Algin with anti-photoaging activity as well as preparation method and application thereof

Abstract

The invention discloses algin with anti-photoaging activity as well as a preparation method and application of the algin, and belongs to the technical field of deep processing of seaweed. The molecular weight of the algin is 10-350kDa, the total sugar content is 40wt%-80wt%, the reducing sugar content is 3wt%-16wt%, and the uronic acid content is 40wt%-80wt%. The preparation method of the algin specifically comprises the following steps: (1) degrading dielectric barrier discharge plasma; and (2) dialyzing and freeze-drying. The algin can significantly improve the inhibition rate of elastase and the cell survival rate of HaCaT cells after ultraviolet irradiation, has significant anti-photoaging activity, and can be used for developing anti-photoaging functional foods and skin care products. Meanwhile, the preparation method adopting the dielectric barrier discharge plasma is simple and convenient to operate, rapid, efficient, green, environment-friendly and free of addition of exogenous chemical substances, and the molecular weight of the algin can be remarkably reduced, so that the additional value of the algin is improved, the application range of the algin is widened, and the algin has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of deep processing of seaweeds, and more specifically to an alginate having anti-photoaging activity, a preparation method thereof, and an application thereof. Background Art

[0002] Alginate mainly exists in the cell walls of seaweeds (such as Sargassum, Laminaria, etc.), belongs to polysaccharide aldose, and the main components are β-D-mannuronic acid (ManA) and α-L-guluronic acid (GulA). In the process of preparing alginate, although the alginates obtained have similar structures due to different raw material sources and extraction methods, the M / G ratio, relative molecular weight, etc. are not the same. Alginate is currently found to have some biological activities, such as antiviral, hypoglycemic, anticoagulant, antioxidant, antitumor, immunomodulatory activity, etc. However, due to the large molecular weight, high viscosity, and strong gel property of alginate, its biological activity is restricted to a certain extent.

[0003] Treatment by degradation means can not only improve the solubility of alginate, but also the obtained products have better biological activities. At present, there are many common degradation means for alginate, such as enzymatic method, chemical method, and physical method, etc. Among them, the enzymatic method for degrading alginate is the most commonly used degradation means for alginate, mainly using alginate lyase to prepare the degradation product of alginate by β-elimination reaction. The chemical methods for degrading alginate include acidolysis, alkali hydrolysis, oxidative degradation, etc. Generally speaking, the process for preparing alginate by chemical method is simple, but the acid and alkali will corrode the equipment during the preparation process, and the use of chemical reagents is also likely to cause environmental pollution. The physical methods for degrading alginate include γ-ray, ultrasonic, hydrothermal treatment, ultraviolet irradiation, high-pressure method, etc. The physical method is environmentally friendly, but has high requirements for equipment and high cost. In addition, the efficiency of the physical method is limited, and its action mechanism is not yet clear. In actual use, the physical method is rarely used alone and is often combined with other methods.

[0004] Plasma is a new type of green processing technology, a combination of physical and chemical methods, environmentally friendly. It has been found that it has good degradation effects on polysaccharides such as chitosan, starch, and cellulose, and can improve the biological activities of the product polysaccharides. Plasma includes dielectric barrier discharge plasma, pulsed discharge plasma, glow discharge plasma, etc., among which dielectric barrier discharge plasma has attracted much attention. However, there is no literature report on preparing alginate by dielectric barrier discharge plasma method and affecting its anti-photoaging activity.

[0005] Therefore, how to prepare alginate having anti-photoaging activity by dielectric barrier discharge plasma method is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide an alginate with anti-photoaging activity, its preparation method and application, so as to solve the deficiencies in the prior art.

[0007] To achieve the above purpose, the present invention adopts the following technical solutions:

[0008] An alginate with anti-photoaging activity, having a molecular weight of 10 - 350 kDa, a total sugar content of 40 wt% - 80 wt%, a reducing sugar content of 3 wt% - 16 wt%, and a uronic acid content of 40 wt% - 80 wt%.

[0009] Furthermore, the molecular weight of the above-mentioned alginate with anti-photoaging activity is 10 - 30 kDa, the total sugar content is 55 wt% - 77 wt%, the reducing sugar content is 4 wt% - 15 wt%, and the uronic acid content is 51 wt% - 73 wt%.

[0010] A preparation method of the above-mentioned alginate with anti-photoaging activity specifically includes the following steps:

[0011] (1) Dielectric barrier discharge plasma degradation

[0012] First, add alginate to pure water and dissolve it to obtain an alginate solution; secondly, place the alginate solution in a dielectric barrier discharge plasma reactor for degradation to obtain a degradation solution; then, evaporate and concentrate the degradation solution to obtain a concentrated solution; finally, centrifuge the concentrated solution and take the supernatant;

[0013] (2) Dialysis and freeze-drying

[0014] Dialyze the supernatant using a dialysis bag, collect the macromolecule retention solution, and vacuum freeze-dry it to obtain the alginate with anti-photoaging activity.

[0015] Furthermore, in the above step (1), the raw material sources of alginate include but are not limited to various seaweeds such as kelp, Sargassum fusiforme, Undaria pinnatifida, etc. The extraction methods of alginate include but are not limited to enzymatic hydrolysis method, acid-base method, ultrasonic method, precipitation method, etc. The molecular weight of alginate is 300 - 800 kDa, the total sugar content is 40 wt% - 60 wt%, the reducing sugar content is 3 wt% - 10 wt%, and the uronic acid content is 50 wt% - 90 wt%.

[0016] Even further, in the above step (1), the molecular weight of alginate is 400 - 600 kDa, the total sugar content is 40 wt% - 60 wt%, the reducing sugar content is 3 wt% - 6 wt%, and the uronic acid content is 70 wt% - 90 wt%.

[0017] Furthermore, in the above step (1), the concentration of the alginate solution is 2 - 10 mg / mL.

[0018] Further, in the above step (1), the dielectric barrier discharge plasma reactor comprises a plasma power supply (Nanjing Suman Plasma Technology Co., Ltd., model: CTP-2000K), a dielectric barrier discharge reactor (Nanjing Suman Plasma Technology Co., Ltd., model: DBD-100), and a dielectric barrier discharge reaction kettle (Nanjing Suman Plasma Technology Co., Ltd., model: DBD-200).

[0019] Further, in the above step (1), the voltage of the dielectric barrier discharge plasma reactor is 50 - 90V, the current is 1 - 4A, and the distance between the upper dielectric and the liquid surface is 2 - 8mm.

[0020] Further, in the above step (1), the degradation time is 5 - 60min.

[0021] Further, in the above step (2), the cut-off molecular weight of the dialysis bag is 100 - 3000Da; the dialysis time is 12 - 54h.

[0022] The present invention also claims the application of the above-mentioned alginate with anti-photoaging activity or the alginate with anti-photoaging activity prepared by the above-mentioned preparation method in the preparation of foods and skin care products with anti-photoaging functions.

[0023] As can be seen from the above technical solutions, compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] The alginate of the present invention can significantly improve the inhibition rate of elastase and the cell survival rate of HaCaT cells after ultraviolet irradiation, has significant anti-photoaging activity, and can be used for the development of anti-photoaging functional foods and skin care products. At the same time, the preparation method using dielectric barrier discharge plasma of the present invention is simple, rapid, efficient, green and environmentally friendly, without the addition of exogenous chemical substances, and can significantly reduce the molecular weight of alginate, thereby improving the added value of seaweed and broadening the application range of seaweed, and has good application prospects. Description of the Drawings

[0025] Figure 1 is the elastase inhibition rate of alginate samples A - E and control sample L;

[0026] Figure 2 is the elastase inhibition rate of alginate samples E - K and control sample L;

[0027] Figure 3 is the cytotoxicity diagram of alginate samples A - E and control sample L; wherein, the mark * represents that the cell survival rate of the alginate sample group has a significant difference from that of the Control group;

[0028] Figure 4Cell survival rate graphs of alginate samples A - E and control sample L after ultraviolet irradiation; among them, the mark * represents that the cell survival rate of the alginate sample group after irradiation has a significant difference from that of the Model group. Detailed implementation manner

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0030] Example 1

[0031] A preparation method of alginate with anti - photoaging activity specifically includes the following steps:

[0032] (1) Dielectric barrier discharge plasma degradation

[0033] First, add alginate to pure water and dissolve it to obtain a 2mg / mL alginate solution; secondly, place the alginate solution in a plasma power supply of model CTP - 2000K, a dielectric barrier discharge reactor of model DBD - 100, and a dielectric barrier discharge reaction kettle of model DBD - 200 produced by Nanjing Suman Plasma Technology Co., Ltd. Set the treatment voltage to 50V, the current to 2A, the distance between the upper dielectric and the liquid surface to 5mm, and degrade for 60min to obtain a degradation solution; then, evaporate and concentrate the degradation solution to 1 / 6 of the original volume to obtain a concentrated solution; finally, centrifuge the concentrated solution and take the supernatant;

[0034] (2) Dialysis and freeze - drying

[0035] Dialyze with a dialysis bag with a molecular weight cut - off of 500Da for 48h, collect the macromolecular retention solution, and vacuum freeze - dry to obtain alginate with anti - photoaging activity (marked as sample A).

[0036] Example 2

[0037] A preparation method of alginate with anti - photoaging activity specifically includes the following steps:

[0038] First, add alginate into pure water and dissolve it to obtain an alginate solution with a concentration of 2 mg / mL. Secondly, place the alginate solution in a plasma power supply model CTP-2000K, a dielectric barrier discharge reactor model DBD-100, and a dielectric barrier discharge reaction kettle model DBD-200 produced by Nanjing Suman Plasma Technology Co., Ltd. Set the treatment voltage to 60 V, the current to 2 A, and the distance between the upper dielectric and the liquid surface to 5 mm, and degrade for 60 min to obtain a degradation solution. Then, evaporate and concentrate the degradation solution to 1 / 6 of the original volume to obtain a concentrated solution. Finally, centrifuge the concentrated solution and take the supernatant;

[0039] (2) Dialysis and freeze-drying

[0040] Dialyze with a dialysis bag with a molecular weight cut-off of 500 Da for 48 h, collect the macromolecule retentate, and vacuum freeze-dry to obtain alginate with anti-photoaging activity (labeled as sample B).

[0041] Example 3

[0042] A preparation method of alginate with anti-photoaging activity specifically includes the following steps:

[0043] (1) Dielectric barrier discharge plasma degradation

[0044] First, add alginate into pure water and dissolve it to obtain an alginate solution with a concentration of 2 mg / mL. Secondly, place the alginate solution in a plasma power supply model CTP-2000K, a dielectric barrier discharge reactor model DBD-100, and a dielectric barrier discharge reaction kettle model DBD-200 produced by Nanjing Suman Plasma Technology Co., Ltd. Set the treatment voltage to 70 V, the current to 2 A, and the distance between the upper dielectric and the liquid surface to 5 mm, and degrade for 60 min to obtain a degradation solution. Then, evaporate and concentrate the degradation solution to 1 / 6 of the original volume to obtain a concentrated solution. Finally, centrifuge the concentrated solution and take the supernatant;

[0045] (2) Dialysis and freeze-drying

[0046] Dialyze with a dialysis bag with a molecular weight cut-off of 500 Da for 48 h, collect the macromolecule retentate, and vacuum freeze-dry to obtain alginate with anti-photoaging activity (labeled as sample C).

[0047] Example 4

[0048] A preparation method of alginate with anti-photoaging activity specifically includes the following steps:

[0049] (1) Dielectric barrier discharge plasma degradation

[0050] First, add alginate into pure water and dissolve it to obtain an alginate solution with a concentration of 2 mg / mL. Secondly, place the alginate solution in a plasma power supply model CTP-2000K, a dielectric barrier discharge reactor model DBD-100, and a dielectric barrier discharge reaction kettle model DBD-200 produced by Nanjing Suman Plasma Technology Co., Ltd. Set the treatment voltage to 90 V, the current to 2 A, the distance between the upper dielectric and the liquid surface to 5 mm, and degrade for 60 min to obtain a degradation solution. Then, evaporate and concentrate the degradation solution to 1 / 6 of its original volume to obtain a concentrated solution. Finally, centrifuge the concentrated solution and take the supernatant;

[0051] (2) Dialysis and freeze-drying

[0052] Dialyze for 48 h using a dialysis bag with a molecular weight cut-off of 500 Da, collect the macromolecular retentate, and vacuum freeze-dry it to obtain alginate with anti-photoaging activity (labeled as sample D).

[0053] Example 5

[0054] A preparation method of alginate with anti-photoaging activity specifically includes the following steps:

[0055] (1) Dielectric barrier discharge plasma degradation

[0056] First, add alginate into pure water and dissolve it to obtain an alginate solution with a concentration of 2 mg / mL. Secondly, place the alginate solution in a plasma power supply model CTP-2000K, a dielectric barrier discharge reactor model DBD-100, and a dielectric barrier discharge reaction kettle model DBD-200 produced by Nanjing Suman Plasma Technology Co., Ltd. Set the treatment voltage to 80 V, the current to 2 A, the distance between the upper dielectric and the liquid surface to 5 mm, and degrade for 60 min to obtain a degradation solution. Then, evaporate and concentrate the degradation solution to 1 / 6 of its original volume to obtain a concentrated solution. Finally, centrifuge the concentrated solution and take the supernatant;

[0057] (2) Dialysis and freeze-drying

[0058] Dialyze for 48 h using a dialysis bag with a molecular weight cut-off of 500 Da, collect the macromolecular retentate, and vacuum freeze-dry it to obtain alginate with anti-photoaging activity (labeled as sample E).

[0059] Example 6

[0060] A preparation method of alginate with anti-photoaging activity specifically includes the following steps:

[0061] (1) Dielectric barrier discharge plasma degradation

[0062] First, add alginate into pure water and dissolve it to obtain an alginate solution with a concentration of 2 mg / mL. Secondly, place the alginate solution in a plasma power supply of model CTP-2000K, a dielectric barrier discharge reactor of model DBD-100, and a dielectric barrier discharge reaction kettle of model DBD-200, all produced by Nanjing Suman Plasma Technology Co., Ltd. Set the treatment voltage to 80 V, the current to 2 A, the distance between the upper dielectric and the liquid surface to 5 mm, and degrade for 5 min to obtain a degradation solution. Then, evaporate and concentrate the degradation solution to 1 / 6 of its original volume to obtain a concentrated solution. Finally, centrifuge the concentrated solution and take the supernatant;

[0063] (2) Dialysis and freeze-drying

[0064] Dialyze for 48 h using a dialysis bag with a molecular weight cut-off of 500 Da, collect the macromolecular retentate, and vacuum freeze-dry it to obtain alginate with anti-photoaging activity (labeled as sample F).

[0065] Example 7

[0066] A preparation method of alginate with anti-photoaging activity specifically includes the following steps:

[0067] (1) Dielectric barrier discharge plasma degradation

[0068] First, add alginate into pure water and dissolve it to obtain an alginate solution with a concentration of 2 mg / mL. Secondly, place the alginate solution in a plasma power supply of model CTP-2000K, a dielectric barrier discharge reactor of model DBD-100, and a dielectric barrier discharge reaction kettle of model DBD-200, all produced by Nanjing Suman Plasma Technology Co., Ltd. Set the treatment voltage to 80 V, the current to 2 A, the distance between the upper dielectric and the liquid surface to 5 mm, and degrade for 10 min to obtain a degradation solution. Then, evaporate and concentrate the degradation solution to 1 / 6 of its original volume to obtain a concentrated solution. Finally, centrifuge the concentrated solution and take the supernatant;

[0069] (2) Dialysis and freeze-drying

[0070] Dialyze for 48 h using a dialysis bag with a molecular weight cut-off of 500 Da, collect the macromolecular retentate, and vacuum freeze-dry it to obtain alginate with anti-photoaging activity (labeled as sample G).

[0071] Example 8

[0072] A preparation method of alginate with anti-photoaging activity specifically includes the following steps:

[0073] (1) Dielectric barrier discharge plasma degradation

[0074] First, add alginate into pure water and dissolve it to obtain a 2 mg / mL alginate solution. Secondly, place the alginate solution in a plasma power supply of model CTP-2000K, a dielectric barrier discharge reactor of model DBD-100, and a dielectric barrier discharge reaction kettle of model DBD-200 produced by Nanjing Suman Plasma Technology Co., Ltd. Set the treatment voltage to 80 V, the current to 2 A, the distance between the upper dielectric and the liquid surface to 5 mm, and degrade for 20 min to obtain a degradation solution. Then, evaporate and concentrate the degradation solution to 1 / 6 of the original volume to obtain a concentrated solution. Finally, centrifuge the concentrated solution and take the supernatant;

[0075] (2) Dialysis and freeze-drying

[0076] Dialyze for 48 h using a dialysis bag with a molecular weight cut-off of 500 Da, collect the macromolecule retentate, and vacuum freeze-dry it to obtain alginate with anti-photoaging activity (labeled as sample H).

[0077] Example 9

[0078] A preparation method of alginate with anti-photoaging activity specifically includes the following steps:

[0079] (1) Dielectric barrier discharge plasma degradation

[0080] First, add alginate into pure water and dissolve it to obtain a 2 mg / mL alginate solution. Secondly, place the alginate solution in a plasma power supply of model CTP-2000K, a dielectric barrier discharge reactor of model DBD-100, and a dielectric barrier discharge reaction kettle of model DBD-200 produced by Nanjing Suman Plasma Technology Co., Ltd. Set the treatment voltage to 80 V, the current to 2 A, the distance between the upper dielectric and the liquid surface to 5 mm, and degrade for 30 min to obtain a degradation solution. Then, evaporate and concentrate the degradation solution to 1 / 6 of the original volume to obtain a concentrated solution. Finally, centrifuge the concentrated solution and take the supernatant;

[0081] (2) Dialysis and freeze-drying

[0082] Dialyze for 48 h using a dialysis bag with a molecular weight cut-off of 500 Da, collect the macromolecule retentate, and vacuum freeze-dry it to obtain alginate with anti-photoaging activity (labeled as sample I).

[0083] Example 10

[0084] A preparation method of alginate with anti-photoaging activity specifically includes the following steps:

[0085] (1) Dielectric barrier discharge plasma degradation

[0086] First, add alginate into pure water and dissolve it to obtain a 2 mg / mL alginate solution. Secondly, place the alginate solution in a plasma power supply of model CTP-2000K, a dielectric barrier discharge reactor of model DBD-100, and a dielectric barrier discharge reaction kettle of model DBD-200 produced by Nanjing Suman Plasma Technology Co., Ltd. Set the treatment voltage to 80 V, the current to 2 A, the distance between the upper medium and the liquid surface to 5 mm, and degrade for 40 min to obtain a degradation solution. Then, evaporate and concentrate the degradation solution to 1 / 6 of the original volume to obtain a concentrated solution. Finally, centrifuge the concentrated solution and take the supernatant.

[0087] (2) Dialysis and freeze-drying

[0088] Dialyze for 48 h using a dialysis bag with a molecular weight cut-off of 500 Da, collect the macromolecular retentate, and vacuum freeze-dry to obtain alginate with anti-photoaging activity (labeled as sample J).

[0089] Example 11

[0090] A preparation method of alginate with anti-photoaging activity specifically includes the following steps:

[0091] (1) Dielectric barrier discharge plasma degradation

[0092] First, add alginate into pure water and dissolve it to obtain a 2 mg / mL alginate solution. Secondly, place the alginate solution in a plasma power supply of model CTP-2000K, a dielectric barrier discharge reactor of model DBD-100, and a dielectric barrier discharge reaction kettle of model DBD-200 produced by Nanjing Suman Plasma Technology Co., Ltd. Set the treatment voltage to 80 V, the current to 2 A, the distance between the upper medium and the liquid surface to 5 mm, and degrade for 50 min to obtain a degradation solution. Then, evaporate and concentrate the degradation solution to 1 / 6 of the original volume to obtain a concentrated solution. Finally, centrifuge the concentrated solution and take the supernatant.

[0093] (2) Dialysis and freeze-drying

[0094] Dialyze for 48 h using a dialysis bag with a molecular weight cut-off of 500 Da, collect the macromolecular retentate, and vacuum freeze-dry to obtain alginate with anti-photoaging activity (labeled as sample K).

[0095] Control

[0096] The alginate before degradation (molecular weight 502 kDa) is named control sample L.

[0097] Performance test

[0098] 1. Influence of dielectric barrier discharge plasma on the molecular weight of alginate

[0099] The alginate samples A - E prepared in Examples 1 - 5 were selected, and the molecular weights of the comparative sample L in the comparative examples were compared.

[0100] Among them, the determination of molecular weight: The molecular weight of polysaccharides was determined by high performance gel permeation chromatography (HPGPC). A Waters 2414 differential refractive index detector was used. The chromatographic columns TSKgel G - 6000PWXL (7.8×300 mm) and TSKgel G - 3000PWXL (7.8×300 mm) were used in series. A 0.02 M KH2PO4 solution was used as the mobile phase. The column oven temperature was set at 35 ± 1 °C, and the flow rate was set at 0.5 mL / min; 30 μL of sample was injected each time. Pullulan polysaccharides with molecular weights of 504, 991, 9800, 22000, 107000, 201000, 739000, and 1450000 Da were used as molecular weight standards respectively. A standard curve was made with the retention time as the abscissa and lgMw as the ordinate. The standards, alginate, and degradation products were all dissolved with the mobile phase. After the concentration was prepared to 2 mg / mL, they were filtered through a 0.22 μm aqueous filter membrane for detection.

[0101] The results are shown in Table 1.

[0102] Table 1 Effects of dielectric barrier discharge plasma on the molecular weight of alginate

[0103] Alginic acid sample Average molecular weight (kDa) Control sample L 502 Sample A 27 Sample B 16 Sample C 13 Sample D 12 Sample E 12

[0104] As can be seen from Table 1, dielectric barrier discharge plasma can reduce the molecular weight of alginate.

[0105] 2. Effects of dielectric barrier discharge plasma on the molecular weight and chemical composition of alginate

[0106] In order to further evaluate the degradation effect of dielectric barrier discharge plasma on alginate, the alginate samples E - K prepared in Examples 5 - 11 were selected, and the molecular weights and chemical compositions (total sugar, reducing sugar, and uronic acid) of the comparative sample L in the comparative examples were compared.

[0107] Among them, the determination of molecular weight: The molecular weight of the polysaccharide was determined by high performance gel permeation chromatography (HPGPC). A Waters 2414 differential refractive index detector was used. The chromatographic columns TSKgel G-6000PWXL (7.8×300 mm) and TSKgel G-3000PWXL (7.8×300 mm) were used in series. A 0.02 M KH2PO4 solution was used as the mobile phase. The column oven temperature was set at 35±1 °C, and the flow rate was set at 0.5 mL / min; 30 μL of sample was injected each time. Pullulan polysaccharides with molecular weights of 504, 991, 9800, 22000, 107000, 201000, 739000, and 1450000 Da were used as molecular weight standards. A standard curve was made with the retention time as the abscissa and lgMw as the ordinate. The standards, alginate, and degradation products were all dissolved in the mobile phase. After the concentration was prepared to 2 mg / mL, it was filtered through a 0.22 μm aqueous filter membrane for detection.

[0108] Determination of total sugar content: The total sugar content of alginate was determined by the phenol-sulfuric acid method, using fucose as the standard.

[0109] Determination of reducing sugar content: The reducing sugar content of alginate was determined by the DNS method (3,5-dinitrosalicylic acid), using fucose as the standard.

[0110] Content of uronic acid: The content of uronic acid in alginate was determined by the m-hydroxybiphenyl method, using mannuronic acid as the standard.

[0111] The results are shown in Table 2.

[0112] Table 2 Effects of dielectric barrier discharge plasma on the molecular weight and chemical composition of alginate

[0113]

[0114]

[0115] As shown in Table 2, the degradation by dielectric barrier discharge plasma can lead to a decrease in the molecular weight and uronic acid content of alginate and an increase in the total sugar and reducing sugar contents.

[0116] 3. Effects of dielectric barrier discharge plasma on the anti-photoaging activity of alginate

[0117] The alginate samples A-K prepared in Examples 1-11 were selected, and the elastase inhibition rate, cytotoxicity, and cell viability of HaCaT cells after ultraviolet irradiation of the control sample L in the control example were compared.

[0118] Among them, the determination of elastase inhibition rate: Add 20 μL of alginate sample solution with concentrations of 125, 250, 500, 750, and 1000 μg / mL and 100 μL of 100 U / mL elastase solution into the 96-well plate. After mixing with an oscillator, incubate at 37 °C for 20 min. Then add 80 μL of 2 mmol / L N-succinyl-Ala-Ala-Ala-pNA solution and incubate at 37 °C for 20 min. The whole reaction is carried out in 100 mM Tris-HCl buffer at pH = 8.0. Each sample is analyzed in triplicate, and the absorbance of the reaction product is recorded at 410 nm. The Tris-HCl buffer is used as a blank control, and the elastase inhibition rate is calculated by the following formula: Elastase inhibition rate % = (1 - (V1 - V0) / (C1 - C0)) × 100%; where C0 is the absorbance value of the control group before the reaction, C1 is the absorbance value of the control group after the reaction, V0 is the absorbance value of the sample group before the reaction, and V1 is the absorbance value of the sample group after the reaction.

[0119] Determination of alginate cytotoxicity: In the 96-well plate, inoculate 1.2×10 4 HaCaT cells per well, and culture in a biological incubator (37 °C, 5% CO2) for 24 h. Then discard the old medium. Use the addition of 100 μL / well of serum-free medium as the control group (Control group), and use the addition of 100 μL / well of alginate solution with different concentrations (prepared with serum-free medium) as the experimental group. The concentrations of each alginate are 31.25, 62.5, and 125 μg / mL respectively. After adding the samples, place them in the incubator for 24 h, then discard the medium. After washing each well with 100 μL of PBS solution, add 100 μL of DMEM complete medium to each well and continue to incubate for 24 h. The MTT method is used to determine cell viability. Add 10 μL of 1×MTT solution to each well, continue to incubate in the incubator for 4 h, take out, discard the MTT, then add 150 μL / well of DMSO solution, pipette evenly, and read the absorbance at a wavelength of 570 nm. Cell survival rate / % = absorbance of the experimental group / absorbance of the control group × 100%.

[0120] Determination of the survival rate of HaCaT cells after ultraviolet irradiation: In the 96-well plate, inoculate 1.2×10 4HaCaT cells were cultured in an incubator (37 °C, 5% CO2) for 24 h, and then the old medium was discarded. 100 μL / well of serum-free medium was added to both the control group (Control group) and the model group (Model group), while 100 μL / well of alginate solutions with different concentrations (prepared with serum-free medium) were added to the experimental groups. The concentrations of each alginate sample were 31.25, 62.5, and 125 μg / mL respectively. After adding the samples, they were placed in the incubator and cultured for another 6 h. Then the medium was discarded. After adding 50 μL of PBS solution to each well, except for the Control group, the other groups were irradiated with ultraviolet light at a dose of 2 mJ / cm 2 , and after the irradiation, the PBS was aspirated and discarded. 100 μL of complete DMEM medium was added to each well and incubated for another 24 h. The cell viability was measured by the MTT method. 10 μL of 1×MTT solution was added to each well, and the incubation was continued in the incubator for 4 h. Then it was taken out, the MTT was discarded, and 150 μL / well of DMSO solution was added, and it was pipetted evenly. The absorbance was measured at a wavelength of 570 nm. Cell survival rate / % = absorbance of the experimental group / absorbance of the control group × 100%.

[0121] The results are as Figures 1 - 4 shown.

[0122] After ultraviolet radiation damages the skin, it often causes a reduction in elastin and further reduces skin elasticity. During the process of elastin reduction, elastase plays a major role. Therefore, the elastase inhibition rate can be used to evaluate the anti-photoaging ability of active substances well. As Figure 1 shown, the elastase inhibition rate of the contrast sample L (alginate before degradation) at a concentration of 1000 μg / mL was only 52.25%, but the alginate samples obtained after dielectric barrier discharge plasma treatment had higher elastase inhibition rates at concentrations of 500 - 1000 μg / mL. Among them, the sample E obtained in Example 5 had an elastase inhibition rate of 95.90% at a concentration of 1000 μg / mL. Figure 2 shows the elastase inhibition rates of alginates degraded for different times at a voltage of 80 V. From Figure 2 it can be seen that except for sample F, the other samples had significantly higher elastase inhibition rates than the contrast sample L at concentrations of 500 - 1000 μg / mL. The above results indicate that the alginate prepared by dielectric barrier discharge plasma treatment has a stronger inhibitory effect on elastase, indicating its better anti-photoaging activity.

[0123] As Figure 3As shown, the cell viability of HaCaT cells treated with the control sample L and samples A - E was all above 80%, indicating that the alginate samples obtained by dielectric barrier discharge plasma treatment had no obvious cytotoxicity, and the sample A obtained in Example 1 had a significant proliferation - promoting effect at concentrations below 62.5 - 250 μg / mL.

[0124] After ultraviolet irradiation, cells will release some cytokines, which may lead to cell death. Therefore, by measuring the cell viability after ultraviolet irradiation, the anti - photoaging activity of the sample can be reflected to a certain extent. As Figure 4 shown, compared with the Control group, after ultraviolet irradiation, the cell viability of the Model group decreased to about 50%, indicating that ultraviolet irradiation could cause the death of HaCaT cells, and the photoaging model was successfully established. Under the intervention of samples B - E at different concentrations, the cell viability was significantly increased compared with the model group. Among them, after treatment with 62.5 μg / mL of sample E, the cell viability after ultraviolet irradiation reached 77.10%, and after treatment with 62.5 μg / mL of sample D, the cell viability after ultraviolet irradiation reached 77.48%. The results showed that the alginate prepared by dielectric barrier discharge plasma treatment could significantly improve the cell viability of HaCaT cells after ultraviolet irradiation and had good anti - photoaging activity.

[0125] Some studies have shown that the contents of reducing sugar and uronic acid play an important role in the exertion of the biological activity of polysaccharides. Combining Table 1 - 2 and Figures 1 - 4 it can be known that alginates with a molecular weight of 10 - 30 kDa, a total sugar content of 55 wt% - 77 wt%, a reducing sugar content of 4 wt% - 15 wt%, and a uronic acid content of 51 wt% - 73 wt% have significant anti - photoaging activity.

[0126] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An alginate with anti-photoaging activity, characterized in that, The molecular weight is 10 - 350 kDa, the total sugar content is 40 wt% - 80 wt%, the reducing sugar content is 3 wt% - 16 wt%, and the uronic acid content is 40 wt% - 80 wt%.

2. The alginate with anti-photoaging activity according to claim 1, wherein The molecular weight is 10 - 30 kDa, the total sugar content is 55 wt% - 77 wt%, the reducing sugar content is 4 wt% - 15 wt%, and the uronic acid content is 51 wt% - 73 wt%.

3. A preparation method of alginate with anti-photoaging activity as described in claim 1, characterized in that, Specifically, it includes the following steps: (1) Degradation by dielectric barrier discharge plasma First, add alginate into pure water and dissolve it to obtain an alginate solution; second, place the alginate solution in a dielectric barrier discharge plasma reactor for degradation to obtain a degradation solution; Then, evaporate and concentrate the degradation solution to obtain a concentrated solution; Finally, centrifuge the concentrated solution and take the supernatant; (2) Dialysis and freeze-drying Dialyze the supernatant using a dialysis bag, collect the macromolecule retention solution, and vacuum freeze-dry it to obtain the alginate with anti-photoaging activity.

4. The preparation method of an alginate having anti-photoaging activity according to claim 3, characterized in that, In step (1), the molecular weight of the alginate is 300 - 800 kDa, the total sugar content is 40 wt% - 60 wt%, the reducing sugar content is 3 wt% - 10 wt%, and the uronic acid content is 50 wt% - 90 wt%.

5. The preparation method of an alginate with anti-photoaging activity according to claim 3, characterized in that, In step (1), the concentration of the alginate solution is 2 - 10 mg / mL.

6. The preparation method of an alginate with anti-photoaging activity according to claim 3, characterized in that, In step (1), the dielectric barrier discharge plasma reactor includes a plasma power supply, a dielectric barrier discharge reactor, and a dielectric barrier discharge reaction kettle.

7. The preparation method of an alginate with anti-photoaging activity according to claim 3, characterized in that, In step (1), the voltage of the dielectric barrier discharge plasma reactor is 50 - 90 V, the current is 1 - 4 A, and the distance between the upper dielectric and the liquid surface is 2 - 8 mm.

8. The preparation method of an alginate with anti-photoaging activity according to claim 3, characterized in that, In step (1), the degradation time is 5 - 60 min.

9. The preparation method of an alginate with anti-photoaging activity according to claim 3, characterized in that, In step (2), the molecular weight cut-off of the dialysis bag is 100 - 3000 Da; the dialysis time is 12 - 54 h.

10. Use of the alginate with anti-photoaging activity according to claim 1 or 2 or the alginate with anti-photoaging activity prepared by the preparation method according to claims 3 - 9 in the preparation of foods and skin care products with anti-photoaging functions.

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