Radix cynanchi bungei total polypeptide as well as preparation method and application thereof

Abstract

The invention provides radix cynanchi bungei total polypeptide, a preparation method and application. The radix cynanchi bungei total polypeptide is extracted from radix cynanchi bungei seeds for the first time. And separating the total polypeptides of the radix cynanchi bungei by ultrafiltration to obtain three micromolecular polypeptides of the radix cynanchi bungei. The total polypeptide of radix cynanchi bungei and the three micromolecule polypeptides obtained through ultrafiltration separation have biological activity, meanwhile have good antioxidant and whitening effects and can be used for preparing cosmetics with the antioxidant and whitening effects, a scientific basis is provided for product development in the field of cosmetics with radix cynanchi bungei as a main raw material, and the application prospect is wide. And a new solution is provided for the waste of traditional Chinese medicine byproducts in industry.

Description

Technical Field

[0001] The invention belongs to the technical field of polypeptide extraction and relates to a total polypeptide of Cynanchum multiflorum, a preparation method and an application thereof. Background Art

[0002] Baishouwu is the dried root of Baishouwu, a plant of the genus Baishouwu in the Apocynaceae family. Baishouwu is mainly distributed in China's subtropical regions, approximately between 34° and 42° north latitude, including Liaoning, Inner Mongolia, Shandong, Hebei, Henan and other provinces. Among them, the planting history of Taishan area in Shandong Province is particularly long, so it is named Taishan Baishouwu, and is one of the four famous medicines of Taishan.

[0003] Bai Shou Wu is a traditional medicinal and edible herb with a long history of application. It is valued for its unique medicinal value and nourishing effect. Bai Shou Wu is rich in a variety of bioactive substances, such as steroidal glycosides, polyphenols, acetophenones and polysaccharides, and also contains rich nutrients such as protein, fat, vitamins and trace elements. Modern pharmacological research has revealed the various effects of Bai Shou Wu, including anti-cancer, antioxidant and immunomodulatory effects, which has led to its wide application in the pharmaceutical, food and cosmetics industries.

[0004] In industrial production, plant proteins from Chinese herbal medicines are usually discarded, which is a waste of resources. A large number of studies have shown that peptide components from Chinese herbal medicines have strong antioxidant activity. Antioxidant peptides, as an important branch of bioactive peptides, have received great attention in recent years. Extracting highly active antioxidant peptides from plant and animal proteins by enzymatic hydrolysis has become a hot topic in the development of natural antioxidants.

[0005] As a traditional anti-aging herb, the seeds of Bai Shou Wu contain rich protein and are an important source for the development of antioxidant active peptides. Currently, the research on Bai Shou Wu mainly focuses on active ingredients such as polysaccharides, steroidal glycosides, and acetophenones. The research on the biological activity of Bai Shou Wu total polypeptide has not yet been carried out and urgently needs to be explored and developed. Summary of the invention

[0006] With regard to the above-mentioned problems to be solved by the present application, the purpose of the present invention is to provide a total polypeptide of Cynanchum multiflorum, a preparation method and an application thereof.

[0007] To achieve the above object, the present invention adopts the following technical solutions: This application proposes for the first time a total polypeptide of Radix Cynoglossi extracted from Radix Cynoglossi seeds. The total polypeptide of Radix Cynoglossi is obtained by enzymatic hydrolysis using Radix Cynoglossi seeds as raw materials to extract proteins; meanwhile, three small-molecule polypeptides of Radix Cynoglossi are obtained by ultrafiltration separation. According to the pharmacological activity test, the polypeptide of Radix Cynoglossi has biological activity and good antioxidant and whitening effects, which provides a scientific basis for the development of cosmetic products using Radix Cynoglossi as the main raw material, and provides a new solution to the waste of Chinese medicine by-products in industry.

[0008] Specifically, the preparation method of the Cynanchum multiflorum total polypeptide comprises: S01: The seeds of Cynanchum multiflorum are crushed and defatted to form defatted powder.

[0009] Select clean and full Radix Cynanchum multiflorum seeds, use a grinder to crush the Radix Cynanchum multiflorum seeds, and pass through a 60-mesh sieve to obtain seed powder. The seed powder is added to cyclohexane at a solid-liquid ratio of 1:5, and a magnetic stirrer is used to stir and defat for multiple times at room temperature to remove the oil in the Radix Cynanchum multiflorum seeds. Stir and defat for 2-3 hours each time until the color of the mixture formed by the seed powder and cyclohexane changes from yellow to colorless. When the color of the mixture becomes colorless, the mixture is filtered and the obtained powder is spread on a tray and placed in a fume hood for ventilation and drying to remove cyclohexane to obtain defatted powder.

[0010] S02: The defatted powder is subjected to ultrasonic extraction with an extraction solvent and centrifugal separation to obtain a protein solution.

[0011] The defatted powder was added to a 30 mg / mL NaCl solution at a solid-liquid ratio of 1:25, so that the NaCl solution was used as an extractant to extract the protein in the seeds of Radix Cynanchum multiflorum. After the defatted powder was mixed with the NaCl solution, the pH was adjusted to 9.0 using a 1 mol / L NaOH solution, so that the defatted powder was in an alkaline environment, thereby facilitating the extraction of protein. After ultrasonic extraction at 25°C for 1 hour, the powder was centrifuged at a centrifugal speed of 4000 rpm for 30 minutes, and the centrifuged solution was filtered to further remove impurities and obtain a protein solution.

[0012] S03: adding a precipitant to the protein solution, allowing it to stand and centrifuging it, then re-dissolving the obtained precipitate, dialyzing it for desalination, and freeze-drying it to obtain a protein freeze-dried powder.

[0013] In an ice bath at 4°C, slowly and repeatedly add ammonium sulfate to the protein solution to make the saturation of ammonium sulfate reach 60%. In the process of adding ammonium sulfate, add a small amount of ammonium sulfate each time to ensure that it is completely dissolved before adding the next time. The entire addition process is completed within 3-10 minutes. After stirring for 10 minutes, centrifuge at 4°C and 4000 rpm for 40 minutes to obtain a precipitate.

[0014] Add 50 mmol / L Tris-HCl buffer solution to the precipitate for redissolution, centrifuge for 20 minutes at a centrifugal speed of 4000 rpm to remove insoluble substances and obtain a purified protein solution. The purified protein solution is placed in a dialysis bag and the bag mouth is clamped with a dialysis clamp. The dialysis bag is immersed in a beaker filled with distilled water and stirred with a magnetic stirrer to promote the ammonium sulfate molecules in the precipitate to seep through the dialysis bag and improve the purity of the protein. During the dialysis process, the distilled water is replaced at 2-4h, 6-8h and 10-14h of dialysis to maintain the dialysis efficiency. At the same time, it is necessary to ensure that the dialysis bag is always completely immersed in distilled water during the entire dialysis process. After the dialysis is completed, the dialysate in the dialysis bag is freeze-dried to obtain protein freeze-dried powder.

[0015] S04: adding distilled water to the protein freeze-dried powder, adjusting the pH, and then adding protease for enzymolysis to obtain an enzymolysis product.

[0016] The protein freeze-dried powder was added to distilled water at a liquid-solid ratio of 100:1 and dissolved by stirring at 50°C. The pH was adjusted to 7.0 using NaOH solution and HCl solution. When the temperature was stable, 8100 U / g flavor protease was added and enzymolysis was performed for 3 hours to obtain the enzymolysis product.

[0017] S05: The enzymatic hydrolysis product is inactivated, cooled, filtered, and freeze-dried to obtain Cynomorium multiflorum total polypeptide.

[0018] After the enzymatic hydrolysis is completed, the enzymatic hydrolysis product is heated at 100°C for 15 minutes for inactivation. When the enzymatic hydrolysis product is cooled to room temperature, it is centrifuged at a centrifugal speed of 4000 rpm for 30 minutes, filtered and separated to obtain a supernatant, which is freeze-dried at -20°C to obtain the total polypeptide of Radix Cynomorii multiflori, which is frozen for storage.

[0019] Furthermore, the total polypeptide of Radix Cynanchum multiflorum was added into distilled water to form a polypeptide solution. The polypeptide solution was placed into an ultrafiltration tube and centrifuged in a refrigerated centrifuge at a speed of 4000 rpm for 30 minutes to obtain three polypeptide components, namely, polypeptide of <3 kDa, polypeptide of 3-10 kDa and polypeptide of >10 kDa. After freeze-drying the three polypeptide components, three polypeptide freeze-dried powders were obtained respectively.

[0020] In addition, the total polypeptide of Cynanchum multiflorum provided in the present application has antioxidant and whitening effects, and is used to prepare cosmetics with antioxidant and whitening effects.

[0021] The present invention has the following beneficial effects: (1) This application proposes for the first time a total polypeptide of Cynanchum multiflorum, which is obtained by extracting protein from Cynanchum multiflorum seeds and performing enzymatic hydrolysis. At the same time, three small-molecule Cynanchum multiflorum polypeptides are obtained by ultrafiltration separation.

[0022] (2) During the extraction of total polypeptides from Radix Polygoni Multiflori, cyclohexane was used to remove the oil from Radix Polygoni Multiflori seeds; NaCl solution was used as an extractant to extract the protein from Radix Polygoni Multiflori seeds; ammonium sulfate was used as a precipitant to precipitate the protein from Radix Polygoni Multiflori seeds; Tris-HCl buffer solution was used for re-dissolution and purification to obtain protein freeze-dried powder; and flavor protease was used to enzymatically hydrolyze the protein freeze-dried powder to obtain total polypeptides from Radix Polygoni Multiflori.

[0023] (3) Ultrafiltration centrifugation was used to separate three small molecule polypeptide components from the total polypeptide of Radix Cynomorii multiflori.

[0024] (4) The total polypeptide of Cynanchum multiflorum and the three small molecule polypeptides separated by ultrafiltration in the present application all have antioxidant and whitening effects, and can be used to prepare cosmetics with antioxidant and whitening effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The antioxidant activity diagram of total polypeptides, polypeptides <3 kDa, polypeptides 3-10 kDa and polypeptides >10 kDa of Radix Cynomorii; Figure 2 The effect of enzymatic hydrolysis time on DPPH free radical scavenging rate and hydrolysis degree of total polypeptide of Cynanchum multiflorum; Figure 3 The effect of enzymatic hydrolysis temperature on DPPH free radical scavenging rate and hydrolysis degree of total polypeptide of Cynanchum multiflorum; Figure 4 The effect of the amount of flavor protease added on the DPPH free radical scavenging rate and hydrolysis degree of total polypeptide of Cynanchum multiflorum; Figure 5 This paper studies the effect of the liquid-solid ratio of protein freeze-dried powder to distilled water on the DPPH radical scavenging rate and hydrolysis degree of Cynomorium multiflorum total polypeptides. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further explained and illustrated by means of specific embodiments below.

[0027] Example 1 The present application embodiment provides a total polypeptide of Radix Cynomorii multiflori, and the preparation method of the total polypeptide of Radix Cynomorii multiflori comprises: S101: Select clean and full Radix Polygoni Multiflori seeds, crush the Radix Polygoni Multiflori seeds with a grinder, and pass through a 60-mesh sieve to obtain seed powder. Add the seed powder to cyclohexane at a solid-liquid ratio of 1:5, and stir and defat for multiple times at room temperature using a magnetic stirrer, stirring and defatting for 2.5 hours each time, until the color of the mixture formed by the seed powder and cyclohexane changes from yellow to colorless. When the color of the mixture changes to colorless, the mixture is filtered, and the obtained powder is spread on a tray and placed in a fume hood for ventilation and drying to remove cyclohexane to obtain defatted powder.

[0028] S102: adding the defatted powder to a 30 mg / mL NaCl solution at a solid-liquid ratio of 1:25, so that the NaCl solution is used as an extractant to extract the protein in the Radix Cynomorii seeds. After the defatted powder is mixed with the NaCl solution, the pH is adjusted to 9.0 using a 1 mol / L NaOH solution, and after ultrasonic extraction at 25°C for 1 hour, the powder is centrifuged at a centrifugal speed of 4000 rpm for 30 minutes, and the centrifuged solution is filtered to obtain a protein solution.

[0029] S103: In an ice bath at 4°C, slowly and repeatedly add 7.22g of ammonium sulfate to 20ml of protein solution to make the saturation of ammonium sulfate reach 60%. In the process of adding ammonium sulfate, add a small amount of ammonium sulfate each time to ensure that it is completely dissolved before adding the next time. The entire addition process is completed within 8 minutes. After stirring for 10 minutes, centrifuge at a temperature of 4°C and a centrifugal speed of 4000rpm for 40 minutes to obtain a precipitate. Add 10ml of Tris-HCl buffer solution with a concentration of 50mmol / L to the precipitate for re-dissolution, and centrifuge at a centrifugal speed of 4000rpm for 20 minutes to obtain a purified protein solution. Put the purified protein solution into a dialysis bag and clamp the bag mouth with a dialysis clamp. Immerse the dialysis bag in a beaker filled with distilled water and stir it with a magnetic stirrer to promote the ammonium sulfate molecules in the precipitate to seep through the dialysis bag and improve the purity of the protein. During the dialysis process, distilled water is replaced at 3h, 7h and 12h to maintain the dialysis efficiency. At the same time, the dialysis bag must be completely submerged in distilled water throughout the dialysis process. After the dialysis is completed, the dialysate in the dialysis bag is freeze-dried to obtain protein freeze-dried powder.

[0030] S104: The protein freeze-dried powder was added to distilled water at a liquid-solid ratio of 100:1, and stirred to dissolve at 50°C. The pH was adjusted to 7.0 using NaOH solution and HCl solution. When the temperature was stable, 8100 U / g flavor protease was added and enzymolysis was performed for 3 hours to obtain an enzymolysis product.

[0031] S105: After the enzymolysis is completed, the enzymolysis product is heated at 100°C for 15 minutes for inactivation. When the enzymolysis product is cooled to room temperature, it is centrifuged at a centrifugal speed of 4000 rpm for 30 minutes, filtered and separated to obtain a supernatant, which is freeze-dried at -20°C to obtain the total polypeptide of Radix Cynomorii Multiflori.

[0032] Example 2 The present application embodiment provides a total polypeptide of Radix Cynomorii multiflori, and the preparation method of the total polypeptide of Radix Cynomorii multiflori comprises: S201: Select clean and full Radix Polygoni Multiflori seeds, crush the Radix Polygoni Multiflori seeds with a grinder, and obtain seed powder after passing through a 60-mesh sieve. Add the seed powder to cyclohexane at a solid-liquid ratio of 1:5, and stir and defat for multiple times at room temperature using a magnetic stirrer, and stir and defat for 2 hours each time until the color of the mixture formed by the seed powder and cyclohexane changes from yellow to colorless. When the color of the mixture changes to colorless, the mixture is filtered, and the obtained powder is spread on a tray and placed in a fume hood for ventilation and drying to remove cyclohexane to obtain defatted powder.

[0033] S202: adding the defatted powder to a NaCl solution with a concentration of 30 mg / mL at a solid-liquid ratio of 1:25, so that the NaCl solution is used as an extractant to extract the protein in the Radix Cynomorii seeds. After the defatted powder is mixed with the NaCl solution, the pH is adjusted to 9.0 using a NaOH solution with a concentration of 1 mol / L, and after ultrasonic extraction at 25°C for 1 hour, the mixture is centrifuged at a centrifugal speed of 4000 rpm for 30 minutes, and the centrifuged solution obtained by centrifugation is filtered to obtain a protein solution.

[0034] S203: In an ice bath at 4°C, slowly and repeatedly add 7.22 g of ammonium sulfate to 20 ml of protein solution until the saturation of ammonium sulfate reaches 60%. In the process of adding ammonium sulfate, add a small amount of ammonium sulfate each time to ensure that it is completely dissolved before adding the next time. The entire addition process is completed within 3 minutes. After continuing to stir for 10 minutes, centrifuge at a temperature of 4°C and a centrifugal speed of 4000 rpm for 40 minutes to obtain a precipitate. Add 10 ml of Tris-HCl buffer solution with a concentration of 50 mmol / L to the precipitate for re-dissolution, and centrifuge at a centrifugal speed of 4000 rpm for 20 minutes to remove insoluble substances to obtain a purified protein solution. Put the purified protein solution into a dialysis bag and clamp the bag mouth with a dialysis clamp. Immerse the dialysis bag in a beaker filled with distilled water and stir it with a magnetic stirrer to promote the ammonium sulfate molecules in the precipitate to seep through the dialysis bag and improve the purity of the protein. During the dialysis process, distilled water is replaced at 2h, 6h and 10h to maintain the dialysis efficiency. At the same time, the dialysis bag must be completely submerged in distilled water throughout the dialysis process. After the dialysis is completed, the dialysate in the dialysis bag is freeze-dried to obtain protein freeze-dried powder.

[0035] S204: The protein freeze-dried powder was added to distilled water at a liquid-solid ratio of 100:1, and stirred to dissolve at 50°C. The pH was adjusted to 7.0 using NaOH solution and HCl solution, and when the temperature was stable, 8100 U / g flavor protease was added and enzymolysis was performed for 3 hours to obtain an enzymolysis product.

[0036] S205: After the enzymolysis is completed, the enzymolysis product is heated at 100°C for 15 minutes for inactivation. When the enzymolysis product is cooled to room temperature, it is centrifuged at a centrifugal speed of 4000 rpm for 30 minutes, filtered and separated to obtain a supernatant, which is freeze-dried at -20°C to obtain the total polypeptide of Radix Cynomorii Multiflori.

[0037] Example 3 The present application embodiment provides a total polypeptide of Radix Cynomorii multiflori, and the preparation method of the total polypeptide of Radix Cynomorii multiflori comprises: S301: Select clean and full Radix Polygoni Multiflori seeds, crush the Radix Polygoni Multiflori seeds with a grinder, and pass through a 60-mesh sieve to obtain seed powder. Add the seed powder to cyclohexane at a solid-liquid ratio of 1:5, and stir and defat for multiple times at room temperature using a magnetic stirrer, stirring and defatting for 3 hours each time, until the color of the mixture formed by the seed powder and cyclohexane changes from yellow to colorless. When the color of the mixture changes to colorless, the mixture is filtered, and the obtained powder is spread on a tray and placed in a fume hood for ventilation and drying to remove cyclohexane to obtain defatted powder.

[0038] S302: adding the defatted powder to a 30 mg / mL NaCl solution at a solid-liquid ratio of 1:25, so that the NaCl solution is used as an extractant to extract the protein in the Radix Cynomorii seeds. After the defatted powder is mixed with the NaCl solution, the pH is adjusted to 9.0 using a 1 mol / L NaOH solution, and after ultrasonic extraction at 25°C for 1 hour, the powder is centrifuged at a centrifugal speed of 4000 rpm for 30 minutes, and the centrifuged solution is filtered to obtain a protein solution.

[0039] S303: In an ice bath at 4°C, slowly and repeatedly add 7.22 g of ammonium sulfate to 20 ml of protein solution until the saturation of ammonium sulfate reaches 60%. In the process of adding ammonium sulfate, add a small amount of ammonium sulfate each time to ensure that it is completely dissolved before adding the next time. The entire addition process is completed within 10 minutes. After continuing to stir for 10 minutes, centrifuge at a temperature of 4°C and a centrifugal speed of 4000 rpm for 40 minutes to obtain a precipitate. Add 10 ml of Tris-HCl buffer solution with a concentration of 50 mmol / L to the precipitate for re-dissolution, and centrifuge at a centrifugal speed of 4000 rpm for 20 minutes to obtain a purified protein solution. Put the purified protein solution into a dialysis bag and clamp the bag mouth with a dialysis clamp. Immerse the dialysis bag in a beaker filled with distilled water and stir it with a magnetic stirrer to promote the ammonium sulfate molecules in the precipitate to seep through the dialysis bag and improve the purity of the protein. During the dialysis process, distilled water is replaced at 4h, 8h and 14h to maintain the dialysis efficiency. At the same time, the dialysis bag must be completely submerged in distilled water throughout the dialysis process. After the dialysis is completed, the dialysate in the dialysis bag is freeze-dried to obtain protein freeze-dried powder.

[0040] S304: Add the protein freeze-dried powder to distilled water at a liquid-to-solid ratio of 1:100, and stir to dissolve at 50° C. Use NaOH solution and HCl solution to adjust the pH to 7.0, and when the temperature is stable, add 8100 U / g flavor protease and perform enzymolysis for 3 hours to obtain an enzymolysis product.

[0041] S305: After the enzymatic hydrolysis is completed, the enzymatic hydrolysis product is heated at 100°C for 15 minutes for inactivation. When the enzymatic hydrolysis product is cooled to room temperature, it is centrifuged at a centrifugal speed of 4000 rpm for 30 minutes, filtered and separated to obtain a supernatant, which is freeze-dried at -20°C to obtain the total polypeptide of Radix Cynomorii Multiflori.

[0042] In order to verify that the total polypeptide of Cynanchum multiflorum prepared in the examples of the present application has whitening and antioxidant effects, the examples of the present application respectively carry out whitening effect evaluation and antioxidant effect evaluation.

[0043] 1. Evaluation of whitening efficacy In the examples of the present application, the inhibition of tyrosinase activity is used to evaluate the whitening effect. Specifically, the total polypeptide of Radix Cynomorii obtained in Example 1 is subjected to ultrafiltration separation to obtain polypeptides <3kDa, 3-10kDa and >10kDa. The total polypeptide of Radix Cynomorii, polypeptide <3kDa, polypeptide 3-10kDa and polypeptide >10kDa are respectively used as test samples, and a PBS solution with a pH of 6.8, L-tyrosine, the test sample and 200U / ml tyrosinase are added to a 96-well plate according to the amounts in Table 1 to form a reaction solution ABCD, with 3-6 parallel wells in each group; the positive control group is a vitamin C solution. The absorbance of the reaction solution is measured at 475nm using an enzyme marker. Among them, the tyrosinase inhibition rate calculation formula is: , where ABCD are the absorbance values ​​of reaction solution ABCD respectively. The inhibition rate calculation results of each group are shown in Table 2.

[0044] Table 1: Reaction solution composition and dosage (μL) Table 2: Inhibition rate data of each group of samples As can be seen from Table 2, the total polypeptide of Radix Cynoglossifolia, polypeptide <3 kDa, polypeptide 3-10 kDa and polypeptide >10 kDa all have certain inhibition rates on tyrosinase, but they are all lower than the inhibition rate of the positive control group. This is because the total polypeptide of Radix Cynoglossifolia is a mixture of polypeptides of various molecular weights. Generally, the smaller the molecular weight, the higher the biological activity of the polypeptide. Therefore, the inhibition rate of polypeptide <3 kDa on tyrosinase is the highest.

[0045] 2. Evaluation of Antioxidant Efficacy In the present application, the antioxidant efficacy was evaluated from the DPPH free radical scavenging ability, ABTS free radical scavenging ability, superoxide anion free radical scavenging ability and ferrous ion chelating ability, and the free radical scavenging rate was calculated to obtain the attached Figure 1 Among them, taking glutathione (GSH) as the control, the determination method and calculation method of each free radical scavenging ability are as follows: 1. Determination of DPPH free radical scavenging ability 50 μL of total polypeptides of Radix Cynanchum multiflorum, polypeptides <3 kDa, polypeptides 3-10 kDa and polypeptides >10 kDa were placed in a 96-well plate, and 200 μL of DPPH solution prepared with 60% ethanol was added to each component and mixed thoroughly. The mixture was reacted at room temperature in the dark for 30 minutes, and the absorbance was determined by using an enzyme marker at a wavelength of 517 nm to be A1. The sample solution was replaced with 60% ethanol as the blank group, and its absorbance was determined to be A0. The control group replaced the DPPH solution with 60% ethanol, and its absorbance was determined to be A2. Among them, the preparation of DPPH solution includes accurately weighing 2 mg, dissolving DPPH in 60% ethanol, and using 60% ethanol to make up to 50 mL volumetric flask, ready for use.

[0046] The calculation formula of DPPH free radical scavenging rate is: .

[0047] 2. Determination of ABTS free radical scavenging ability Prepare 7mM ABTS solution and 2.5mM K2S2O8 solution respectively, mix them in a volume ratio of 1:1, place the mixture in the dark at room temperature for 36 hours, then dilute it 50 times with distilled water to prepare ABTS working solution, and store it at room temperature. Take 50μL of total polypeptide of Radix Cynanchum multiflorum, polypeptide of <3kDa, polypeptide of 3-10kDa and polypeptide of >10kDa respectively and place them in a 96-well plate, add 200μL ABTS working solution to each component, mix well, react in the dark at room temperature for 10min, and measure the absorbance value at a wavelength of 734nm as A1. Use distilled water instead of the sample to be tested as the blank group, and measure its absorbance value as A0. Use distilled water instead of the ABTS working solution as the control group, and measure its absorbance value as A2.

[0048] The calculation formula of ABTS free radical scavenging rate is: .

[0049] 3. Superoxide anion free radical scavenging ability Take 30 μL of total polypeptides of Radix Cynanchum multiflorum, polypeptides <3 kDa, polypeptides 3-10 kDa and polypeptides >10 kDa, add 900 μL of Tris-HCl buffer with a concentration of 50 mM and pH 8.0, mix well and let stand at room temperature for 20 min, add 90 μL of 60 mM pyrogallol solution, mix well, keep warm at 25 °C for 5 min, then quickly add 300 μL of concentrated hydrochloric acid, mix well and measure the absorbance of the sample at a wavelength of 420 nm as A. Use distilled water instead of the sample solution as the blank group, and measure its absorbance value as A0. Use distilled water instead of the pyrogallol solution as the control group, and measure its absorbance value as A1.

[0050] The calculation formula of superoxide anion radical scavenging rate is: .

[0051] 4. Ferrous ion chelating ability Prepare 250μM FeCl2 solution and 500μM feroxine solution. Take 200μL of total polypeptides of Radix Cynanchum multiflorum, polypeptides <3kDa, polypeptides 3-10kDa and polypeptides >10kDa, add 100μL FeCl2 solution respectively, mix well, keep warm at 50℃ for 30min, take out and cool to room temperature, add 500μL distilled water and 200μL feroxine solution in turn, mix well and let stand at room temperature for 10min, and measure the absorbance of the sample at 562nm wavelength as A. Use distilled water instead of sample solution as blank group, and measure its absorbance as A0.

[0052] The calculation formula of ferrous ion chelation rate is: .

[0053] By the attached Figure 1 It can be seen that in terms of DPPH free radical scavenging ability, ABTS free radical scavenging ability and ferrous ion chelating ability, the ability order of the three components obtained after ultrafiltration is (<3kDa polypeptide)>(3-10kDa polypeptide)>(>10kDa polypeptide), that is, the polypeptide component with a molecular weight less than 3kDa has the highest DPPH free radical scavenging rate, ABTS free radical scavenging rate and ferrous ion chelating rate, which are 80.01±1.45%, 91.35±2.67% and 84.12±2.38% respectively, which are significantly higher than the activity of total polypeptides of Cynomorium multiflorum (P<0.05).

[0054] In terms of superoxide anion free radical scavenging ability, the ability order of the three components obtained after ultrafiltration was (3-10kDa polypeptide) > (<3kDa polypeptide) > (>10kDa polypeptide), that is, the 3-10kDa polypeptide had the strongest superoxide anion free radical scavenging ability, which was 55.67±1.76%, significantly higher than the activity of total polypeptides of Cynomorium multiflorum (P<0.05).

[0055] This shows that there is a correlation between the molecular weight of a polypeptide and its activity. Usually, polypeptides with smaller molecular weights have higher biological activity, which may be because smaller molecular polypeptides have simpler structures, thereby increasing their activity, and because of their good solubility, permeability and stability, these small molecules are easier to be absorbed and utilized by organisms, and bind more effectively to substrates, thereby accelerating the reaction rate and increasing their activity. Therefore, low molecular weight ultrafiltration components usually exhibit superior antioxidant effects.

[0056] In the examples of the present application, in order to verify the effects of enzymatic hydrolysis time, enzymatic hydrolysis temperature, the amount of flavor protease added, and the liquid-solid ratio of protein freeze-dried powder to distilled water on the DPPH free radical scavenging ability and hydrolysis degree (DH) of total polypeptide of Radix Cynomorii multiflori, the protein freeze-dried powder prepared in Example 1 was taken as an example, and experiments were carried out according to different enzymatic hydrolysis times, enzymatic hydrolysis temperatures, the amount of flavor protease added, and the liquid-solid ratio of protein freeze-dried powder to distilled water, as described below.

[0057] 1. Effect of enzymatic hydrolysis time The protein freeze-dried powder was added into distilled water at a liquid-to-solid ratio of 50:1, and stirred to dissolve at 50°C. The pH was adjusted to 7.0 using NaOH solution and HCl solution. When the temperature stabilized, 6000U / g flavor protease was added, and enzymolysis was performed at 50°C for 1h, 2h, 3h, 4h and 5h to obtain enzymolysis products. After the enzymolysis was completed, the enzymolysis products were heated at 100°C for 15min for inactivation. After the enzymolysis products were cooled to room temperature, they were centrifuged at a speed of 4000rpm for 30min, filtered and separated to obtain the supernatant. The DPPH free radical scavenging rate and degree of hydrolysis of the supernatant were measured to obtain the attached Figure 2 .

[0058] By the attached Figure 2 It can be seen that as the enzymolysis time is extended, the DPPH radical scavenging rate and the degree of hydrolysis both increase gradually. When the enzymolysis time is greater than 3h, the DPPH radical scavenging rate and the degree of hydrolysis no longer increase. Therefore, the optimal enzymolysis time in this application is 3h.

[0059] 2. Effect of enzymatic hydrolysis temperature The protein freeze-dried powder was added into distilled water at a liquid-to-solid ratio of 50:1, and stirred to dissolve at 50°C. The pH was adjusted to 7.0 using NaOH solution and HCl solution. When the temperature stabilized, 6000U / g flavor protease was added, and enzymolysis was performed at 30°C, 40°C, 50°C, 60°C, and 70°C for 3 hours to obtain enzymolysis products. After the enzymolysis was completed, the enzymolysis products were heated at 100°C for 15 minutes for inactivation. After the enzymolysis products were cooled to room temperature, they were centrifuged at a speed of 4000rpm for 30 minutes, filtered and separated to obtain the supernatant. The DPPH free radical scavenging rate and degree of hydrolysis of the supernatant were measured to obtain the attached Figure 3 .

[0060] By the attached Figure 3 It can be seen that with the increase of enzymolysis temperature, the DPPH free radical scavenging rate and the degree of hydrolysis both increase first and then decrease. When the enzymolysis temperature is 50°C, the DPPH free radical scavenging rate and the degree of hydrolysis are the highest. Therefore, the optimal enzymolysis temperature in this application is 3h.

[0061] 3. Effect of the amount of flavor protease added The protein freeze-dried powder was added into distilled water at a liquid-to-solid ratio of 50:1, and stirred to dissolve at 50°C. The pH was adjusted to 7.0 using NaOH solution and HCl solution. When the temperature stabilized, 4000 U / g, 6000 U / g, 8000 U / g, 10000 U / g, and 12000 U / g flavor proteases were added, respectively, and enzymolysis was performed at 50°C for 3 hours to obtain enzymolysis products. After the enzymolysis was completed, the enzymolysis products were heated at 100°C for 15 minutes for inactivation. After the enzymolysis products were cooled to room temperature, they were centrifuged at a speed of 4000 rpm for 30 minutes, filtered and separated to obtain the supernatant. The DPPH free radical scavenging rate and degree of hydrolysis of the supernatant were measured to obtain the attached Figure 4 .

[0062] By the attached Figure 4 It can be seen that as the amount of flavor protease added increases, the DPPH radical scavenging rate and the degree of hydrolysis both increase first and then decrease. When the amount of flavor protease added is 8000U / g, the DPPH radical scavenging rate and the degree of hydrolysis are the highest. Therefore, the optimal amount of flavor protease added in this application is 8000U / g.

[0063] 4. Effect of liquid-solid ratio of protein freeze-dried powder and distilled water The protein freeze-dried powder was added into distilled water at a liquid-to-solid ratio of 25:1, 50:1, 100:1, 150:1, and 200:1, and stirred to dissolve at 50°C. The pH was adjusted to 7.0 using NaOH solution and HCl solution. When the temperature stabilized, 8000U / g flavor protease was added and enzymolysis was performed at 50°C for 3 hours to obtain an enzymolysis product. After the enzymolysis was completed, the enzymolysis product was heated at 100°C for 15 minutes for inactivation. After the enzymolysis product was cooled to room temperature, it was centrifuged at a speed of 4000rpm for 30 minutes, filtered and separated to obtain a supernatant. The DPPH free radical scavenging rate and degree of hydrolysis of the supernatant were measured to obtain the attached Figure 5 .

[0064] By the attached Figure 5 It can be seen that, with the increase of liquid-solid ratio, DPPH radical scavenging rate and degree of hydrolysis both increase first and then remain basically unchanged. When the liquid-solid ratio is 100:1, DPPH radical scavenging rate and degree of hydrolysis remain basically unchanged. Therefore, the optimal liquid-solid ratio of protein freeze-dried powder and distilled water in this application is 100:1.

[0065] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing total polypeptides of Radix Cynanchum multiflorum, characterized in that: include: The seeds of Cynanchum multiflorum are crushed and defatted to form defatted powder; The defatted powder is subjected to ultrasonic extraction with an extraction solvent and centrifugal separation to obtain a protein solution; Adding a precipitant to the protein solution, allowing it to stand and centrifuge, re-dissolving the obtained precipitate, dialyzing and desalting, and freeze-drying to obtain a protein freeze-dried powder; Distilled water is added to the protein freeze-dried powder, and after adjusting the pH, protease is added to perform enzymolysis to obtain an enzymolysis product; The enzymatic hydrolysis product is inactivated, cooled, filtered and freeze-dried to obtain the Cynomorium multiflorum total polypeptide.

2. The method for preparing the total polypeptide of Radix Cynanchum multiflorum according to claim 1, characterized in that: The pulverization and defatting of Cynanchum multiflorum seeds to form defatted powder comprises: Select clean and full Cynanchum multiflorum seeds, crush them into 60 meshes, and obtain seed powder; The seed powder is added to cyclohexane at a solid-liquid ratio of 1:5, and stirred for defatting until the color of the mixture becomes colorless; The powder obtained by suction filtration of the mixture is air-dried to obtain defatted powder.

3. The method for preparing the total polypeptide of Radix Cynanchum multiflorum according to claim 1, characterized in that: The defatted powder is subjected to ultrasonic extraction with an extraction solvent and centrifugal separation to obtain a protein solution comprising: The defatted powder was added to a NaCl solution at a solid-liquid ratio of 1:25, the pH was adjusted to 9.0 using a NaOH solution, and then ultrasonically extracted at 25° C. for 1 h; After the ultrasound was completed, the mixture was centrifuged at 4000 rpm for 30 min, and the centrifuge liquid was filtered to obtain a protein solution.

4. The method for preparing the total polypeptide of Radix Cynomorii according to claim 1, characterized in that: Adding a precipitant to the protein solution, standing and centrifuging the solution comprises: Under ice bath conditions at 4° C., slowly and repeatedly add ammonium sulfate to the protein solution to make the saturation of the ammonium sulfate reach 60%; After stirring for 10 minutes, the mixture was centrifuged at 4°C and 4000 rpm for 40 minutes to obtain a precipitate.

5. The method for preparing the total polypeptide of Radix Cynomorii according to claim 1, characterized in that: The precipitate is redissolved, dialyzed for desalination, and freeze-dried to obtain a protein freeze-dried powder, which comprises: Adding Tris-HCl buffer solution to the precipitate for redissolution, and centrifuging at 4000 rpm for 20 min to obtain a purified protein solution; The purified protein solution is placed in a dialysis bag placed in distilled water for dialysis to obtain a dialysate; The dialyzate is freeze-dried to obtain freeze-dried protein powder.

6. The method for preparing the total polypeptide of Radix Cynanchum multiflorum according to claim 1, characterized in that: Distilled water is added to the protein freeze-dried powder, and after adjusting the pH, protease is added for enzymolysis to obtain an enzymolysis product including: The protein freeze-dried powder was added into distilled water at a liquid-to-solid ratio of 100:1, and stirred to dissolve at 50°C; After adjusting the pH to 7.0, 8100 U / g flavor protease was added and enzymolysis was performed for 3 h to obtain an enzymolysis product.

7. The method for preparing the total polypeptide of Radix Cynomorii multiflori according to claim 1, characterized in that: The enzymatic hydrolysis product is inactivated, cooled, filtered, and freeze-dried to obtain the total polypeptide of Cynanchum multiflorum, which includes: The enzymatic hydrolysate was heated at 100°C for 15 minutes to be inactivated, cooled to room temperature, centrifuged at 4000 rpm for 30 minutes, filtered and separated to obtain a supernatant; the supernatant was freeze-dried at -20°C to obtain the total polypeptide of Radix Cynomorii Multiflori.

8. The method for preparing the total polypeptide of Cynanchum multiflorum according to claim 1, characterized in that: The method further comprises: Adding the Cynanchum multiflorum total polypeptide to distilled water to form a polypeptide solution; The polypeptide solution was placed in an ultrafiltration tube and centrifuged in a refrigerated centrifuge at a rotation speed of 4000 rpm for 30 minutes to obtain polypeptides of less than 3 kDa, polypeptides of 3-10 kDa and polypeptides of greater than 10 kDa.

9. A total polypeptide of Cynanchum multiflorum, characterized in that Prepared according to any method of claims 1-8.

10. The total polypeptide of Cynanchum multiflorum prepared by any method of claims 1-8 is used for preparing cosmetics with antioxidant and whitening effects.

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