finished, 200 mu L of liquid to be detected is respectively put on an enzyme label plate, and the absorbance of the liquid is measured at the wavelength of 540 nm.

(3) BSA as standard protein, dissolved in ddH ₂ And O is used as a standard curve.

(4) Protein concentrations were calculated for each set of data before and after centrifugation as BSA standard curves.

In industrial applications, protein solubility is one of the important functional factors. Temperature, glycosylation modification can affect the structure of whey protein isolate, affecting its solubility. The main reasons for the changes in protein solubility during glycosylation are the following: 1. original hydrophobic groups of the protein are combined, and the solubility is increased due to the reduction of hydrophobicity; 2. the connection of sugar molecules increases the steric hindrance of the protein, so that the glycated protein is not easy to aggregate, thereby causing the increase of the solubility. 3. The reduced contact sites for water molecules and the exposure of hydrophobic groups make the protein more susceptible to aggregation, resulting in reduced solubility. The above conclusions indicate that: after the glycosylation reaction, the protein surface becomes less hydrophobic. However, as can be seen from fig. 5, the WPI solubility did not increase due to the decrease in surface hydrophobicity. The reason for this may be that protein cross-linking causes reduced contact sites between protein and water molecules, and reduced hydration; and the glycosylation reaction can affect the content and proportion of each amino acid, influence the polarity of WPI, and simultaneously change the charge to cause different aggregation conditions, and the factors influence the WPI together, thereby finally causing the reduction of the solubility of the WPI. In addition, as can be seen from figure 5, the addition of rosemary alcohol extract resulted in a recovery of WPI solubility. Therefore, to a certain extent, the rosemary extract can inhibit glycosylation reaction, and reduce the influence of glycosylation reaction on WPI solubility.

Example 6: effect of Rosemary alcohol extracts on protein digestibility variation in the model System

Protein digestibility is one of the indicators of protein nutritional value, and protein cross-linking affects digestibility. It is believed that the cross-linking reaction between protein molecules will reduce the digestion capacity of the protein in vitro, and the sugar group is introduced to facilitate the action of digestive enzyme. In addition, glycosylation modification does not change the secondary structure of the protein too much, but often changes the tertiary structure of the protein, so that the protein forms a 'fused' partially denatured structure, which is also beneficial to the digestion of the protein by protease. Protein cross-linking and the incorporation of sugar groups can have an effect on protein digestibility. Intramolecular and intermolecular cross-linking of proteins may result in tighter protein structures and hidden digestion sites, thereby reducing protein digestibility; the insertion of glycosyl group will often cause the extension of partial molecular structure of protein and increase of hydrophilicity, which is beneficial to the hydrolysis of protein and thus causes the increase of digestibility. Under the combined influence of these two factors, therefore, protein digestibility varies differently under different conditions.

The method comprises the following steps:

(1) Sample system A was prepared by taking 2mL of sample per tube, adjusting pH to 1.5, incubating at 37 deg.C for 10min, adding 90. Mu.l of pepsin, and reacting at 37 deg.C for 30min.

(2) Taking 30 microliters of the sample A in the step (1), adding 600 microliters of OPA solution, processing at 37 ℃ for 2min, and then carrying out absorbance determination at 370 nm;

(3) Treating the rest sample B of the sample A in the step (1) at 37 ℃ for 30min, taking 30 microliter of sample, adding 600 microliter of OPA solution, treating at 37 ℃ for 2min, and then carrying out absorbance determination at 370 nm;

(4) Adjusting the pH of the rest sample to 7, adding trypsin, reacting at 37 deg.C for 30min, adding 30 microliter sample into 600 microliter OPA solution, treating at 37 deg.C for 2min, and measuring absorbance at 370 nm; the procedure was repeated for the remaining samples at each time of the current step, thus obtaining absorbance values of the samples at five time points in total (30 min, 60min, 90min, 120min, 180 min).

TABLE 4 protein digestibility in the simulated system

Note: a. b represents the significance comparison of the values in the same treatment time, the same letter has no significance difference (P > 0.05), and different letters have significance difference (P < 0.05).

As can be seen from table 4, the digestibility of each treatment group increased with time. The WPI digestibility decreased after glycosylation reaction for 30-60 min (hydrolysis with pepsin), and increased after glycosylation reaction for 90-180 min (hydrolysis with trypsin). The reason is presumed to be that when pepsin is hydrolyzed, the electrostatic repulsive force is reduced due to the pH condition of pepsin, protein aggregation crosslinking is promoted, and the digestion site of pepsin is hidden, so that the digestibility is reduced; and when the trypsin is hydrolyzed, electrostatic repulsion is increased under the pH condition of the trypsin to prevent protein aggregation, and the protein structure is extended to be beneficial to the trypsin to play a role due to the access of glycosyl, so that the WPI can be better digested by the trypsin. In addition, as shown in table 3, since the effect of adding rosemary alcohol extract on digestibility is approximately opposite to that of glycosylation reaction (group F), the addition of rosemary alcohol extract has a certain effect of suppressing glycosylation reaction. However, significantly, neither glycosylation reaction nor addition of rosemary alcohol extract has significant influence on protein digestibility.

Claims

1. Application of rosemary alcohol extract in inhibiting glycosylated end product AGEs induced by glyoxal GO.

2. Use according to claim 1, wherein the rosemary alcohol extract is prepared by a process comprising:

3. The use according to claim 2, wherein the time for each reflux extraction is 1 hour.

4. Use according to claim 2, wherein the volume ratio of the air-dried and disintegrated rosemary to ethanol is 1.

5. The use according to claim 2, wherein the ethanol is present in a volume fraction of 95%.

6. An inhibitor of glycosylated end products AGEs induced by glyoxal GO, characterized by comprising rosemary alcohol extract.

7. The inhibitor of AGEs (advanced glycation end products) induced by glyoxal GO (GO) according to claim 6, wherein said rosemary alcohol extract is prepared by the process comprising:

8. The inhibitor for AGEs (advanced glycation end products) induced by glyoxal GO according to claim 7, wherein the time for each reflux extraction is 1 hour and the volume ratio of air-dried crushed rosemary to ethanol is 1.

9. The inhibitor of glyoxal GO-induced glycosylation end products AGEs according to claim 8, wherein the volume fraction of ethanol is 95%.