Preparation method of pachyrhizus flower polysaccharide and application of pachyrhizus flower polysaccharide in anti-glycation and antioxidation

Abstract

The application discloses a preparation method of Pueraria lobata flower polysaccharide and application of the Pueraria lobata flower polysaccharide in anti-glycation and antioxidation, relates to the technical field of traditional Chinese medicine extraction, and is mainly applied to the medical, food and beauty industries. The Pueraria lobata dried flower is crushed into fine powder by using an impact type pulverizer, is sieved, is soaked and stirred for 30 min by using purified water, is adjusted to pH 3-5 by using dilute hydrochloric acid, is enzymolyzed at 40-55 DEG C for 60 min, is then raised to 80 DEG C to inactivate the enzyme, is filtered, the filtrate is collected, is concentrated by using a rotary evaporator, is precipitated by adding ethanol, is placed at 2-8 DEG C overnight, is filtered, the precipitate is collected, the precipitate is washed 2-3 times by using anhydrous ethanol, and is then dried under reduced pressure to obtain the Pueraria lobata flower polysaccharide. The application provides an enzymatic extraction process of Pueraria lobata flower polysaccharide. The Pueraria lobata flower polysaccharide prepared by using the method not only has an inhibiting effect on the generation of advanced glycation end products AGES, but also has a remarkable antioxidation effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine extraction, and particularly relates to a preparation method of Pachyrhizus flower polysaccharide and application of the Pachyrhizus flower polysaccharide in anti-glycation and anti-oxidation. BACKGROUND

[0002] With the development of society and the continuous improvement of people's living standards, the diet of Chinese residents has undergone tremendous changes, which has accelerated the generation of advanced glycation end products (AGES). Strictly speaking, AGEs can be mainly divided into two categories: endogenous AGEs and exogenous AGEs. Endogenous AGEs are mainly formed by the glycation reaction of biological macromolecules such as proteins in the body and reducing sugars. This type of glycation reaction is part of normal human metabolism. In other words, endogenous AGEs are continuously synthesized in the life process of organisms. However, the content of endogenous AGEs generated by normal metabolism of the human body is very limited. The level of AGEs in the normal human body tends to slowly increase with age. Exogenous AGEs are AGEs that are taken into the body from the external environment through various pathways.

[0003] When the generation of endogenous AGEs and the intake of exogenous AGEs exceed the maximum limit of the excretion capacity of the human body, AGEs will eventually accumulate in multiple organs and tissues of the body, including the skin, blood vessels, lens, retina, kidney, lung, liver, etc., and further cause a series of diseases. SUMMARY

[0004] The present application aims to at least solve one of the technical problems in the prior art, a preparation method of Pachyrhizus flower polysaccharide and application thereof.

[0005] The present application provides a preparation method of Pachyrhizus flower polysaccharide in the first aspect, comprising the following steps:

[0006] A preparation method of Pachyrhizus flower polysaccharide, comprising the following steps:

[0007] 1) Drying and crushing: dry the Pachyrhizus flowers, crush them into fine powder, sieve, and obtain Pachyrhizus flower powder;

[0008] 2) Enzymolysis: add purified water to the Pachyrhizus flower powder of step 1) according to a certain amount of liquid-to-solid ratio, soak and stir for 30 min, adjust the pH to 3-5 with a hydrochloric acid solution, add a compound enzyme, heat to 40-50℃, and enzymolysis for 60 min to obtain Pachyrhizus flower enzymolysis liquid;

[0009] 3) Enzyme inactivation: continue to heat the Pachyrhizus flower enzymolysis liquid obtained in step 2) to 70-80℃, heat for 30-60 min, and obtain enzyme inactivation extraction liquid;

[0010] 4) Microwave-assisted extraction: the enzyme-inactivated extraction solution of step 3) is subjected to microwave-assisted extraction for 30-60 min, repeated for 2-4 times, filtered, and the filtrates are combined to obtain the extract of Pueraria lobata flowers;

[0011] 5) Concentration: the extract of Pueraria lobata flowers obtained in step 4) is further concentrated under reduced pressure to a certain volume to obtain a concentrated extract of Pueraria lobata flowers;

[0012] 6) The concentrated extract of Pueraria lobata flowers obtained in step 5) is dissolved in ethanol and placed at 2-8℃ overnight, filtered, and the precipitate is washed with ethanol for 2-3 times;

[0013] 7) Drying: the precipitate obtained in step 6) is dried to obtain the powder of Pueraria lobata flower polysaccharides.

[0014] Preferably, in step 1), the sieving uses a sieve with a mesh size of 60-100.

[0015] Preferably, the ratio of the added purified water to the solution is 1:8-10 (g / ml).

[0016] Preferably, the complex enzyme is cellulase and protease in a mass ratio of 1:1-2.

[0017] Preferably, in step 2), the amount of the added complex enzyme is 0.2-0.5 wt%.

[0018] Preferably, in step 4), the filtration is a 100-200 um flat plate filtration.

[0019] Preferably, in step 5), the concentration volume is 1 / 3-1 / 4 times of the original solution; and / or;

[0020] The ethanol is 75-90% (v / v) ethanol; and / or;

[0021] In step 7), the drying is vacuum reduced pressure drying.

[0022] The present application also provides a powder of Pueraria lobata flower polysaccharides prepared by any of the above preparation methods.

[0023] The present application also provides a powder of Pueraria lobata flower polysaccharides which has an inhibitory effect on the generation of advanced glycation end products (AGES).

[0024] The present application also discloses the above-mentioned powder of Pueraria lobata flower polysaccharides in the application of antioxidant.

[0025] The present application has the following beneficial effects:

[0026] The pueraria flower polysaccharide of the present application is obtained by water extraction of dried pueraria flower, addition of appropriate amount of enzyme and enzyme hydrolysis followed by water extraction and alcohol precipitation. The process can not only increase the content of polysaccharide, but also has lower reaction temperature and shorter extraction time. The pueraria flower polysaccharide prepared by the method has inhibitory effect on the generation of advanced glycation end products (AGES) and significant antioxidant effect. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The infrared spectrum of the pueraria flower polysaccharide obtained in Example 1 is shown in Figure 1.

[0028] Figure 2 The standard curve for determination of the content of the pueraria flower polysaccharide obtained in Example 1 is shown in Figure 2.

[0029] Figure 3 The DPPH scavenging capacity of the pueraria flower polysaccharide obtained in Example 1 is shown in Figure 3.

[0030] Figure 4 The standard curve for determination of AGES of the pueraria flower polysaccharide obtained in Example 1 is shown in Figure 4. DETAILED DESCRIPTION

[0031] The present application will be described in detail below with reference to specific examples. The following examples will help those skilled in the art to further understand the present application, but do not limit the present application in any form. It should be noted that those skilled in the art can make several changes and improvements without departing from the concept of the present application. These all belong to the protection scope of the present application.

[0032] DPPH mentioned in the specification refers to 1,1-diphenyl-2-trinitrophenylhydrazine.

[0033] Example 1

[0034] The present embodiment provides a preparation method of pueraria flower polysaccharide, comprising the following steps:

[0035] Step 1: The pueraria flower is first dried and then crushed into fine powder with an impact crusher, and sieved through a No. 4 sieve (65 mesh) to obtain pueraria flower powder.

[0036] Step 2: The pueraria flower powder is added to purified water at a solid-liquid ratio of 1:8 (g / ml), soaked and stirred for 30 min, then the pH is adjusted to 3 with dilute hydrochloric acid (15 wt%), and 0.2 wt% of a composite enzyme (cellulase and protease at a weight ratio of 1:1) is added. The mixture is heated to 40℃ for enzyme hydrolysis for 60 min, and then the enzyme hydrolysis liquid is further heated to 70℃ for 60 min to inactivate the enzyme.

[0037] Step 3: The inactivated pueraria flower enzyme hydrolysis liquid is subjected to microwave-assisted extraction for 30 min, repeated twice, and then filtered through a 100 um flat membrane to obtain the pueraria flower extract.

[0038] Step 4 The obtained kudzu flower extract solution was further concentrated under reduced pressure to 1 / 3 of the original volume to obtain a kudzu flower concentrate.

[0039] Step 5 The kudzu flower concentrate was dissolved in 80% (v / v) ethanol at 2°C and allowed to stand overnight, filtered, and the precipitate washed with ethanol twice.

[0040] Step 6 The precipitate was dried under reduced pressure to obtain a kudzu flower polysaccharide extract.

[0041] Example 2

[0042] Step 1 The dried kudzu flower was first ground into fine powder using an impact grinder, and sieved through a No. 5 sieve (80 mesh) to obtain kudzu flower powder.

[0043] Step 2 The kudzu flower powder was added to purified water at a solid-to-liquid ratio of 1:10 (g / ml), soaked and stirred for 30 min, and then adjusted to a pH of 4 using dilute hydrochloric acid (15 wt%), and 0.3 wt% of a composite enzyme (cellulase and protease at a weight ratio of 1:2) was added, and the mixture was heated to 45°C for enzymatic hydrolysis for 60 min to obtain a kudzu flower enzymatic hydrolysate, which was further heated to 75°C for 45 min to inactivate the enzyme.

[0044] Step 3 The kudzu flower enzymatic hydrolysate after enzyme inactivation was subjected to microwave-assisted extraction for 45 min, repeated twice, and filtered through a 200 um flat membrane, and the filtrate was combined to obtain a kudzu flower extract solution.

[0045] Step 4 The obtained kudzu flower extract solution was further concentrated under reduced pressure to 1 / 3 of the original volume to obtain a kudzu flower concentrate.

[0046] Step 5 The kudzu flower concentrate was dissolved in 90% (v / v) ethanol at 4°C and allowed to stand overnight, filtered, and the precipitate washed with ethanol three times.

[0047] Step 6 The precipitate was dried under reduced pressure to obtain a kudzu flower polysaccharide extract.

[0048] Example 3

[0049] Step 1 The dried kudzu flower was first ground into fine powder using an impact grinder, and sieved through a No. 6 sieve (100 mesh) to obtain kudzu flower powder.

[0050] Step 2 The kudzu flower powder was added to purified water at a solid-to-liquid ratio of 1:9 (g / ml), soaked and stirred for 30 min, and then adjusted to a pH of 3.5 using dilute hydrochloric acid (15 wt%), and 0.4 wt% of a composite enzyme (cellulase and protease at a weight ratio of 1:2) was added, and the mixture was heated to 50°C for enzymatic hydrolysis for 60 min to obtain a kudzu flower enzymatic hydrolysate, which was further heated to 80°C for 60 min to inactivate the enzyme.

[0051] Step 3 The enzyme-inactivated Pueraria lobata flower enzymatic hydrolysate was microwave-assisted extracted for 45 min, repeated twice, filtered through a 200 um flat membrane, and the filtrate was combined to obtain the Pueraria lobata flower extract.

[0052] Step 4 The obtained Pueraria lobata flower extract was further concentrated under reduced pressure to 1 / 3 of the original volume to obtain a Pueraria lobata flower concentrate.

[0053] Step 5 The Pueraria lobata flower concentrate was dissolved in 95% (v / v) ethanol at 4°C overnight, filtered, and the precipitate was washed with ethanol for 3 times.

[0054] Step 6 The precipitate was dried under reduced pressure to obtain the Pueraria lobata polysaccharide extract.

[0055] Comparative Example 1

[0056] Step 1 The dried Pueraria lobata flower was first crushed into fine powder using an impact crusher, sieved through a No. 4 sieve to obtain Pueraria lobata flower powder.

[0057] Step 2 The Pueraria lobata flower powder was added to purified water at a solid-liquid ratio of 1:8 (g / ml), heated to reflux at 90-100°C for 1 h, repeated twice, filtered through a 100 um flat membrane, and the filtrate was combined to obtain the Pueraria lobata flower extract.

[0058] Step 3 The obtained Pueraria lobata flower extract was further concentrated under reduced pressure to 1 / 3 of the original volume to obtain a Pueraria lobata flower concentrate.

[0059] Step 4 The Pueraria lobata flower concentrate was dissolved in 80% (v / v) ethanol at 2°C overnight, filtered, and the precipitate was washed with ethanol for 2 times.

[0060] Step 5 The precipitate was dried under reduced pressure to obtain the Pueraria lobata polysaccharide extract.

[0061] Comparative Example 2

[0062] Step 1 The dried Pueraria lobata flower was first crushed into fine powder using an impact crusher, sieved through a No. 5 sieve (80 mesh) to obtain Pueraria lobata flower powder.

[0063] Step 2 The Pueraria lobata flower powder was added to purified water at a solid-liquid ratio of 1:10 (g / ml), heated to reflux at 90-100°C for 1 h, repeated twice, filtered through a 100 um flat membrane, and the filtrate was combined to obtain the Pueraria lobata flower extract.

[0064] Step 3 The obtained Pueraria lobata flower extract was further concentrated under reduced pressure to 1 / 3 of the original volume to obtain a Pueraria lobata flower concentrate.

[0065] Step 4 The Pueraria lobata flower concentrate was dissolved in 90% (v / v) ethanol at 4°C overnight, filtered, and the precipitate was washed with ethanol for 3 times.

[0066] Step 5 The precipitate was dried under reduced pressure to obtain the Pueraria lobata polysaccharide extract.

[0067] Comparative Example 3

[0068] Step 1 The dried flower of Pueraria lobata was first crushed into fine powder by using an impact crusher, and then sieved through a No. 6 sieve (100 mesh) to obtain the Pueraria lobata flower powder.

[0069] Step 2 The Pueraria lobata flower powder was added to purified water at a solid-to-liquid ratio of 1:9 (g / ml), and heated to reflux at 90-100°C for 1 hour, repeated twice, and then filtered through a 100-μm flat filter membrane to obtain the Pueraria lobata flower extract.

[0070] Step 3 The Pueraria lobata flower extract was further concentrated under reduced pressure to 1 / 3 of the original volume to obtain the Pueraria lobata flower concentrate.

[0071] Step 5 The Pueraria lobata flower concentrate was dissolved in 95% (v / v) ethanol at 4°C overnight, filtered, and the precipitate was washed with ethanol three times.

[0072] Step 6 The precipitate was dried under reduced pressure to obtain the Pueraria lobata flower polysaccharide extract.

[0073] The Pueraria lobata flower polysaccharide extraction rates in the above examples and comparative examples are shown in Table 1.

[0074] Table 1 Pueraria lobata flower polysaccharide extraction rates

[0075] Group Extraction rate (%) Example 1 16.1 Example 2 16.7 Example 3 17.3 Comparative Example 1 12.2 Comparative Example 2 11.2 Comparative Example 3 12.5

[0076] Note: The extraction rate calculation formula is: Extraction rate = (mass of Pueraria lobata flower polysaccharide / mass of Pueraria lobata flower powder) * 100%

[0077] Test Example 1

[0078] I. Infrared Spectroscopy Analysis

[0079] 5 mg of the Pueraria lobata flower polysaccharide was mixed with a certain amount of dry KBr powder, and then ground uniformly and pressed into a tablet. The prepared tablet was placed in a Fourier transform infrared spectrometer, and infrared scanning was performed in the range of 4000-400 cm -1 -1. The obtained infrared absorption spectrum was analyzed. Generally, the absorption peak in the range of 3600-3200 cm -1 is the stretching vibration of the intramolecular hydroxyl O-H bond. The stretching vibration absorption peak of the C-H bond is in the range of 3000-2800 cm -1 . These two regions are characteristic absorption peaks of sugars. The region of 1200-800 cm -1 is the fingerprint region of polysaccharides, which can be used to determine the type and configuration of sugars. The results are shown in Table 1. Figure 1 -1 A broad and strong O-H absorption peak appeared at 3408 cm -1 ​The stretching vibration peak of C-H appeared, the appearance of the two peaks indicated that the Pueraria lobata flower polysaccharide contained the characteristic absorption peak of polysaccharide compounds, 1642 cm -1 and 1339 cm -1 The characteristic absorption peaks were C-O absorption peak and C-H variable angle vibration absorption peak, respectively; the three peaks appeared at 1152-1025 cm -1 The stretching vibration of the pyran ring was caused, which indicated that the Pueraria lobata flower polysaccharide sugar chain contained pyran-type sugar ring.

[0080] Test Example 2

[0081] II. Determination of total content of Pueraria lobata flower polysaccharide - phenol-sulfuric acid method

[0082] 1. Preparation of standard curve: precisely take 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 ml of D-anhydrous glucose control solution (concentration: 0.2 mg / ml) into 20 ml test tubes with stoppers, add water to 1.0 ml for the volume less than 1.0 ml, then add 2.0 ml of 5% (v / v) phenol-sulfuric acid solution, add 7.0 ml of concentrated sulfuric acid, shake slowly, take out immediately after heating in boiling water for 15 min, cool in cold water, and detect by ultraviolet spectrophotometry at 486 nm (ultraviolet spectrophotometer, model: SHIMADZU UV-2550), take 2.0 ml of water as blank by the same color developing operation, take absorbance as vertical coordinate and control product concentration as horizontal coordinate, and draw the standard curve as shown in Figure 2 .

[0083] 2. Determination of sample content

[0084] Precisely take 20 mg of the product into a conical flask, add 100 ml of water, weigh, treat by different ultrasonic time and different treatment time, cool, add water to the previous weight, take 0.5 ml into a 20 ml test tube with stopper, add water to 1.0 ml, then add 2.0 ml of 5% (v / v) phenol-sulfuric acid solution, add 7.0 ml of concentrated sulfuric acid, shake slowly, take out immediately after heating in boiling water for 15 min, cool in cold water, and detect by ultraviolet spectrophotometry at 486 nm. The results are shown in Table 2

[0085] Table 2 Content of Pueraria lobata flower polysaccharide

[0086] Group Polysaccharide content (%) Example 1 65.42 Example 2 67.29 Example 3 69.12 Comparative Example 1 62.11 Comparative Example 2 60.17 Comparative Example 3 59.93

[0087] Note: The polysaccharide content calculation formula is calculated according to the standard curve:

[0088] Standard curve Y = kx + B;

[0089]

[0090] C is the final concentration of the sample;

[0091] V is the volume of the sample taken.

[0092] As can be seen from Table 1 and Table 2, the extraction rate and content of the Pueraria lobata flower polysaccharide of the examples are higher than those of the comparative examples. The main reason may be that the enzyme hydrolysis method is used before ethanol precipitation in the examples. Compared with the traditional process, the enzyme hydrolysis method has the characteristics of mild extraction conditions, easy removal of impurities, low energy consumption, and lower damage to polysaccharides. The enzyme used in this case is a composite enzyme (cellulase-protease), and the extraction rate of composite enzyme is usually higher than that of single enzyme method. Moreover, the cell wall of most traditional Chinese medicinal materials is composed of cellulose, and the effective components are often wrapped in the cell wall. Cellulase acts on the medicinal plant cells, degrades the cellulose material in the cell wall and intercellular substance, destroys the dense structure of the cell wall, causes local loosening, swelling, and collapse of the cell wall and intercellular substance structure, reduces the mass transfer resistance of the cell wall and intercellular substance to the diffusion of effective components from the cell to the extraction medium, and truly promotes the improvement of the extraction rate of effective components from the mass transfer point of view. In the traditional extraction process, the protein in the medicinal material is easy to coagulate and starch gelatinize during decoction, which affects the decoction of effective components and makes separation difficult. The protease can purify the clarity of the extraction liquid. Therefore, by selecting appropriate enzymes and through mild enzymatic reaction, the impurity components in the liquid preparation can be decomposed and removed, thereby improving the clarity of the traditional Chinese medicine extraction and improving the quality of the finished product. The ethanol precipitation of the concentrated liquid can better remove water and impurities while retaining the effective components of the medicine, so this method improves the extraction rate and content of the Pueraria lobata flower polysaccharide.

[0093] Test Example 3

[0094] III. Test of the DPPH free radical scavenging ability of Pueraria lobata flower polysaccharide

[0095] 1. Preparation of DPPH- standard solution

[0096] 15 mg of DPPH reagent was accurately weighed into a 50 mL volumetric flask, diluted to the mark with anhydrous ethanol, and then 10 mL was transferred into a 100 mL volumetric flask, diluted to the mark with anhydrous ethanol, to prepare a DPPH- standard solution of 30 ug / mL.

[0097] 2. Preparation of control and sample solutions

[0098] 2.1 Take an appropriate amount of VC and dilute it to a 1 mg / ml stock solution with anhydrous ethanol. Before use, dilute it with anhydrous ethanol to prepare control solutions containing VC at concentrations of 50, 100, 150, 200, and 300 uL / ml for testing.

[0099] 2.2 Take the prepared Dioscorea opposita Thunb. flower polysaccharide in an appropriate amount, dilute it with anhydrous ethanol to a stock solution of 1 mg / ml, and dilute it with anhydrous ethanol to sample solutions containing Dioscorea opposita Thunb. flower polysaccharide at concentrations of 50, 100, 150, 200, and 300 uL / ml for testing.

[0100] 3. Accurately pipette 2 mL of the DPPH standard solution, each concentration of VC control solution, and each concentration of Dioscorea opposita Thunb. flower polysaccharide solution, mix each with 2 mL of anhydrous ethanol, and then measure the absorbance values of each mixture at a wavelength of 517 nm using an ultraviolet spectrophotometer. The measured results are shown in Table 1, and it is found that the Dioscorea opposita Thunb. flower polysaccharide has a good free radical scavenging effect. Figure 3

[0101] Note:

[0102] Among them,

[0103] AS: the absorbance value at a wavelength of 517 nm of 2 mL of each concentration of VC control solution and Dioscorea opposita Thunb. flower polysaccharide solution mixed with 2 mL of DPPH solution;

[0104] A B : the absorbance value at a wavelength of 517 nm of 2 mL of each concentration of VC control solution and Dioscorea opposita Thunb. flower polysaccharide solution mixed with 2 mL of anhydrous ethanol solvent;

[0105] A D : the absorbance value at a wavelength of 517 nm of 2 mL of DPPH standard solution mixed with 2 mL of anhydrous ethanol solvent.

[0106] Test Example 4

[0107] Four, Dioscorea opposita Thunb. flower polysaccharide has an inhibitory effect on the generation of advanced glycation end products (AGES)

[0108] 1. Preparation of the drug

[0109] A suitable amount of Dioscorea opposita Thunb. flower polysaccharide was accurately weighed and dissolved with ultrapure water, and administered at a dose of 200 mg / kg.

[0110] 2. Grouping and administration of experimental animals

[0111] During the feeding of experimental animals, 12 h of day and night alternation, temperature maintained at 25±1℃, humidity maintained at 55-65%, and air circulation maintained. After one week of adaptive feeding, the animals were randomly numbered, and the db / db mice were randomly divided into two groups, a blank group (normal saline) and a drug administration group (Dioscorea opposita Thunb. flower polysaccharide group), with 6 animals in each group. The animals were administered by gavage at a dose of 200 mg / kg. The administration period was 5 weeks (D1-D35).

[0112] 3. Collection of serum samples

[0113] ​The mice were fasted for 12 hours before blood sampling on the first day (D1). The mice were tail-vein bled and the supernatant plasma was centrifuged and stored at -80°C. After the completion of the dosing period, the mice were fasted overnight before serum sample collection. The next day, the mice were tail-vein bled, the supernatant plasma was centrifuged and stored for use.

[0114] 4. AGES test

[0115] 4.1 Instruments

[0116] Microplate reader;

[0117] Electronic balance (1 / 10,000);

[0118] Pipette (10ul, 50ul, 200ul);

[0119] 4.2 Experimental reagents and reagents

[0120] Human advanced glycation end products (AGEs) enzyme-linked immunoassay kit;

[0121] Purified water from Wahaha;

[0122] Methanol;

[0123] 4.3 Experimental operation process

[0124] Preparation of standard curve

[0125] 480 pg / ml Standard No. 5 150 μl of the original standard was added to 150 μl of standard diluent 240 pg / ml Standard No. 4 150 μl of Standard No. 5 was added to 150 μl of standard diluent 120 pg / ml Standard No. 3 150 μl of Standard No. 4 was added to 150 μl of standard diluent 60 pg / ml Standard No. 2 150 μl of Standard No. 3 was added to 150 μl of standard diluent 30 pg / ml Standard No. 1 150 μl of Standard No. 2 was added to 150 μl of standard diluent

[0126] 4.2 Preparation of test sample solution

[0127] Take 10ul of plasma from the blank group and the dosing group, add 200ul of methanol, vortex and mix well, then centrifuge to precipitate the protein, and take the supernatant for use.

[0128] 4.3 Microplate reader detection

[0129] After the preparation of the test sample and the control sample, the following steps are performed:

[0130] 4.3.1 Sample addition: Set up blank wells (blank control wells do not add samples and enzyme-labeled reagents, and the rest of the steps are the same), standard wells, and sample wells. Add 50ul of standard to the enzyme-labeled coating plate, and then add 10ul of sample to the sample well (the final dilution of the sample is 5 times). Add the sample to the bottom of the enzyme-labeled plate well, try not to touch the well wall, and mix gently.

[0131] 4.3.2 Incubation: After sealing the plate with a sealing film, incubate at 37°C for 30 minutes.

[0132] 4.3.3 Reconstitution: Reconstitute 30X concentrated wash solution with 30X distilled water and store at room temperature.

[0133] 4.3.4 Wash: Carefully remove the plate seal and discard the liquid. Wash the plate 5 times by adding 200 μl of wash solution to each well, incubating for 30 seconds, and discarding the wash solution. Tap the plate dry.

[0134] 4.3.5 Add enzyme: Add 50 μl of enzyme reagent to each well, except the blank wells.

[0135] 4.3.6 Incubate: Incubate the plate as described in 4.3.2.

[0136] 4.3.7 Wash: Wash the plate as described in 4.3.4.

[0137] 4.3.8 Develop: Add 50 μl of color reagent A to each well, followed by 50 μl of color reagent B. Gently mix the plate.

[0138] Incubate the plate at 37°C for 10 minutes in the dark.

[0139] 4.3.9 Stop: Add 50 μl of stop solution to each well to stop the reaction (the blue color will change to yellow).

[0140] 4.3.10 Measure: Measure the absorbance (OD) of each well at 450 nm using the blank wells as a reference.

[0141] Measure the absorbance within 15 minutes after adding the stop solution.

[0142] 5.2 Test Results

[0143] The standard curve is shown in Table 3 and Figure 4 ;

[0144] Table 3 Standard curve for the determination of AGES by using the Kudzu flower polysaccharide

[0145] Absorbance 0.615499996 0.347299996 0.1624 0.077200003 0.033 Concentration C (pg / ml) 480 240 120 60 30

[0146] The test results for the samples are shown in Table 4.

[0147] Table 4 Absorbance values for the blank group and the administration group at Dl and D35

[0148]

[0149]

[0150] From the above results, it can be seen that the OD values of the blank group and the administration group decreased after D35, but the decrease in the administration group was significantly higher than that in the blank group, which proves that the Kudzu flower polysaccharide has a significant inhibitory effect on AGES.

[0151] The above merely illustrates the embodiments of the present application, and does not constitute any limitation on the protection scope of the present application. Any technical scheme formed by equivalent exchange or equivalent replacement falls within the protection scope of the present application.

Claims

1. The application of a kudzu flower polysaccharide in anti-glycation drugs, characterized in that, The kudzu flower polysaccharide can inhibit the formation of advanced glycation end products (AGEs). The preparation method of the kudzu flower polysaccharide includes the following steps: 1) Drying and pulverizing: After drying the kudzu flowers, pulverize them into fine powder, sieve them, and obtain kudzu flower powder; 2) Enzymatic hydrolysis: Add the kudzu flower powder from step 1) to purified water at a certain material-to-liquid ratio, soak and stir, then adjust the pH to 3-5 with hydrochloric acid solution, add a compound enzyme, heat to 40-50℃ for 60 minutes to obtain kudzu flower enzymatic hydrolysate; the amount of the compound enzyme added is 0.2-0.5 wt%. 3) Enzyme inactivation: Continue heating the kudzu flower enzymatic hydrolysate obtained in step 2) to 70-80℃ for 30-60 minutes to obtain an enzyme-inactivated extract; 4) Microwave-assisted extraction: The enzyme-inactivated extract from step 3) is subjected to microwave-assisted extraction for 30-60 minutes, repeated 2-4 times, filtered, and the filtrates are combined to obtain kudzu flower extract; 5) Concentration: The kudzu flower extract obtained in step 4) is further concentrated under reduced pressure to a certain volume to obtain kudzu flower concentrate; 6) Dissolve the kudzu flower concentrate obtained in step 5) in ethanol, let it stand overnight at 2-8℃, filter, and wash the precipitate with ethanol 2-3 times. 7) Drying: The precipitate obtained in step 6) is dried to obtain kudzu flower polysaccharide; In step 2), the complex enzyme is a combination of cellulase and protease in a mass ratio of 1:1-2.

2. The application according to claim 1, characterized in that, In step 1), the sieving is done using a 60-100 mesh sieve.

3. The application according to claim 1, characterized in that, In step 1), the material-to-liquid ratio is 1:8-10 (g / ml).

4. The application according to claim 1, characterized in that, In step 4), the filtration is a 100-200um flat plate filter.

5. The application according to claim 1, characterized in that, In step 5), the concentrated volume is 1 / 3 to 1 / 4 of the original solution; and / or; The ethanol mentioned is 75-90% (v / v) ethanol; and / or; In step 7), the drying is vacuum drying.

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