Polygonatum odoratum sugar-binding protein as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of cosmetics, and particularly relates to a polygonatum odoratum sugar-binding protein and a preparation method and application thereof. The invention provides the preparation method of the polygonatum odoratum sugar-binding protein, the polygonatum odoratum sugar-binding protein with a specific structure can be obtained by utilizing the preparation method, and the obtained polygonatum odoratum sugar-binding protein has the activity of agglutinating sheep red blood cells, can inhibit the activity of hyaluronidase and elastase, can control inflammatory reaction and anaphylactic reaction, and can be used for preparing the polygonatum odoratum sugar-binding protein. The skin barrier repair is promoted. Results of the embodiment show that the skin cream prepared from the polygonatum odoratum sugar binding protein has a protective effect on cells damaged by ultraviolet rays, and the polygonatum odoratum sugar binding protein can protect skin cells, improve the tolerance of skin to ultraviolet rays and relieve skin aging caused by light damage, so that the anti-aging effect is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of cosmetics, and particularly relates to a yuzutose-binding protein and a preparation method and application thereof. Background Art

[0002] Aging is an irreversible natural phenomenon of organisms as time goes by and age increases. In the process of skin aging, inflammatory reactions are usually caused. The proinflammatory factors released by inflammatory cells can promote the activation of hyaluronidase, collagenase and elastase, which in turn leads to the hydrolysis of hyaluronic acid, collagen and elastin, causing the dermis to lose elasticity and tension. In addition, hyaluronidase is activated when allergic reactions occur, and its activation is positively correlated with the release of histamine. Therefore, one of the strategies for anti-skin aging is to use inhibitors of hyaluronidase and elastase to achieve the purpose of controlling inflammatory reactions and allergic reactions and promoting skin barrier repair.

[0003] Polygonatum odoratum (Mill.) Druce is a traditional Chinese medicinal and edible plant. In the field of cosmetics, extracts of Polygonatum odoratum are often used as raw materials for cosmetics. Biomolecules with hemagglutination can recognize the sugar chain structure on the surface of blood cells and promote cell mitosis. However, to date, there has been no report on a Polygonatum odoratum active ingredient with lectin erythrocyte properties. Summary of the invention

[0004] The purpose of the present invention is to provide a yuzunosan binding protein and a preparation method and application thereof. The yuzunosan binding protein prepared by the preparation method of the present invention has the activity of agglutinating sheep red blood cells, can inhibit the activity of hyaluronidase and elastase, and has an anti-aging effect.

[0005] The present invention provides a method for preparing a yuzu sugar-binding protein, comprising the following steps: adding (NH 4 ) 2 SO 4 To (NH 4 ) 2 SO 4 The saturation of the polymer is 60% to 80%, and precipitation is performed, and solid components are collected to obtain a precipitate of baicalin binding protein; the precipitate of baicalin binding protein is first dissolved and then dialyzed, and a first retentate is collected to obtain a crude extract of baicalin binding protein; the crude extract of baicalin binding protein is secondly dissolved and then affinity chromatography is performed, and an eluate is collected; the eluate is secondly dialyzed, and a second retentate is collected to obtain baicalin binding protein;

[0006] The cut-off of the first dialysis is 10000Da;

[0007] The affinity chromatography column is a cross-linked ovalbumin-porcine gastric mucin affinity chromatography column;

[0008] The elution solvent of the affinity chromatography is 1.5M NaCl solution;

[0009] The cut-off of the second dialysis was 10000Da.

[0010] As a preferred solution, the preparation of the water extract of Polygonatum odoratum comprises: extracting Polygonatum odoratum with physiological saline to obtain the water extract of Polygonatum odoratum; the solid-liquid ratio of the Polygonatum odoratum to the physiological saline is 1 g:5 mL.

[0011] As a preferred embodiment, the water extraction time is 12 to 16 hours.

[0012] As a preferred embodiment, the solvent used for the first dissolution is a 0.15M NaCl solution; the solid-liquid ratio of the polysaccharide binding protein precipitate to the 0.15M NaCl solution is 1 g:10 mL.

[0013] As a preferred solution, the solvent used in the second dissolution is 0.01-0.1M Tris-HCl buffer; the pH of the Tris-HCl buffer is 6-9.

[0014] As a preferred embodiment, after collecting the second retentate, the method further comprises: dissolving the second retentate for a third time and eluting it using a Sephadex G-100 molecular sieve chromatography column, collecting OD 280 The eluent of the absorption peak.

[0015] The present invention also provides a yuzusu-binding protein prepared by the preparation method described in the above scheme, wherein the molecular weight of the yuzusu-binding protein is 15000Da; the N-terminus of the yuzusu-binding protein is: NH 2 -As p-Asn-Ser-Leu-Thr-Ser-Pro-Asn-Ser-Leu-; in the secondary structure of the polysaccharide binding protein, the proportion of α-helix is ​​20.20%, the proportion of β-fold is 24.12%, the proportion of β-turn is 14.83%, and the proportion of random coil is 40.85%.

[0016] The present invention also provides the yuzusan binding protein prepared by the preparation method described in the above scheme or the use of the yuzusan binding protein described in the above scheme in the preparation of hyaluronidase and / or elastase inhibitors.

[0017] The present invention also provides the yuzusan-binding protein prepared by the preparation method described in the above scheme or the use of the yuzusan-binding protein described in the above scheme in the preparation of cosmetics.

[0018] The present invention also provides a cosmetic, wherein the active ingredient of the cosmetic includes the yuzunosan-binding protein prepared by the preparation method described in the above scheme or the yuzunosan-binding protein described in the above scheme.

[0019] Beneficial effects:

[0020] The present invention provides a method for preparing a yuzu sugar-binding protein, comprising the following steps: adding (NH 4 ) 2 SO 4 To (NH 4 ) 2 SO 4 The saturation of the saturation is 60% to 80%, and precipitation is performed, and the solid components are collected to obtain a yuzhuose binding protein precipitate; the yuzhuose binding protein precipitate is first dissolved and then dialyzed for the first time, and the first retentate is collected to obtain a yuzhuose binding protein crude extract; the yuzhuose binding protein crude extract is secondly dissolved and then affinity chromatography is performed, and the eluate is collected; the eluate is dialyzed for the second time, and the second retentate is collected to obtain a yuzhuose binding protein. The preparation method of the present invention can obtain a yuzhuose binding protein with a specific structure, and the obtained yuzhuose binding protein has the activity of agglutinating sheep red blood cells, and can inhibit the activity of hyaluronidase and elastase, control inflammatory reactions and allergic reactions, and promote skin barrier repair. The results of the embodiment show that the skin cream prepared by using the yuzhuose binding protein of the present invention has a protective effect on ultraviolet damaged cells. The yuzhuose binding protein can protect skin cells, improve the skin's tolerance to ultraviolet rays, and reduce skin aging caused by light damage, thereby achieving an anti-aging effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0022] Figure 1 This is the elution profile of the Sephadex G-100 molecular sieve chromatography column in Example 1;

[0023] Figure 2 This is the SDS polyacrylamide gel electrophoresis diagram of the yuzuose binding protein in Example 1; wherein POL is yuzuose binding protein;

[0024] Figure 3 The circular dichroism spectrum of the polygonatumose binding protein in Example 1;

[0025] Figure 4 This is a graph showing the effect of the yuzu sugar binding protein agglutinating sheep red blood cells in Application Example 1;

[0026] Figure 5This is a diagram showing the effect of the inhibition of the aggregation of sheep red blood cells by pig gastric mucin in Application Example 1 by the polygonatum odoratum binding protein;

[0027] Figure 6 This is the hyaluronidase inhibition curve of the yuzuose binding protein in Application Example 2;

[0028] Figure 7 This is the curve of the inhibition of elastase by the polygonatum odoratum binding protein in Application Example 2. DETAILED DESCRIPTION

[0029] The present invention provides a method for preparing a yuzu sugar-binding protein, comprising the following steps: adding (NH 4 ) 2 SO 4 To (NH 4 ) 2 SO 4 The saturation of the polymer is 60% to 80%, and precipitation is performed, and solid components are collected to obtain a precipitate of baicalin binding protein; the precipitate of baicalin binding protein is first dissolved and then dialyzed, and a first retentate is collected to obtain a crude extract of baicalin binding protein; the crude extract of baicalin binding protein is secondly dissolved and then affinity chromatography is performed, and an eluate is collected; the eluate is secondly dialyzed, and a second retentate is collected to obtain baicalin binding protein;

[0030] The cut-off of the first dialysis is 10000Da;

[0031] The affinity chromatography column is a cross-linked ovalbumin-porcine gastric mucin affinity chromatography column;

[0032] The elution solvent of the affinity chromatography is 1.5M NaCl solution;

[0033] The cut-off of the second dialysis was 10000Da.

[0034] The first dialysis, the second dialysis, the first dissolution, and the second dissolution of the present invention are only for distinction and have no particular limiting effect.

[0035] The invention crushes and sieves the polygonatum odoratum to obtain polygonatum odoratum powder. As an embodiment, the polygonatum odoratum includes polygonatum odoratum rhizomes; as an embodiment, the particle size of the polygonatum odoratum powder is 30 meshes.

[0036] After obtaining the radix polygonatum powder, the present invention uses physiological saline to extract the radix polygonatum powder to obtain a radix polygonatum water extract. As an embodiment, the water extraction also includes solid-liquid separation; the solid-liquid ratio of the radix polygonatum powder to the physiological saline is 1g:5mL. As an embodiment, the water extraction time is 12 to 16h. The present invention uses physiological saline as an extraction solvent, which can promote the dissolution of radix polygonatum sugar-binding protein and improve the extraction efficiency.

[0037] The present invention adds (NH 4 ) 2 SO 4 To (NH 4 ) 2 SO 4 The saturation of the mixture is 60% to 80%, and precipitation is performed to collect the solid components to obtain a precipitate of yuzusulan binding protein. In a specific embodiment of the present invention, the (NH 4 ) 2 SO 4 The saturation of can be any value between 60% and 80%. 4 ) 2 SO 4 The saturation of the yuzhuanose binding protein and most of the impurity protein components in the yuzhuanose water extract can be precipitated, which is beneficial to the subsequent separation and purification of the yuzhuanose binding protein.

[0038] The present invention performs a first dialysis after the first dissolution of the yuzhu sugar protein precipitate, collects the first retentate, and obtains a crude extract of yuzhu sugar binding protein. As an embodiment, the solvent used in the first dissolution is a 0.15M NaCl solution; the solid-liquid ratio of the yuzhu sugar protein precipitate and the 0.15M NaCl solution is 1g:10mL. As an embodiment, the interception of the first dialysis is 10000Da. As an embodiment, the present invention freeze-dries the first retentate to obtain a crude extract of yuzhu sugar binding protein. The present invention performs a first dialysis on the yuzhu sugar binding protein precipitate to remove small molecule impurities such as ammonium sulfate and improve the purity of yuzhu sugar binding protein.

[0039] The present invention performs affinity chromatography after the second dissolution of the crude extract of yuzhuanose binding protein, and collects the eluate. As an embodiment, the solvent used in the second dissolution is 0.01-0.1M Tris-HCl buffer; the pH of the Tris-HCl buffer is 6-9. As an embodiment, the chromatographic column of the affinity chromatography is a cross-linked ovalbumin-porcine gastric mucin affinity chromatography column; as an embodiment, the elution solvent is 1.5M NaCl solution. In a specific embodiment of the present invention, the affinity chromatography includes: adding the dissolved yuzhuanose binding protein crude extract to the cross-linked ovalbumin-porcine gastric mucin affinity chromatography column; after adding, first use 10-20 times the column volume of pH9, 0.1M Tris-HCl buffer for washing; after washing, use 3-5 times the column volume of 1.5M NaCl solution for elution, and collect the NaCl eluate. In a specific embodiment of the present invention, the cross-linked ovalbumin-porcine gastric mucin affinity chromatography column is prepared according to patent 201310393629.2. The present invention uses a cross-linked ovalbumin-porcine gastric mucin affinity chromatography column for affinity chromatography, and the sugar chain structure of porcine gastric mucin can be combined with yuzhunose binding protein by spatial connection, thereby specifically adsorbing yuzhunose binding protein. In addition, the adsorbed yuzhunose binding protein is easily eluted by NaCl eluent to obtain a yuzhunose binding protein eluent with specific structural functions. The results of the embodiment show that the yuzhunose binding protein prepared by the method of the present invention can be inhibited by porcine gastric mucin in agglutinating sheep red blood cells.

[0040] The present invention performs a second dialysis on the eluate to collect a second retentate; the retentate of the second dialysis is 10000Da. The present invention performs a second dialysis on the eluate to effectively remove impurities and improve the purity of the yuzusan binding protein.

[0041] As an embodiment, after collecting the second retentate, the present invention further comprises: freeze-drying the second retentate to obtain a freeze-dried powder. After freeze-drying the second retentate, the present invention facilitates storage and prolongs the shelf life. As an embodiment, the present invention performs a third dissolution of the freeze-dried powder and applies it to a Sephadex G-100 molecular sieve chromatography column to collect OD 280 The eluate of the absorption peak is used to obtain the yuzuose binding protein. As an embodiment, the solvent used in the third dissolution is 0.01-0.1M Tris-HCl buffer; the pH of the Tris-HCl buffer is 6-9. As an embodiment, the elution solution used in the Sephadex G-100 molecular sieve chromatography column is a pH 9, 0.1M Tris-HCl solution. The present invention utilizes a Sephadex G-100 molecular sieve chromatography column to collect the eluate of a specific absorption peak, thereby improving the purity of the yuzuose binding protein.

[0042] The present invention also provides a yuzusu-binding protein prepared by the preparation method described in the above scheme, wherein the molecular weight of the yuzusu-binding protein is 15000Da; the N-terminus of the yuzusu-binding protein is: NH 2 -As p-Asn-Ser-Leu-Thr-Ser-Pro-Asn-Ser-Leu-; in the secondary structure of the polysaccharide binding protein, the proportion of α-helix is ​​20.20%, the proportion of β-fold is 24.12%, the proportion of β-turn is 14.83%, and the proportion of random coil is 40.85%.

[0043] The present invention also provides the yuzusu-binding protein prepared by the preparation method described in the above scheme or the use of the yuzusu-binding protein described in the above scheme in the preparation of hyaluronidase and / or elastase inhibitors. The yuzusu-binding protein described in the present invention can inhibit the activity of hyaluronidase and elastase, and has concentration dependence, can control inflammatory reactions and allergic reactions, and promote skin barrier repair.

[0044] The present invention also provides the yuzusu-binding protein prepared by the preparation method described in the above scheme or the use of the yuzusu-binding protein described in the above scheme in the preparation of cosmetics. As an embodiment, the cosmetics include anti-aging cosmetics. The yuzusu-binding protein described in the present invention can protect skin cells, improve the skin's tolerance to ultraviolet rays, reduce skin aging caused by light damage, and play an anti-aging role.

[0045] The present invention also provides a cosmetic, wherein the active ingredient of the cosmetic includes the yuzusu-binding protein prepared by the preparation method described in the above scheme or the yuzusu-binding protein described in the above scheme. As an embodiment, the active ingredient of the cosmetic is the yuzusu-binding protein. As an embodiment, the mass proportion of the yuzusu-binding protein in the cosmetic is 0.1% to 1.0%.

[0046] In order to further illustrate the present invention, a yuzuose binding protein provided by the present invention and its preparation method and application are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0047] Example 1

[0048] (1) Preparation of Polygonatum tiliaceum Binding Protein:

[0049] A. ① Grind the rhizome of Polygonatum odoratum with a grinder, and sieve it through a 30-mesh sieve to obtain Polygonatum odoratum powder; ② Mix the Polygonatum odoratum powder with 0.9% (w / v) physiological saline at a solid-liquid ratio of 1g:5mL, and soak overnight; ③ After soaking, centrifuge at 9000rpm for 20min and take the supernatant.

[0050] B. ① Add (NH 4 ) 2 SO 4 To (NH 4 ) 2 SO 4 The saturation of the cellulose is 80%, precipitation is carried out, and the solid components are collected to obtain a cellulose protein precipitate; ② The cellulose protein precipitate is dissolved in a 0.15M NaCl solution at a solid-liquid ratio of 1g:10mL; ③ The dissolved cellulose protein precipitate is placed in a dialysis bag, and a first dialysis is performed in deionized water to collect a first retentate, wherein the retention amount of the first dialysis is 10000Da; ④ The first retentate obtained in step ③ is freeze-dried to obtain a crude extract of cellulose binding protein.

[0051] C. ① The crude extract of yuzhuanose binding protein in step B was dissolved in 0.1M Tris-HCl buffer according to a solid-liquid ratio of 1g:10mL, and the pH of the Tris-HCl buffer was 9; ② ​​A cross-linked ovalbumin-porcine gastric mucin affinity chromatography column was prepared with reference to Chinese Patent 201310393629.2, and the dissolved yuzhuanose binding protein crude extract was added to the cross-linked ovalbumin-porcine gastric mucin affinity chromatography column for affinity chromatography, first washing the chromatography column with 15 column volumes of pH9, 0.1M Tris-HCl buffer, and then eluting with 3 column volumes of 1.5MNaCl solution, and collecting the NaCl eluate; ③ The NaCl eluate collected in step ② was subjected to a second dialysis to collect a second retentate, and the interception of the second dialysis was 10000Da; ④ The second retentate in step ③ was freeze-dried to obtain a freeze-dried powder.

[0052] D. ① Dissolve the lyophilized powder obtained in step C in pH 9, 0.1M Tris-HCl buffer; ② Add the dissolved lyophilized powder to a Sephadex G-100 molecular sieve chromatography column, elute with pH 9, 0.1M Tris-HCl, and collect the OD 280 The eluted solutions in the above-mentioned collection tubes are mixed with the chromatographic peaks absorbed, that is, the eluted solutions with agglutination activity are mixed to obtain the yuzuose binding protein. Figure 1 shown.

[0053] (2) Detection of Polygonatum officinale Binding Protein

[0054] A. The obtained lectin-binding protein was subjected to SDS polyacrylamide gel electrophoresis. The operation steps were referred to the literature (Qu M, Tong C, Kong L, Yan X, Chernikov O, Lukyanov P, Jin Q, Li W. Purification of asecreted lectin from Andrias davidianus skin and its anti bacterial activity. Comp. Biochem. Physiol. 2015, 167C: 140-146.). SDS polyacrylamide gel electrophoresis was performed as follows: Figure 2 shown.

[0055] according to Figure 2 It can be seen that the molecular weight of the yuzusu-binding protein prepared by the present invention is 15,000 Daltons. 2 -Asp-Asn-Ser-Leu-Thr-Ser-Pro-Asn-Ser-Leu-.

[0056] B. The yuzhuanose binding protein prepared in step (1) was prepared into a 1.0 mg / mL solution, and the circular dichroism spectrum of the yuzhuanose binding protein sample was measured using a circular dichroism spectrometer. The sample was scanned with a step length of 0.2 nm and a bandwidth of 2.0 nm; the spectral range was 190 to 250 nm, and the scanning speed was 200 nm / min; the baseline spectrum of the buffer was subtracted from each spectrum, and the obtained value was converted into molar ellipticity (θ) using the analysis function built into the instrument supporting software. The circular dichroism results are shown in FIG. Figure 3 shown.

[0057] According to the circular dichroism spectrometer test results, the secondary structure of the polysaccharide binding protein of the present invention has an α-helix ratio of 20.20%, a β-fold ratio of 24.12%, a β-turn ratio of 14.83%, and a random coil ratio of 40.85%.

[0058] Application Example 1

[0059] (1) Experiment on the activity of yuzusu sugar binding protein in agglutinating blood cells

[0060] ① Suspend sterile sheep red blood cells (Nanjing Maojie Microbiological Technology Co., Ltd.) in 10 mL of 0.1 mol / L Tris-HCl buffer to prepare a 2% volume fraction of sheep red blood cell suspension.

[0061] ② The yuzhuanose binding protein prepared in Example 1 was configured into a 2 mg / mL solution, which was used as the mother liquor, denoted as 1; it was diluted in multiples using 0.01 mol / L Tris-HCl buffer to obtain yuzhuanose binding protein solutions with mother liquor concentrations of 1, 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64, 1 / 128, 1 / 256, 1 / 512 and 1 / 1024, respectively; 25 μL of the above-mentioned yuzhuanose binding protein solutions of different concentrations were taken respectively and added to a U-shaped 96-well plate, denoted as the experimental group; 25 μL of 0.01 mol / L Tris-HCl buffer was added to each well of the control group.

[0062] ③ In the experimental group and the control group in the U-shaped 96-well plate, 25 μL of stirred 2% (v / v) sheep red blood cell suspension was added, and the hemagglutination results were visually observed after 30 minutes. The results are shown in Figure 3.

[0063] according to Figure 4 It can be seen that the sheep erythrocytes in the control group did not agglutinate, and sank to the bottom of the U-shaped well in the "U"-shaped 96-well plate to form a small red dot; while a red plaque that diffused like a network was formed between the erythrocytes in the experimental group, proving that the yuzhuan-binding protein of the present invention has the activity of agglutinating sheep erythrocytes.

[0064] (2) The activity of yuzusulan binding protein in agglutinating blood cells was inhibited by pig gastric mucin

[0065] ① Prepare a 2 mg / mL solution of porcine gastric mucin and use it as a mother solution, denoted as 1; use 0.01 mol / L Tris-HCl buffer to perform serial dilutions to obtain porcine gastric mucin solutions with mother solution concentrations of 1, 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64 and 1 / 128 in sequence; take 25 μL of the above porcine gastric mucin solutions of different concentrations and add them to a U-shaped 96-well plate; ② Add 25 μL of the 2 mg / mL polysaccharide binding protein solution in step (1) to each well and react for 30 minutes; ③ After the reaction is completed, add 25 μL of a stirred sheep red blood cell suspension with a volume fraction of 2%, and visually observe the hemagglutination results after 30 minutes. The results are as follows: Figure 5 shown.

[0066] The yuzusu-binding protein of the present invention can specifically bind to porcine gastric mucin, thereby inhibiting the aggregation activity of the yuzusu-binding protein. Figure 5 It can be seen that the red blood cells sink to the bottom of the U-shaped wells in the U-shaped 96-well plate to form a small red dot. This shows that the agglutination of sheep red blood cells by the polysaccharide binding protein of the present invention can be inhibited by porcine gastric mucin.

[0067] Application Example 2

[0068] (1) Determination of the inhibitory activity of hyaluronidase by pyrotein-binding protein

[0069] ① After the yuzu sugar-binding protein prepared in Example 1 was configured into a 5 mg / mL solution, deionized water was added for gradient dilution to obtain yuzu sugar-binding protein solutions with concentrations of 5 mg / mL, 4 mg / mL, 3 mg / mL, 2 mg / mL and 1 mg / mL for later use.

[0070] ② Group A1: a. Take 50 μL of the different concentrations of the polysaccharide binding protein solution in step ①, add 50 μL of hyaluronidase solution (the hyaluronidase solution is prepared with pH 5.4 0.2 mol / L acetic acid buffer, and the concentration of the hyaluronidase solution is 500 U / mL), and react at 37°C for 20 min. b. After the reaction, add 10 μL of 2.5 mol / L CaCl 2 Solution, react at 37℃ for 20min. c. After the reaction, add 50μL of 0.5mg / mL potassium hyaluronate solution respectively, react at 37℃ for 40min; then react at room temperature (25℃) for 10min. d. After the reaction, add 50μL of acetylacetone solution (3.5mL acetylacetone dissolved in 50mL 0.1mol / L sodium carbonate solution, prepared and used immediately), 10μL of 5mol / L NaOH solution, 50μL of deionized water respectively, react in a boiling water bath for 15min, and react in a cold water bath for 10min after cooling. e. After cooling, add 100μL of P-DAB color developer (0.8g DBMA mixed with 15mL concentrated hydrochloric acid and 15mL anhydrous ethanol) respectively and shake thoroughly. f. After shaking, add 350μL of anhydrous ethanol respectively, color develop at room temperature for 30min, and measure OD 530 value.

[0071] Group A2: The operation method is the same as that of Group A1, except that the hyaluronidase solution in step a and the potassium hyaluronate solution in step c of Group A1 are replaced with an equal volume of acetic acid buffer.

[0072] Group B1: The operation method is the same as that of Group A1, except that the yuzusan binding protein solution in step a is replaced with an equal volume of deionized water.

[0073] Group B2: The operation method is the same as that of Group A1, except that the yuzusan binding protein solution in step a is replaced with an equal volume of deionized water, and the hyaluronidase solution in step a and the potassium hyaluronate solution in step c are replaced with an equal volume of acetic acid buffer.

[0074] The hyaluronidase inhibition rate of different concentrations of yuzunosan binding protein was calculated according to the hyaluronidase inhibition rate formula. The results are shown in Table 1 and Figure 6 As shown, the hyaluronidase inhibition rate formula is:

[0075] Hyaluronidase inhibition rate (%) = (1-(A1-A2) / (B1-B2)) × 100%; A1 is the OD measured in group A1 530 A2 is the OD value of group A2. 530 value, B1 is the OD measured by group B1 530 value, B2 is the OD measured by group B2 530 value.

[0076] Table 1 Inhibition rate of hyaluronidase by yuzusulan binding protein

[0077]

[0078] (2) Determination of the inhibitory activity of yuzusulan binding protein on elastase

[0079] ① After the yuzu sugar-binding protein prepared in Example 1 was configured into a 50 mg / mL solution, deionized water was added for gradient dilution to obtain yuzu sugar-binding proteins with concentrations of 50 mg / mL, 40 mg / mL, 30 mg / mL, 20 mg / mL and 10 mg / mL for later use.

[0080] ② Group A1: a. Take 50 μL of the different concentrations of yuzusu-binding protein solution in step ①, add 50 μL of 0.5 U / mL elastase solution and 50 μL of 0.05 mol / L pH 8.5 Tris-HCl buffer, and react at 37°C for 15 min. b. After the reaction, add 50 μL of 1 mmol / L N-succinyl-Ala-Ala-Val-Ala p-nitroaniline solution, react at 37°C for 20 min, and measure OD 410 value.

[0081] Group A2: The operation method is the same as that of Group A1, except that the elastase solution in step a and the 1mmol / L N-succinyl-Ala-Ala-Val-Ala p-nitroaniline solution in step b are replaced with an equal volume of 0.05mol / L pH 8.5 Tris-HCl buffer;

[0082] Group B1: The operation method is the same as that of Group A1, except that the yuzusan binding protein solution in step a is replaced with an equal volume of 0.05 mol / L pH 8.5 Tris-HCl buffer;

[0083] Group B2: The operation method is the same as that of Group A1, except that the yuzusan binding protein solution in step a and the 1mmol / L N-succinyl-Ala-Ala-Val-Ala p-nitroaniline solution in step b are replaced with an equal volume of 0.05mol / L pH 8.5 Tris-HCl buffer.

[0084] The transelastinase inhibition rate of different concentrations of yuzusulan binding protein was calculated according to the transelastinase inhibition rate formula. The results are shown in Table 2 and Figure 7 As shown, the formula for the inhibition rate of elastase is:

[0085] Elastase inhibition rate (%) = (1-(A1-A2) / (B1-B2)) × 100%; A1 is the OD measured in group A1 410 A2 is the OD value of group A2. 410 value, B1 is the OD measured by group B1 410 value, B2 is the OD measured by group B2 410 value.

[0086] Table 2 Inhibition rate of yuzu sugar binding protein on elastase

[0087]

[0088] According to Table 1 and Figure 6 It can be seen that the yuzhuanose binding protein showed good inhibition of hyaluronidase activity, which was concentration-dependent. At a concentration of 5 mg / mL, the inhibition rate was 67.68%. Figure 7 It can be seen that yuzhunose binding protein also showed good inhibition of elastase activity, which was concentration-dependent. At a concentration of 50 mg / mL, the inhibition rate was 78.53%. It can be seen that yuzhunose binding protein has the function of controlling inflammatory reactions and allergic reactions, promoting skin barrier repair, and thus has anti-aging effects.

[0089] Application Example 3

[0090] (1) Preparation of skin cream containing yuzu sugar binding protein

[0091] ① Weigh propylparaben, glyceryl monostearate, stearic acid, cetyl alcohol, propylparaben and dimethicone according to the proportion of each component in Table 3. ② Mix and heat the raw materials weighed in step ① to 80°C under constant stirring, maintain for 20 minutes, and sterilize as the oil phase. ③ Weigh glycerol and triethanolamine according to the proportion of each component in Table 3, add them to distilled water, stir and heat to 80°C, maintain for 20 minutes to sterilize, and cool as the water phase. ④ Weigh the yuzhusuan binding protein prepared in Example 1 according to the ratio of yuzhusuan binding protein: skin cream = 1g: 1000g. ⑤ Slowly add the yuzhusuan binding protein of step ④ to the aqueous phase of step ③ and stir and emulsify to obtain an aqueous phase containing yuzhusuan binding protein. ⑥ After the emulsification is completed, add the aqueous phase containing yuzhusuan binding protein in step ⑤ to the oil phase of step ②, add the essence when the temperature drops to 45°C, continue stirring and cooling, and obtain a skin cream containing yuzhusuan binding protein.

[0092] Table 3 Content of skin cream components

[0093] Element Mass percentage (%) Element Mass percentage (%) Glyceryl Monostearate 8 Propylparaben 0.5 Stearic acid 8 Glycerol 10 Cetyl Alcohol 1 Triethanolamine 1 Propylparaben 0.1 Distilled water 69.9 Dimethicone 1 essence 0.5

[0094] Take 0.5g of skin cream containing yuzu sugar binding protein and dissolve it in 2.5mL of distilled water. Centrifuge at 10000rpm for 20min, take the supernatant, and filter it through a 0.2μm filter membrane to obtain a skin cream sample containing yuzu sugar binding protein, which is recorded as sample A and stored at 4℃ for future use.

[0095] (2) Preparation of Polygonatum odoratum Binding Protein Samples:

[0096] The yuzuose binding protein prepared in Example 1 was prepared into a 2 μg / mL yuzuose binding protein solution with distilled water, centrifuged at 10,000 rpm for 20 min, and the supernatant was filtered through a 0.2 μm filter membrane to obtain a yuzuose binding protein sample, recorded as sample B, and stored at 4°C for later use.

[0097] (3) Preparation of control samples

[0098] ① Prepare a skin cream according to step (1), except that no yuzu sugar binding protein is added to obtain a skin cream. ② Dissolve 0.5 g of skin cream in 2.5 mL of distilled water, centrifuge at 10000 rpm for 20 min, take the supernatant, filter through a 0.2 μm filter membrane, obtain a skin cream sample, record it as sample C, and store it at 4°C for later use.

[0099] (4) Protective effect on HaCaT cells

[0100] Human immortalized keratinocytes HaCaT are often used to replace human primary keratinocytes for in vitro experiments. HaCaT cells were selected to observe the protective effect of yuzusan binding protein on cells.

[0101] ① Use DMEM medium (containing 10% fetal bovine serum FBS, penicillin 100U / ml and streptomycin 100μg / ml) at 37℃ and 5% CO 2 HaCaT cells were cultured in an incubator with DMEM medium regularly replaced. When the cells adhered to the wall by nearly 90%, they were digested with 0.25% (w / v) trypsin solution. When about 80% of the cells began to suspend under a microscope, cell culture medium was added to terminate the digestion. The cells were centrifuged at 1000 rpm for 5 min, and the cells were collected and prepared with culture medium to a density of 1×10 4 / mL HaCaT cell suspension.

[0102] ② The HaCaT cell suspension prepared in step ① was inoculated into a 96-well plate at a dose of 200 μL / well and placed at 37°C with 5% CO 2The cells were incubated in an incubator and set up sample A, sample B, sample C and control groups. After incubation for 48 h, the old culture medium was aspirated and 200 μL of sample A, sample B and sample C were added to the sample A, sample B and sample C groups respectively. 200 μL of distilled water was added to the control group. The cells were placed at 37 °C with 5% CO 2 After 6 hours of incubation, the old culture medium was aspirated, the quartz plate was covered, and ultraviolet light was irradiated for 20 minutes. After the irradiation, fresh culture medium was added, and after another 24 hours of incubation, 20 μL MTT (5 mg / mL) was added to each well and incubated at 37°C with 5% CO 2 Continue incubating in the incubator; after incubation for 4 hours, discard the culture medium, add 180μL DMSO, oscillate on a constant temperature oscillator for 60 seconds, and then detect the absorbance at a wavelength of 570nm using an enzyme reader.

[0103] Three parallel samples were set for each sample solution, and the cell viability was calculated based on the measured absorbance values. The results are shown in Table 4. The cell viability calculation formula is:

[0104] Cell survival rate = [(absorbance value of sample group - absorbance value of control group) / absorbance value of control group]

[0105] ×100%.

[0106] Table 4 Relative survival rate of HaCaT cells

[0107]

[0108] According to Table 4, the skin cream sample containing yuzu sugar binding protein (sample A) has a protective effect on cells damaged by ultraviolet rays. Yuzu sugar binding protein can protect skin cells, improve the skin's tolerance to ultraviolet rays, and reduce skin aging caused by photodamage, thereby achieving an anti-aging effect.

[0109] Application Example 4

[0110] Evaluation of the long-lasting firming and anti-aging effects of yuzu sugar combined with protein skin cream samples

[0111] A randomized, open, self-controlled before-after test method was used to test 40 female subjects aged 30 to 45 years old. After cleaning their faces, the subjects used the skin cream containing yuzu sugar binding protein prepared in Application Example 3 at a dosage of 0.2 mL / cm 2 .

[0112] Facial skin wrinkles, UV spots and brown spots were detected using VISIA skin detector. The efficacy was evaluated and adverse reactions were recorded on the first visit day and 30 days after using the product. The results are shown in Table 5.

[0113] Table 5 Comparison of the numerical values ​​of various indicators detected by VISIA

[0114] project Texture Brown Area Red Zone Before treatment 797.43±166.70 257.13±76.37 822.31±311.07 After treatment 688.3±157.65 198.22±86.32 724.44±254.66 p-value 0.017 0.015 0.314

[0115] According to Table 5, after using the skin cream containing yuzu sugar binding protein for 30 days, the skin texture and brown area of ​​the volunteers were significantly improved (P<0.05), and the VISIA value of the red area after treatment was lower than the VISIA value before treatment. The results show that the skin cream containing yuzu sugar binding protein has a long-lasting firming and anti-aging effect, and no adverse reactions were observed in the subjects.

[0116] In summary, the preparation method of the present invention can be used to obtain a yuzusan-binding protein with a specific structure. The obtained yuzusan-binding protein has the activity of agglutinating sheep red blood cells, and can inhibit the activities of hyaluronidase and elastase, control inflammatory reactions and allergic reactions, promote skin barrier repair, and enhance the skin's tolerance to ultraviolet rays, reduce skin aging caused by light damage, thereby achieving an anti-aging effect.

[0117] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing a polysaccharide-binding protein, characterized in that: The following steps are involved: Add (NH4)2SO4 to the water extract of Polygonatum odoratum until the saturation of (NH4)2SO4 is 60% to 80%, perform precipitation, collect solid components, and obtain a precipitate of zedoary sugar binding protein; perform a first dialysis after dissolving the precipitate of zedoary sugar binding protein, collect a first retentate, and obtain a crude extract of zedoary sugar binding protein; perform a second dissolution of the crude extract of zedoary sugar binding protein, perform affinity chromatography, and collect an eluate; perform a second dialysis on the eluate, collect a second retentate, and obtain zedoary sugar binding protein; The cut-off of the first dialysis is 10000Da; The affinity chromatography column is a cross-linked ovalbumin-porcine gastric mucin affinity chromatography column; The elution solvent of the affinity chromatography is 1.5M NaCl solution; The cut-off of the second dialysis was 10000Da.

2. The preparation method according to claim 1, characterized in that: The preparation of the water extract of Polygonatum odoratum comprises: extracting Polygonatum odoratum with physiological saline to obtain the water extract of Polygonatum odoratum; the material-liquid ratio of the Polygonatum odoratum to the physiological saline is 1 g:5 mL.

3. The preparation method according to claim 2, characterized in that: The water extraction time is 12 to 16 hours.

4. The preparation method according to claim 1, characterized in that: The solvent used in the first dissolution is a 0.15M NaCl solution; the solid-liquid ratio of the polysaccharide binding protein precipitate to the 0.15M NaCl solution is 1 g:10 mL.

5. The preparation method according to claim 1, characterized in that: The solvent used in the second dissolution is 0.01-0.1M Tris-HCl buffer; the pH of the Tris-HCl buffer is 6-9.

6. The preparation method according to claim 1, characterized in that: After collecting the second retentate, the method further comprises: dissolving the second retentate for a third time and eluting it using a Sephadex G-100 molecular sieve chromatography column, collecting OD 280 The eluent of the absorption peak.

7. The polysaccharide-binding protein prepared by the preparation method according to any one of claims 1 to 6, characterized in that: The molecular weight of the yuzuose binding protein is 15000Da; the N-terminus of the yuzuose binding protein is: NH2-Asp-Asn-Ser-Leu-Thr-Ser-Pro-Asn-Ser-Leu-; the proportion of α-helix in the secondary structure of the yuzuose binding protein is 20.20%, the proportion of β-fold is 24.12%, the proportion of β-turn is 14.83%, and the proportion of random coil is 40.85%.

8. Use of the zedoarylose binding protein prepared by the preparation method according to any one of claims 1 to 6 or the zedoarylose binding protein according to claim 7 in the preparation of hyaluronidase and / or elastase inhibitors.

9. Use of the yuzusan binding protein prepared by the preparation method according to any one of claims 1 to 6 or the yuzusan binding protein according to claim 7 in the preparation of cosmetics.

10. A cosmetic, characterized in that: The active ingredient of the cosmetic comprises the zedoarylose-binding protein prepared by the preparation method according to any one of claims 1 to 6 or the zedoarylose-binding protein according to claim 7.

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