CN111007135B - Method for evaluating anti-saccharification effect of plant extract - Google Patents
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- CN111007135B CN111007135B CN201911365050.9A CN201911365050A CN111007135B CN 111007135 B CN111007135 B CN 111007135B CN 201911365050 A CN201911365050 A CN 201911365050A CN 111007135 B CN111007135 B CN 111007135B
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Abstract
The invention discloses a method for evaluating the anti-saccharification effect of a plant extract, which comprises a method for evaluating the prevention effect of protein saccharification crosslinking and a method for evaluating the inhibition effect of protein saccharification crosslinking. The invention can scientifically and effectively evaluate the anti-saccharification property of the plant extract; in the method for evaluating the prevention effect of protein saccharification and crosslinking, protein and reducing sugar are incubated to accurately simulate the saccharification process of protein in a human body environment, and the prevention effect of the plant extract on protein saccharification and crosslinking can be evaluated by comparing the relative molecular mass of a sample and a blank reference substance; in the evaluation method of the protein saccharification cross-linking inhibition effect, the structural characteristics of the protein saccharification of the skin tissue in the aging stiffness state are simulated by incubating the protein and the active aldehyde, and the saccharification cross-linking degree of a sample added with the plant extract is compared with that of a blank reference substance containing an anti-saccharification standard substance, so that the inhibition effect of the plant extract on the protein saccharification can be better evaluated.
Description
Technical Field
The invention relates to the technical field of biochemical engineering, in particular to an evaluation method for the anti-saccharification effect of a plant extract.
Background
Glycation is a non-enzymatic reaction of free amino groups in proteins with reducing sugars, also known as maillard reaction. In the skin, Glycation reactions produce new residues or cross-linking in the extracellular matrix of the dermis, producing Advanced Glycation End Products (AGEs). AGEs are a glycated protein whose formation and accumulation in human tissues is inevitable with age. The intermolecular glycation cross-linking of aging skin tissue causes the skin to age more rapidly and gradually lose elasticity, and the skin becomes stiff and dull. After the collagen in the dermis layer is saccharified, the elasticity of collagen fibers is reduced, so that the skin tension is lost, the skin is loosened, and wrinkles are formed.
At present, many anti-glycation functional ingredients are used in the cosmetic industry, but the method for evaluating the anti-glycation effect is not clear, and it is often difficult to distinguish the anti-oxidation function and the anti-glycation function of the functional ingredients. Therefore, aiming at the diversified demands of the market on the cosmetic products, the invention explores a method for evaluating the anti-saccharification effect of the plant extract so as to provide technical support for the development of anti-saccharification cosmetics.
Disclosure of Invention
In order to solve the above-mentioned drawbacks and disadvantages of the prior art, the present invention is directed to a method for evaluating the anti-glycation effect of a plant extract.
In order to realize the purpose, the invention adopts the technical scheme that:
a method for evaluating the anti-glycation effect of a plant extract, which comprises a method for evaluating the preventive effect of glycation cross-linking of a protein and a method for evaluating the inhibitory effect of glycation cross-linking of a protein.
The method for evaluating the preventive effect of glycation cross-linking of a protein comprises the following steps:
a 1: dissolving protein and reducing sugar in a solvent, adding a plant extract, uniformly mixing, and carrying out oscillation incubation for 15-30 days at 36-40 ℃ under the aseptic and anaerobic condition to obtain a sample;
a 2: preparing a blank control without the plant extract by the method of step a 1;
a 3: respectively taking equal amount of sample and blank control, performing SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis), and determining the relative molecular mass χ of protein in the sample 1 And the relative molecular mass χ of the protein in the blank 2 (ii) a Ruo X 1 Is less than x 2 Indicating that said plant extract has the effect of preventing cross-linking of glycation of proteinsOtherwise, it has no effect of preventing glycated crosslinking of proteins.
Preferably, in the step a1, the mass ratio of reducing sugar to protein (reducing sugar: protein) is more than 0 and less than or equal to 5. Experiments show that when the protein and the reducing sugar are in the ratio, the saccharification process of a human body can be simulated more truly, so that the prevention effect of the plant extract on protein saccharification crosslinking can be evaluated more accurately, and otherwise, the reliability is lower.
The method for evaluating the inhibition effect of protein glycation crosslinking comprises the following steps:
b 1: dissolving protein and active aldehyde in a solvent, adding a plant extract, uniformly mixing, and carrying out oscillation incubation for 15-30 days at 36-40 ℃ under the aseptic and anaerobic condition to obtain a sample;
b 2: dissolving protein and active aldehyde in a solvent, adding an anti-saccharification standard substance with the same mass as the plant extract, uniformly mixing, and carrying out oscillation incubation for 15-30 days at 36-40 ℃ under the sterile and anaerobic condition to obtain a blank reference substance;
b 3: respectively taking equal amount of sample and blank control, performing SDS-PAGE electrophoretic analysis, and determining relative molecular mass у of protein in the sample 1 And the relative molecular mass of the protein in the blank у 2 ；y 1 The closer to y 2 It is indicated that the plant extract has better effect of inhibiting the protein glycation cross-linking, otherwise the effect of inhibiting the protein glycation cross-linking is worse.
Preferably, in the step b1, the mass ratio of the active aldehyde to the protein (active aldehyde: protein) is greater than 0 and less than or equal to 10. Experiments show that when the protein and the active aldehyde are in the proportion, the process of protein saccharification of skin tissues in a human body aging rigid state can be simulated more truly, so that the inhibition effect of the plant extract on the protein saccharification can be evaluated more accurately, and otherwise, the reliability is lower.
Preferably, the method for evaluating the preventive effect on glycation cross-linking of protein further comprises: evaluating the strength of the preventive effect of glycation cross-linking by calculating a preventive index for glycation cross-linking, the larger the preventive index for glycation cross-linking, the stronger the preventive effect of glycation cross-linking, and the formula for the preventive index for glycation cross-linking includes:
in the formula, x 1 Is the relative molecular mass, χ, of the proteins in the sample 2 Relative molecular mass of the protein in the blank control.
Preferably, the method for evaluating the inhibitory effect on glycation cross-linking of a protein further comprises: evaluating the strength of the inhibition effect of the protein saccharification and crosslinking by calculating the inhibition index of the protein saccharification and crosslinking, wherein the greater the inhibition index of the protein saccharification and crosslinking, the greater the inhibition effect of the protein saccharification and crosslinking, and the calculation formula of the inhibition index of the protein saccharification and crosslinking comprises the following steps:
in the formula, у 1 Is the relative molecular mass of the proteins in the sample, у 2 Relative molecular mass of the protein in the blank control.
The protein and the reducing sugar are mixed for incubation, so that the saccharification process of the protein in the human body environment can be accurately simulated. After the protein is saccharified and cross-linked and polymerized, the relative molecular mass of the protein is increased, so that the content of SDS-extractable protein is reduced, and the content of non-extractable protein is increased, therefore, the comparison of chi 1 Hexix- 2 To evaluate whether the plant extract has the effect of preventing the glycated cross-linking of proteins.
The active aldehyde can accelerate the saccharification of protein, and the protein and the active aldehyde adopted by the invention are mixed and incubated, so that the structural characteristics of the protein saccharification of skin tissues in a human body aging and stiffness state can be simulated. Also, the protein was subjected to saccharification cross-linking polymerization, and the relative molecular mass of the protein was increased, so that the content of SDS-extractable protein was decreased and the content of non-extractable protein was increased, and thus у was compared 1 And у 2 To evaluate the inhibitory effect of plant extracts on protein glycation.
Preferably, in the method for evaluating the effect of preventing glycation cross-linking, the concentration of the plant extract is not less than 5% by mass.
Preferably, in the method for evaluating the inhibitory effect on protein glycation cross-linking, the mass percentage concentration of the plant extract is not less than 5%, and the mass percentage concentration of the anti-glycation standard substance is not less than 0.1%.
Preferably, in the method for evaluating a preventive effect on glycated protein cross-linking and the method for evaluating an inhibitory effect on glycated protein cross-linking, the plant extract is added in an amount, based on the volume of the solvent, of: less than or equal to 1g/mL or less than or equal to 1 mL/mL; otherwise the reliability of the evaluation method is low.
Preferably, the solvent used in the method for evaluating the preventive effect on protein glycation cross-linking and the method for evaluating the inhibitory effect on protein glycation cross-linking is Tris-HCl buffer solution at a pH of 6.8 and a concentration of 0.01 mol/L.
Preferably, the protein used in the method for evaluating the effect of preventing glycation cross-linking and the method for evaluating the effect of inhibiting glycation cross-linking is at least one of bovine serum albumin, human serum albumin, collagen, lactalbumin, casein and ovalbumin. The protein can better simulate various proteins in human blood and skin tissues, and is closer to the actual use condition.
Preferably, the reducing sugar used in the method for evaluating the effect of preventing glycation cross-linking is at least one of glucose, fructose, galactose, lactose and maltose. The reducing sugar is reducing sugar with high exposure rate in human diet or environment.
Preferably, the active aldehyde used in the method for evaluating the inhibitory effect on glycation cross-linking of proteins is at least one of glyoxal, methylglyoxal, glyceraldehyde and deoxyglucuronone. The above active aldehydes are active aldehydes widely present in the diet or human body, and are also the root cause of glycation.
Preferably, the anti-glycation standard substance used in the method for evaluating the effect of inhibiting the glycated protein by using is at least one of aminoguanidine, a compound related to aminoguanidine (including metformin), pyridoxamine, 2-isopropylidenediazino-4-oxothiazolidin-5-ylacetanilide (Invitrogen: 2-isopolylidenehydrno-4-oxo-thiazolidine-5-ylacetanilide, having a trade name of OPB-9195, CAS No. 163107-50-4), a benzothiazolium derivative (Invitrogen: Phenylthiazolium Derivatives), tenixitan, a flavonoid, ethanediase (Invitrogen: Glassase), Amadorases, Fructosamine-3-kinase (Invitrogen: F3-kinase, FN3K), and erythritol.
Preferably, the method for SDS-PAGE electrophoretic analysis in the method for evaluating the preventive effect on protein glycation cross-linking and the method for evaluating the inhibitory effect on protein glycation cross-linking is: diluting a sample or a blank reference substance to a protein content of 0.1-0.5 mg/mL, adding 1mL of buffer solution, uniformly mixing, standing at room temperature for 1h, centrifuging at 8000rpm for 10min, placing supernatant in a boiling water bath, heating for 5min, cooling, and injecting a sample to perform SDS-PAGE analysis; preferably, the buffer solution consists of the following components in percentage by mass: 10% SDS, 10% glycerol, 0.1% bromophenol blue and the balance Tris-HCl buffer at a concentration of 0.01mol/L, the pH of the Tris-HCl buffer being 6.8. According to the invention, the sample or blank reference substance is diluted to the protein content of 0.1-0.5 mg/mL, so that the requirement of SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis can be met, the experimental result is reliable, and if the concentration is too high, the band is too thick, and the reliable experimental result is difficult to obtain.
Compared with the prior art, the invention has the beneficial effects that: the method for evaluating the anti-saccharification effect of the plant extract can accurately evaluate the anti-saccharification effect of the plant extract. In the method for evaluating the prevention effect of protein saccharification and crosslinking, protein and reducing sugar are incubated to accurately simulate the saccharification process of the protein in a human body environment, different plant extracts are added, and a sample without the plant extracts is used as a blank control. SDS-PAGE results showed that the degree of glycated cross-linking of the proteins in the blank was high (i.e., greater relative molecular mass), while the degree of glycated cross-linking of the proteins in the samples with the plant extracts was lower (i.e., less relative molecular mass). Thus, the prevention effect of plant extracts on protein glycation cross-linking can be assessed by comparing the relative molecular mass sizes of the sample and the blank. Meanwhile, in the method for evaluating the inhibition effect of the protein saccharification cross-linking, the structural characteristics of the protein saccharification of the skin tissue in the aging and rigid state are simulated by incubating the protein and the active aldehyde, and different plant extracts are added, and an anti-saccharification standard substance is added as a blank control, and the SDS-PAGE result shows that the saccharification cross-linking of the protein in the blank control containing the anti-saccharification standard substance is greatly inhibited, so that the inhibition effect of the plant extracts on the protein saccharification can be better evaluated by comparing the saccharification cross-linking degree of a sample added with the plant extracts with that of the blank control containing the anti-saccharification standard substance.
Drawings
FIG. 1 is a schematic diagram of the glycated crosslinking of collagen or elastin to AGEs.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention is further illustrated by the following examples. It is apparent that the following examples are only a part of the embodiments of the present invention, and not all of them. It should be understood that the embodiments of the present invention are only for illustrating the technical effects of the present invention, and are not intended to limit the scope of the present invention.
Example 1
A method for evaluating the anti-glycation effect of a plant extract, comprising: (1) a method for evaluating the effect of preventing glycation crosslinking; (2) a method for evaluating the inhibitory effect of glycation cross-linking of a protein.
(1) A method for evaluating the preventive effect of glycation cross-linking of a protein, comprising the steps of:
a 1: accurately weighing 8.00mg of bovine serum albumin and 8.00mg of glucose, dissolving the bovine serum albumin and the glucose in 1mL of Tris-HCl buffer solution with the pH value of 6.8 and the concentration of 0.01mol/L, adding 1g of plant extract, uniformly mixing, adding 1 mu L of toluene or chloroform to prevent the growth of microorganisms, removing air in a container by using nitrogen or argon, and then oscillating and incubating for 15 days at 36-40 ℃ (40 ℃ in the embodiment) to prepare a sample;
a 2: preparing a blank control without adding plant extracts by the method of step a 1;
a 3: respectively taking an equal amount of sample and a blank control, diluting the sample and the blank control until the protein content is 0.5mg/mL, adding 1mL of buffer solution (0.01mol/L Tris-HCl buffer solution, pH 6.8, containing 10% SDS, 10% glycerol and 0.1% bromophenol blue), mixing uniformly, standing at room temperature for 1h, centrifuging at 8000rpm for 10min, heating the supernatant in a boiling water bath for 5min, cooling, introducing sample for SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoresis analysis, and determining the relative molecular mass chi of the protein in the sample 1 And the relative molecular mass χ of the protein in the blank 2 (ii) a Evaluating the strength of the protein saccharification crosslinking prevention effect by calculating a protein saccharification crosslinking prevention index, wherein the larger the protein saccharification crosslinking prevention index is, the stronger the protein saccharification crosslinking prevention effect is; the formula for calculating the glycation cross-linking prevention index of the protein comprises the following steps:
(2) a method for evaluating the inhibitory effect of glycation cross-linking of a protein, comprising the steps of:
b 1: accurately weighing 8.00mg of bovine serum albumin, dissolving the bovine serum albumin in 1mL of Tris-HCl buffer solution with the pH value of 6.8 and the concentration of 0.01mol/L, adding 0.2mL of glyoxal solution with the mass concentration of 40% and 1g of plant extract, uniformly mixing, adding 1 mu L of toluene or chloroform to prevent the breeding of microorganisms, removing the air in a container by using nitrogen or argon, and then carrying out oscillation incubation for 15 days at 36-40 ℃ (40 ℃ in the embodiment) to prepare a sample;
b 2: accurately weighing 8.00mg of bovine serum albumin, dissolving the bovine serum albumin in 1mL of Tris-HCl buffer solution with the pH value of 6.8 and the concentration of 0.01mol/L, adding 0.2mL of 40% glyoxal solution and 1g of aminoguanidine hydrochloride, uniformly mixing, adding 1 mu L of toluene or chloroform to prevent the breeding of microorganisms, removing the air in a container by using nitrogen or argon, and then oscillating and incubating for 15 days at 36-40 ℃ (40 ℃ in the embodiment) to prepare a blank control;
b 3: respectively taking equal amount of sample and blank control, and diluting the sample and blank control to obtain proteinThe content is 0.5mg/mL, then adding 1mL buffer solution (0.01mol/L Tris-HCl buffer solution, pH 6.8, containing 10% SDS, 10% glycerol and 0.1% bromophenol blue), mixing well, standing at room temperature for 1h, centrifuging at 8000rpm for 10min, heating the supernatant in boiling water bath for 5min, cooling, introducing sample for SDS-PAGE electrophoretic analysis, and determining the relative molecular mass of protein in the sample у 1 And the relative molecular mass of the protein in the blank у 2 (ii) a Evaluating the strength of the inhibition effect of the protein saccharification and crosslinking by calculating the inhibition index of the protein saccharification and crosslinking, wherein the greater the inhibition index of the protein saccharification and crosslinking, the greater the inhibition effect of the protein saccharification and crosslinking, and the calculation formula of the inhibition index of the protein saccharification and crosslinking comprises the following steps:
comparative example 1
The reliability of the evaluation method of example 1 was verified using a complete enzymatic method and an instrumental analysis method.
The method comprises the following steps:
(1) a method for evaluating the preventive effect of glycation cross-linking of a protein, comprising the steps of:
a 1: accurately weighing 8.00mg of bovine serum albumin and 8.00mg of glucose, dissolving in 1mL of Tris-HCl buffer solution with pH of 6.8 and concentration of 0.01mol/L, adding 1g of plant extract, uniformly mixing, adding 1 mu L of toluene or chloroform to prevent microorganism breeding, removing air in a container by using nitrogen or argon, and then oscillating and incubating for 15 days at 40 ℃ to prepare a sample;
a 2: preparing a blank control without adding plant extracts by the method of step a 1;
a 3: respectively taking an equal amount of sample and a blank reference substance, carrying out full enzymolysis on the sample and the blank reference substance by using pepsin, trypsin and aminopeptidase, purifying the sample and the blank reference substance by adopting a solid-phase extraction technology after centrifugation, and detecting the pentosan content in the purified sample and the blank reference substance by using liquid-phase mass spectrometry.
(2) A method for evaluating the inhibitory effect of glycation cross-linking of a protein, comprising the steps of:
b 1: accurately weighing 8.00mg of bovine serum albumin, dissolving the bovine serum albumin in 1mL of Tris-HCl buffer solution with the pH value of 6.8 and the concentration of 0.01mol/L, adding 0.2mL of glyoxal solution with the mass concentration of 40% and 1g of plant extract, uniformly mixing, adding 1 mu L of toluene or chloroform to prevent the breeding of microorganisms, removing the air in a container by using nitrogen or argon, and then oscillating and incubating for 15 days at the temperature of 40 ℃ to prepare a sample;
b 2: accurately weighing 8.00mg of bovine serum albumin, dissolving the bovine serum albumin in 1mL of Tris-HCl buffer solution with the pH value of 6.8 and the concentration of 0.01mol/L, adding 0.2mL of 40% glyoxal solution and 1g of aminoguanidine hydrochloride, uniformly mixing, adding 1 mu L of toluene or chloroform to prevent the breeding of microorganisms, removing the air in a container by using nitrogen or argon, and then oscillating and incubating for 15 days at 40 ℃ to prepare a blank reference substance;
b 3: respectively taking an equal amount of sample and a blank reference substance, carrying out full enzymolysis on the sample and the blank reference substance by using pepsin, trypsin and aminopeptidase, purifying the sample and the blank reference substance by adopting a solid-phase extraction technology after centrifugation, and detecting the pentosan content in the purified sample and the blank reference substance by using liquid-phase mass spectrometry.
Comparative example 2
Comparative example 2 is also a method for evaluating the anti-glycation effect of a plant extract, which is different from example 1 only in that: in the method for evaluating the preventive effect on glycated protein and crosslinking in comparative example 2, the amount of glucose added was 50.00 mg. In the method for evaluating the inhibitory effect on glycated protein cross-linking in comparative example 2, the amount of glyoxal solution whose mass concentration is 40% added is 0.5 mL.
Comparative example 3
Comparative example 3 is also a method for evaluating the anti-glycation effect of a plant extract, which is different from example 1 only in that: in the method for evaluating the effect of preventing the glycated protein from being crosslinked according to comparative example 3, the amount of the plant extract added was 2 g. In the method for evaluating the inhibitory effect on glycated protein cross-linking in comparative example 3, the amount of the plant extract added was 2 g.
Blueberry extract with the mass percentage concentration of 30% and tea extract with the mass percentage concentration of 30% are taken as plant extract samples. The glycation cross-linking inhibition index and the glycation cross-linking inhibition index of the two plant extracts were obtained by the methods of example 1 and comparative examples 1 to 3, respectively, and the results are shown in the following tables 1 to 3.
TABLE 1
As can be seen from the results in table 1 above, the blueberry extract and the tea extract both have excellent anti-glycation effects, and when the Duncan multiple comparison analysis method is used to compare the method of the present invention (i.e., example 1) with the instrumental analysis method (i.e., comparative example 1), the comparison shows that the glycation cross-linking prevention indexes of the proteins of example 1 and comparative example 1 are not significantly different (P > 0.05), and the glycation cross-linking inhibition indexes of the proteins of example 1 and comparative example 1 are not significantly different (P > 0.05), which indicates that the evaluation method of the present invention has high reliability and can accurately evaluate the anti-glycation effects of the plant extracts.
TABLE 2
Comparing example 1 and comparative example 2 using the Duncan multiple comparative analysis, it was found that example 1 and comparative example 2 have significant difference (P < 0.05) in the protein glycation cross-linking prevention index and example 1 and comparative example 2 have significant difference (P < 0.05) in the protein glycation cross-linking inhibition index, indicating that the reliability of the evaluation method of comparative example 2 is low, which also proves that: in the method of evaluating the preventive effect of glycation crosslinking of protein, when the mass ratio of reducing sugar to protein (reducing sugar: protein) is more than 5, the reliability of the evaluation method is low. In the method of evaluating the inhibitory effect on glycated protein cross-linking, when the mass ratio of the active aldehyde to the protein (active aldehyde: protein) is greater than 10, the reliability of the evaluation method is low. The method of the invention has higher reliability when used for evaluation.
TABLE 3
Comparing example 1 and comparative example 3 by means of Duncan multiple comparative analysis, it was found that the protein glycation cross-linking prevention index of example 1 is significantly different from that of comparative example 3 (P < 0.05), and the cross-linking prevention index of comparative example 3 is higher than that of example 1; the cross-linking inhibition index of protein glycation is significantly different from that of comparative example 3 in example 1 (P < 0.05), and the cross-linking prevention index of comparative example 3 is lower than that of example 1. It is shown that the evaluation method of comparative example 3 gives results with greater variability and lower reliability. This also demonstrates that: when preparing the sample, the addition amount of the plant extract should be less than or equal to 1g/mL or less than or equal to 1mL/mL, otherwise the reliability of the evaluation method is low.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (8)
1. A method for evaluating the anti-glycation effect of a plant extract, which comprises a method for evaluating the preventive effect of glycation cross-linking of a protein and a method for evaluating the inhibitory effect of glycation cross-linking of a protein;
the method for evaluating the effect of preventing glycation cross-linking of protein comprises the following steps a 1-a 3:
a 1: dissolving protein and reducing sugar in a solvent, adding a plant extract, uniformly mixing, and carrying out oscillation incubation for 15-30 days at 36-40 ℃ under the aseptic and anaerobic condition to obtain a sample;
a 2: preparing a blank control without the plant extract by the method of step a 1;
a 3: respectively taking equal amount of sample and blank control, performing SDS-PAGE electrophoretic analysis, and determining the relative molecular mass chi of protein in the sample 1 And the relative molecular mass χ of the protein in the blank 2 (ii) a Ruo X 1 Is less than x 2 Indicating that the plant extract has an effect of preventing glycated protein cross-linking and otherwise has no effect of preventing glycated protein cross-linking;
the method for evaluating the protein glycation crosslinking inhibition effect comprises the following steps b 1-b 3:
b 1: dissolving protein and active aldehyde in a solvent, adding a plant extract, uniformly mixing, and carrying out oscillation incubation for 15-30 days at 36-40 ℃ under the aseptic and anaerobic condition to obtain a sample;
b 2: dissolving protein and active aldehyde in a solvent, adding an anti-saccharification standard substance with the same amount as the plant extract, uniformly mixing, and carrying out oscillation incubation for 15-30 days at 36-40 ℃ under the sterile and anaerobic condition to obtain a blank reference substance;
b 3: respectively taking equal amount of sample and blank control, performing SDS-PAGE electrophoretic analysis, and determining relative molecular mass у of protein in the sample 1 And the relative molecular mass of the protein in the blank у 2 ；y 1 The closer to y 2 The better the plant extract can inhibit the protein saccharification crosslinking, otherwise, the lower the protein saccharification crosslinking inhibition effect;
in the step a1, the mass ratio of reducing sugar to protein (reducing sugar: protein) is more than 0 and less than or equal to 5; in the step b1, the mass ratio of the active aldehyde to the protein (active aldehyde: protein) is: greater than 0 and less than or equal to 10.
2. The method of evaluating an anti-glycation effect of a plant extract according to claim 1, wherein the method of evaluating a glycation cross-linking prevention effect of a protein further comprises: evaluating the strength of the preventive effect of glycation cross-linking by calculating a preventive index for glycation cross-linking, the larger the preventive index for glycation cross-linking, the stronger the preventive effect of glycation cross-linking, and the formula for the preventive index for glycation cross-linking includes:
in the formula, x 1 Is the relative molecular mass, χ, of the proteins in the sample 2 Relative molecular mass of the protein in the blank control.
3. The method of evaluating an anti-glycation effect of a plant extract according to claim 1, wherein the method of evaluating a glycation cross-linking inhibition effect of a protein further comprises: evaluating the strength of the inhibition effect of the protein saccharification and crosslinking by calculating the inhibition index of the protein saccharification and crosslinking, wherein the greater the inhibition index of the protein saccharification and crosslinking, the greater the inhibition effect of the protein saccharification and crosslinking, and the calculation formula of the inhibition index of the protein saccharification and crosslinking comprises the following steps:
in the formula, у 1 Is the relative molecular mass of the proteins in the sample, у 2 Relative molecular mass of the protein in the blank control.
4. The method of evaluating an anti-glycation effect of a plant extract according to claim 1, wherein a solvent used in the method of evaluating a preventive effect on protein glycation cross-linking and the method of evaluating an inhibitory effect on protein glycation cross-linking is Tris-HCl buffer solution at a pH of 6.8 and a concentration of 0.01 mol/L.
5. The method of claim 1, wherein the protein used in the method of assessing the effect of preventing glycation cross-linking of proteins and the method of assessing the effect of inhibiting glycation cross-linking of proteins is at least one of bovine serum albumin, human serum albumin, collagen, lactalbumin, casein, and ovalbumin.
6. The method of evaluating an anti-glycation effect of a plant extract according to claim 1, wherein a reducing sugar used in the method of evaluating an effect of preventing glycation cross-linking is at least one of glucose, fructose, galactose, lactose, and maltose.
7. The method of claim 1, wherein the active aldehyde used in the method of evaluating the effect of inhibiting glycation cross-linking of proteins is at least one of glyoxal, methylglyoxal, glyceraldehyde, and deoxyglucuronone.
8. The method for evaluating an anti-glycation effect of a plant extract according to claim 1, wherein the anti-glycation standard substance used in the method for evaluating an inhibitory effect on glycation cross-linking of a protein is at least one of aminoguanidine, an aminoguanidine-related compound, pyridoxamine, 2-isopropylidenediazinyl-4-oxothiazolidin-5-ylacetanilide, a benzothiazolium derivative, tenixitan, a flavonoid, glyoxalase, amadoriase, fructosamine-3-kinase, and erythritol.
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