A natural source polysaccharide composition, its preparation method and use

Abstract

The application belongs to the technical field of skin care product preparation, and discloses a natural polysaccharide composition, a preparation method and application thereof. The natural polysaccharide composition is composed of the following components in percentage by mass: Pseudoalteromonas ferment product extract 50-70%, locust bean gum 10-15%, cassia gum 5-10%, and psyllium seed gum 15-25%. The Pseudoalteromonas ferment product extract refers to extracellular polysaccharide produced by fermentation of Pseudoalteromonas mucosa DSM15203; the locust bean gum is obtained by extraction and mannanase enzymolysis of seeds of locust trees; the cassia gum is obtained by extraction and mannanase enzymolysis of seed endosperm of cassia plants; and the psyllium seed gum is obtained by extraction and xylanase enzymolysis of seed husks of psyllium.

Description

Technical Field

[0001] The present invention belongs to the technical field of skin care product preparation, and particularly relates to a natural polysaccharide composition, a preparation method and an application thereof. Background Art

[0002] Polysaccharides are formed by the condensation and dehydration of multiple monosaccharide molecules. They are a type of carbohydrate with a complex and bulky molecular structure. All carbohydrates and their derivatives that meet the concept of high molecular weight compounds are called polysaccharides. Polysaccharides are sugar chains connected by glycosidic bonds. They are polymeric sugars composed of more than 10 monosaccharides and are high molecular weight carbohydrates. They can be represented by the general formula (C6H 10 O5) n Polysaccharides composed of the same monosaccharides are called homopolysaccharides, such as starch, cellulose, and glycogen. Polysaccharides composed of different monosaccharides are called heteropolysaccharides, such as gum arabic, which is composed of pentose and galactose. Polysaccharides are not pure chemical substances, but mixtures of substances with varying degrees of polymerization. There are nearly 400 known natural polysaccharides, which are widely found in plants, animals, and microbial tissues and have many important functions.

[0003] Currently, single polysaccharide technology is relatively mature. However, the same polysaccharide can vary significantly in molecular weight and function after enzymatic digestion or other manipulations. Polysaccharides formed by the condensation of a single monosaccharide molecule are called homogeneous polysaccharides. The most abundant homogeneous polysaccharides in nature are starch, glycogen, and cellulose. Polysaccharides formed by the condensation of different monosaccharide molecules are called heterogeneous polysaccharides. Common examples include hyaluronic acid and chondroitin sulfate. Some heterogeneous polysaccharides are composed of a series of repeating disaccharides containing glycosaminoglycans, known as glycosaminoglycans (GAGs), also known as mucopolysaccharides.

[0004] The application of polysaccharides in existing technologies has the following limitations: (1) The efficacy of a single polysaccharide is limited. (2) The influence of the properties of polysaccharides on each other is unclear. (3) The control of polysaccharide molecular weight is not precise enough. Summary of the Invention

[0005] In order to overcome the above-mentioned shortcomings and deficiencies in the prior art, the primary purpose of the present invention is to provide a natural polysaccharide composition.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned natural polysaccharide composition.

[0007] Another object of the present invention is to provide an application of the above-mentioned natural polysaccharide composition.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A natural polysaccharide composition, comprising the following components in percentage by mass:

[0010] Pseudoalteromonas fermentation product extract 50-70%

[0011] Locust bean gum 10-15%

[0012] Cassia gum 5-10%

[0013] Plantain seed gum 15-25%.

[0014] The Pseudoalteromonas fermentation product extract refers to the exopolysaccharide produced by fermentation of Pseudoalteromonas marinus DSM15203.

[0015] The Pseudoalteromonas fermentation product extract is prepared specifically according to the following steps: adding Pseudoalteromonas maritima DSM15203 to a culture medium, placing the culture medium in a constant temperature shaker, and culturing the culture medium at a temperature of 10-15°C and a rotation speed of 200-230 r / min for 36-48 hours; then sequentially performing inactivation, coarse filtration, centrifugal separation of impurities, protease deproteinization, inactivation, decolorization, passing through a UF ultrafiltration membrane, concentration, separation, drying, and pulverization to obtain a Pseudoalteromonas fermentation product extract whose main active ingredient is exopolysaccharide; the culture medium comprises the following components: 150g-200g / L of mixed algae powder consisting of brown algae, red algae, and copper algae, 2.0-3.0g / L of ammonium sulfate, 25.2-37.3g / L of seawater, and the balance being water, and the pH value of the culture medium is 7.0-7.5.

[0016] The locust bean gum is extracted from the seeds of the locust tree and is prepared specifically according to the following steps: selecting dried locust beans, and sequentially subjecting the beans to peeling, germ removal, powdering, screening, decolorization, hydration, filtration, centrifugation, deproteinization, inactivation, mannanase hydrolysis, UF ultrafiltration membrane, concentration, separation, drying and pulverization to obtain locust bean gum whose main active ingredient is a medium- and low-molecular-weight polysaccharide of 50,000-100,000D.

[0017] The cassia gum is extracted from the seed endosperm of the cassia plant and is prepared specifically according to the following steps: selecting dried cassia seeds, and sequentially subjecting them to peeling, removing the embryo, powdering, screening, decoloring, hydration, filtration, centrifugation, deproteinization, inactivation, mannanase hydrolysis, UF ultrafiltration membrane, concentration, separation, drying and pulverization to obtain cassia gum whose main active ingredient is a medium- and low-molecular-weight polysaccharide of 50,000-100,000D.

[0018] The psyllium seed gum is extracted from the seed coat of psyllium or its similar plants, and is prepared according to the following steps: selecting dry psyllium seeds, sequentially performing the following treatments: removing the core, taking off the shell, powdering, sieving, decolorizing, hydrating, filtering, centrifuging, deproteinizing, inactivating, xylanase enzymolysis, UF ultrafiltration membrane filtration, concentrating, separating, drying and crushing, to obtain the psyllium seed gum with 50,000-100,000D low-molecular-weight polysaccharides as main active substances.

[0019] The preparation method of the polysaccharide composition of natural origin, comprising the following operation steps:

[0020] The Pseudomonas alternanans fermentation product extract, locust bean gum, cuttlefish gelatin and psyllium seed gum are weighed and dissolved in water to obtain aqueous solutions with a mass percentage of 2%, and the obtained aqueous solutions of the Pseudomonas alternanans fermentation product extract, locust bean gum, cuttlefish gelatin and psyllium seed gum are mixed to obtain a polysaccharide composition solution; the polysaccharide composition solution is vacuum freeze-dried and crushed to obtain the polysaccharide composition of natural origin.

[0021] The polysaccharide composition of natural origin is applied to the preparation of cosmetics with skin repair effect, antioxidant effect, anti-inflammatory effect and skin barrier repair effect.

[0022] The present application has the following advantages and beneficial effects compared with the prior art:

[0023] (1) The present application uses different polysaccharides to make up for the functional defects of single polysaccharides, and the polysaccharide composition has more comprehensive functions and a wider application range than single polysaccharides.

[0024] (2) The present application uses the property differences between different polysaccharides to change the original properties and form new structures with changes in space and chemical configuration.

[0025] (3) The present application uses enzyme cutting technology to precisely control the enzyme cutting point, reduce the molecular weight of the main chain, and then control the molecular weight through filter membrane screening to achieve the effect of high structural similarity within a certain molecular weight range; the polysaccharide composition obtained by the method of the present application has diversified structure and better function.

[0026] (4) The present application prepares the polysaccharide composition of natural origin, which has batch stability, high repeatability and excellent quality. DETAILED DESCRIPTION

[0027] Figure 1 is a scratch image display diagram.

[0028] Figure 2 is a cell scratch migration rate column chart.

[0029] Figure 3 is a DPPH clearance rate trend chart.

[0030] Figure 4 This is a trend chart of DPPH clearance in the positive control group.

[0031] Figure 5 This is a bar graph showing the relative inhibition rate of the inflammatory factor NO in macrophage RAW264.7 cells.

[0032] Figure 6 It is a bar graph showing the inhibition rate of inflammatory factor TNF-a in macrophage RAW264.7.

[0033] Figure 7 It is a histogram of transepidermal water loss.

[0034] Figure 8 It is a bar graph of stratum corneum water content. DETAILED DESCRIPTION

[0035] The present invention is further described below with reference to specific examples, but these examples should not be construed as limiting the present invention.

[0036] The raw materials used in the following examples were prepared according to the following method:

[0037] (1) The extract of the fermentation product of Pseudoalteromonas mariniglutinosa DSM15203 refers to the extracellular polysaccharide produced by the fermentation of Pseudoalteromonas mariniglutinosa DSM15203, which is prepared by the following steps: adding Pseudoalteromonas mariniglutinosa DSM15203 to a culture medium, placing it in a constant temperature shaker, and culturing it at a temperature of 10-15°C and a speed of 200-230 r / min for 36-48 hours. Then, the extract is sequentially subjected to inactivation, coarse filtration, centrifugal separation of impurities, protease deproteinization, inactivation, decolorization, passing through a UF ultrafiltration membrane, concentration, separation, drying and pulverization to obtain the extract of the fermentation product of Pseudoalteromonas mariniglutinosa, wherein the main active ingredient is the extracellular polysaccharide; the culture medium comprises the following components: 150g-200g / L of mixed algae powder consisting of brown algae, red algae and copper algae, 2.0-3.0g / L of ammonium sulfate, 25.2-37.3g / L of seawater, and the balance is water, and the pH value of the culture medium is 7.0-7.5. The extracellular polysaccharides obtained by fermentation form a protective film on the skin surface, which has a significant moisturizing and repairing effect.

[0038] (2) Locust bean gum is extracted from the seeds of the locust tree and is prepared according to the following steps: dry locust beans are selected and sequentially subjected to peeling, germ removal, powdering, screening, decolorization, hydration, filtration, centrifugation, deproteinization, inactivation, mannanase hydrolysis, UF ultrafiltration membrane, concentration, separation, drying and crushing to obtain locust bean gum whose main active ingredient is 50,000-100,000D medium and low molecular weight polysaccharides.

[0039] The chemical structure of locust bean gum consists of a main chain of D-mannose linked by β-1,4-glycosidic bonds, with side chains consisting of single D-galactose linked to the mannose main chain by α-1,6-glycosidic bonds. The degree of branching in locust bean gum is relatively low, with a molar ratio of galactose to mannose of approximately 1:4. Analysis of the hydrolyzed fragments through partial hydrolysis revealed an uneven distribution of galactose along the main chain, indicating the presence of both "smooth" and "burry" segments within the locust bean gum molecule. The molecular weight of the locust bean gum after enzymatic hydrolysis with the present mannanase is significantly reduced, and its antioxidant capacity is significantly improved compared to unhydrolyzed locust bean gum.

[0040] (3) Cassia gum is extracted from the seed endosperm of the Cassia plant and is prepared according to the following steps: select dried Cassia seeds, and sequentially process them through peeling, degerming, powdering, screening, decolorization, hydration, filtration, centrifugation, deproteinization, inactivation, mannanase hydrolysis, UF ultrafiltration membrane, concentration, separation, drying and crushing to obtain Cassia gum whose main active ingredient is 50,000-100,000D medium and low molecular weight polysaccharides.

[0041] Cassia gum is a typical galactomannan, a neutral polysaccharide widely found in nature. Its structure is generally composed of linear chains branched by 1,4-β-D-mannopyranosyl units and 1,6-α-D-galactopyranosyl units. After enzymatic hydrolysis with the mannanase of the present invention, the main chain structure of Cassia gum is severed, while the retained branches significantly enhance its anti-inflammatory properties.

[0042] (4) Plantago seed gum is extracted from the outer skin of seeds (egg-shaped, 1-3 mm long, 0.5-1.5 mm wide) of Plantago or its similar plants (Plantago asiatica, Plantago ovata, Plantago striata). It is prepared according to the following steps: select dried Plantago seeds, remove the core and shell, grind, sieve, decolorize, hydrate, filter, centrifuge, deproteinize, inactivate, hydrolyze with xylanase, pass through UF ultrafiltration membrane, concentrate, separate, dry and crush to obtain plantago seed gum whose main active ingredient is 50,000-100,000D medium and low molecular weight polysaccharides.

[0043] Plantago seed gum has a β-(1→4) linked xylan backbone structure, with the residues on the side chains connected to the backbone via O-2 or O-3, resulting in a high degree of branching. The side chains contain a large number of β-terminally linked xylopyranosyl groups, α-terminally linked furanosyl groups, α-terminally linked glucuronopyranosyl groups, β-(1→3) linked xylopyranosyl groups, and α-(1→3) linked furanosyl groups. In addition, there are also small amounts of other types of residues, such as (1→4) linked glucopyranosyl groups and (1→3) linked rhamnopyranosyl groups. After enzymatic hydrolysis with the xylanase of the present invention, the main chain of the plantago seed gum is cut, and the rich branched structure retained significantly improves the skin barrier and immunity.

[0044] The preparation method of the natural polysaccharide composition is as follows:

[0045] The above-obtained Pseudoalteromonas fermentation product extract, locust bean gum, Cassia gum and Plantago seed gum are weighed as raw materials, and respectively dissolved in water to obtain aqueous solutions with a mass percentage concentration of 2%. The obtained aqueous solutions of Pseudoalteromonas fermentation product extract, locust bean gum, Cassia gum and Plantago seed gum are mixed and stirred evenly to obtain a polysaccharide composition solution; the polysaccharide composition solution is vacuum freeze-dried and pulverized to obtain a natural-source polysaccharide composition.

[0046] The raw material ratios of the natural polysaccharide compositions of Examples 1-3 are shown in Table 1:

[0047] Table 1 Ratio of raw materials of natural polysaccharide compositions of Examples 1-3

[0048]

[0049] Example 1 is a polysaccharide composition 1 obtained according to the above preparation method and the raw material ratio shown in Table 1;

[0050] Example 2 is a polysaccharide composition 2 obtained according to the above preparation method and the raw material ratio shown in Table 1;

[0051] Example 3 is a polysaccharide composition 3 obtained according to the above preparation method and the raw material ratio shown in Table 1;

[0052] Comparative Example 1 is the same as Example 3 in terms of other raw materials and preparation method, except that the extract of the fermentation product of Pseudoalteromonas is replaced with xanthan gum to obtain polysaccharide composition 4;

[0053] Comparative Example 2 is the same as Example 3 in terms of other raw materials and preparation method, except that locust bean gum is replaced with locust bean gum obtained without the mannanase enzymatic hydrolysis process, and finally a polysaccharide composition 5 is obtained;

[0054] Comparative Example 3 is the same as Example 3 in terms of other raw materials and preparation method, except that Cassia gum is replaced with Cassia gum obtained without the mannanase enzymatic hydrolysis process, and finally obtains Polysaccharide Composition 6;

[0055] Comparative Example 4 is the same as Example 3 in terms of other raw materials and preparation method, except that the psyllium seed gum is replaced with psyllium seed gum obtained without the xylanase enzymatic hydrolysis process, and finally the polysaccharide composition 7 is obtained;

[0056] Comparative Example 5 is prepared by the same method as Example 3, except that all raw materials are replaced with extracts obtained without the special treatment of the present invention, wherein the Pseudoalteromonas fermentation product extract is obtained by fermenting Pseudoalteromonas mariniglutinosa DSM15203 on a medium without mixed algal powder (Beijing Luqiao Pseudomonas agar basal medium / CN agar), locust bean gum is obtained without mannanase enzymatic hydrolysis treatment, cassia gum is obtained without mannanase enzymatic hydrolysis treatment, and psyllium seed gum is obtained without xylanase enzymatic hydrolysis treatment, ultimately obtaining polysaccharide composition 8;

[0057] Comparative Example 6 is the same as Example 3 in terms of other raw materials and preparation method, except that the Pseudoalteromonas fermentation product extract is removed and the other three raw materials are supplemented in equal proportions to 100%, to obtain polysaccharide composition 9;

[0058] Comparative Example 7 is the same as Example 3 in terms of other raw materials and preparation method, except that locust bean gum is removed and the other three raw materials are supplemented in equal proportions to 100%, to obtain polysaccharide composition 10;

[0059] Comparative Example 8 is the same as Example 3 in terms of other raw materials and preparation method, except that Cassia gum is removed and the other three raw materials are supplemented in equal proportions to 100%, to obtain polysaccharide composition 11;

[0060] Comparative Example 9 is the same as Example 3 in terms of other raw materials and preparation method, except that the psyllium seed gum is removed and the other three raw materials are supplemented in equal proportions to 100%, thereby obtaining a polysaccharide composition 12.

[0061] The efficacy testing protocol is as follows:

[0062] The polysaccharide compositions 1-12 prepared in Examples 1-3 and Comparative Examples 1-9 were used as active ingredients to prepare essences to verify their efficacy. Essence 1 was prepared according to the formula shown in Table 2.

[0063] Table 2 Composition of polysaccharide essence formula

[0064]

[0065] Essences 2-12 were prepared by replacing polysaccharide composition 1 with polysaccharide compositions 2-12, respectively, and essence 13 was prepared by removing the polysaccharide composition from the formula.

[0066] 1. Comparison of repair functions

[0067] Essence 1-13 was used as the test substance, and HaCat cells were selected to test the cell scratch experiment.

[0068] 1) First, screen the concentration of the test substance suitable for the scratch test, that is, detect the cytotoxic concentration of the test substance.

[0069] The MTT assay was used to detect cell viability and screen for the maximum safe concentration of cell samples. A blank control (culture medium) and a zero-adjustment well (PBS) were set up for the experiment. The sample concentrations were set to have eight gradient concentrations per sample, with six replicate wells for each concentration. The specific steps are as follows:

[0070] (1) Complete culture medium was used to prepare test substances at different concentrations. Eight concentrations were set for each test substance: 20%, 9.3%, 4.32%, 2.01%, 0.93%, 0.432%, 0.201% and 0.093% (all concentrations are in percentage by mass).

[0071] (2) Inoculation: Take cells in the logarithmic growth phase, digest them and inoculate them into 96-well plates. Place the culture plates in a 37°C, 5% CO2 incubator and incubate for 18-24 hours.

[0072] (3) Sample addition: discard the supernatant from the culture plate after the above incubation, add the culture medium containing different concentrations of the test substance prepared in step (1), and place the culture plate in a 37°C, 5% CO2 incubator for further incubation for 18-24 hours.

[0073] (4) Detection: Discard the supernatant from the culture plate after the above incubation, add the prepared and filtered MTT (0.5 mg / mL), shake gently to mix, and incubate at 37°C in the dark for 4 h. After the incubation, discard the supernatant, add 100 μL of DMSO to each well, shake for 10 min, and read the OD490nm value using a microplate reader.

[0074] (5) Calculation formula for relative cell survival rate:

[0075] Relative cell viability = [(OD of the test sample well - OD of the zero-adjustment well) / (OD of the blank control well - OD of the zero-adjustment well)] × 100%

[0076] Based on the results of relative cell viability, the concentration of the test substance with no cytotoxicity was screened out to be 4.32% (0.0432% when converted into the concentration of the polysaccharide composition), and the subsequent cell scratch test was performed based on this test substance concentration.

[0077] 2) HaCaT cell-based scratch test.

[0078] (1) Inoculation: First, use a pen to draw two vertical lines on the back of a 24-well plate using a ruler. Inoculate the cells into the 24-well plate and incubate in a 37°C, 5% CO2 incubator overnight.

[0079] (2) Scratching: After the cells have been plated and grown in a 24-well plate for 18-24 hours, use a 1 mL pipette tip to measure the ruler and make two scratches perpendicular to the horizontal line on the back of each well. Discard the supernatant and wash three times with PBS to remove the scratched cells.

[0080] (3) Liquid preparation: Essences 1-12 were prepared into 4.32% mass concentration solutions using complete culture medium as samples. Human epidermal growth factor hEGF was prepared into 10 ng / mL solution using complete culture medium as the positive group. A blank group (complete culture medium) was also set up.

[0081] (4) Dosing and photography: Add the prepared samples to the corresponding positions of the 24-well plate for dosing. Take photos under a 4× microscope at 0 h (immediately). Place the culture plate at 37°C and 5% CO2 for another 48 h.

[0082] (5) Photography and image analysis: After 48 hours of cell culture, take another photo under a 4× microscope. The photo position should be the same as at 0 hours. Then use Image J to analyze the migration area.

[0083] (6) Calculation of cell migration rate: Calculate the cell migration rate according to the following formula:

[0084] Cell migration rate (%) = [(A0-A 48 ) / A0]×100%

[0085] A0 refers to the blank area when taking the photo at 0 h, A 48 Refers to the blank area of ​​the photo taken at 48 hours.

[0086] (7) Data processing: All data obtained in the experiment were processed and plotted using Excel software. Statistical analysis was performed using SPSS 17.0. One-way analysis of variance (ANOVA) was used for comparisons between groups. A significant difference (*) was considered when P < 0.05, and an extremely significant difference (**) was considered when P < 0.01.

[0087] (8) The scratch images of the Essence 3 sample group and the blank group are shown as follows Figure 1 As shown in Table 3; The cell migration rate in this experiment is shown in Table 3 and Figure 2 As shown:

[0088] Table 3 Cell scratch migration rate

[0089]

[0090] From Table 3 and Figure 2It can be seen that when the actual addition amount of the polysaccharide composition is 0.0432% by mass, the polysaccharide composition of the present invention is significantly better than the comparative example in terms of repair effect, which mainly depends on the combination of raw materials and the enzyme cleavage process to reduce the molecular weight of the polysaccharide. In addition to the functional enhancement caused by the key process of the raw materials themselves, the re-combination of the raw materials under a specific production process also achieves the effect of 1+1>2. For example, the effect of the polysaccharide composition obtained in Examples 1-3 on cell repair is better than that of the polysaccharide composition obtained in Comparative Examples 1-5, which is mainly due to the better function caused by the difference in the extraction process of the materials; the effect of the polysaccharide composition obtained in Examples 1-3 on cell repair is better than that of the polysaccharide composition obtained in Comparative Examples 6-9, which is mainly due to the better function caused by the difference in material combination.

[0091] 2. Comparison of Antioxidant Function

[0092] The T / SHRH 006-2018 Cosmetics-Free Radical (DPPH) Scavenging Test Method was used to test the antioxidant capacity of essences 1-13.

[0093] 1. Liquid preparation

[0094] (1) Positive control solution: Vitamin E was dissolved and diluted with 95% ethanol to form a series of gradient concentration solutions of 0.16 mg / mL, 0.08 mg / mL, 0.04 mg / mL, 0.02 mg / mL, and 0.01 mg / mL to verify the test system.

[0095] (2) DPPH ethanol solution: Weigh 12 mg of 1,1-diphenyl-2-trinitrophenylhydrazine into a 250 mL beaker, add 100 mL of 95% by volume ethanol, and stir with a glass rod until dissolved to obtain DPPH ethanol solution.

[0096] (3) Test substance sample solution: Essences 1-13 were used as test substances and diluted with water to prepare samples with multiple concentration levels. Five concentrations were set for each test substance: 0.93%, 2.01%, 4.32%, 9.3% and 20% (all concentrations are percentage by mass).

[0097] 2. Add sample

[0098] Refer to Table 4. Use 10 mL test tubes to set up sample tubes, sample background tubes, DPPH tubes, and solvent background tubes. For each concentration of each test substance, three parallel tubes should be set up, and three parallel tubes should also be set up for the DPPH tubes.

[0099] First, add 0.5 mL of sample solution of the same concentration to each of the sample tube and the sample background tube. Then, add solvent to all test tubes: water for water-soluble samples and 95% ethanol by volume for oil-soluble samples, to a total volume of 1.5 mL, and mix thoroughly. Next, add 0.5 mL of DPPH ethanol solution to the sample tube and DPPH tube, and 0.5 mL of 95% ethanol by volume to the sample background tube and solvent background tube. Gently shake and let stand at room temperature for 5 minutes.

[0100] Table 4 Sample addition requirements

[0101]

[0102] 3. Detection

[0103] The added test tubes were transferred into a 96-well plate and the absorbance was measured at 517 nm.

[0104] DPPH free radical scavenging rate calculation formula:

[0105] Clearance rate (%) = [1-(T-T0) / (C-C0)] × 100%

[0106] Where: T is the absorbance of the solution in the sample tube, that is, the absorbance of the solution after the sample reacts with DPPH;

[0107] T0 is the absorbance value of the solution in the sample background tube;

[0108] C is the average of three absorbance values ​​of the solution in the DPPH tube, that is, the absorbance value of the DPPH solution when no sample is added;

[0109] C0 is the absorbance of the solution in the solvent background tube.

[0110] 4. Based on the free radical (DPPH) scavenging test results

[0111] The results of the DPPH clearance rate test of the sample group essence are shown in Table 5, and the results of the DPPH clearance rate test of the positive control group are shown in Table 6. The trend of the DPPH clearance rate of the essence is as follows: Figure 3 As shown in the figure, the change trend of DPPH clearance rate in the positive control group is as follows Figure 4 shown.

[0112] Table 5 DPPH free radical scavenging rate of essence

[0113]

[0114] Table 6 DPPH clearance rate in the positive control group

[0115]

[0116] From the above data, it can be concluded that the antioxidant effect of the polysaccharide composition of the present invention is significant, and it shows dose dependence in the range of actual addition amount of 0.0093%-0.2%, which mainly depends on the combination of raw materials, the control of molecular weight by production process and the enhancement of polysaccharide complementary antioxidant capacity. In addition to the functional enhancement caused by the key process of the raw materials themselves, the re-combination of raw materials under a specific production process also achieves the effect of 1+1>2. For example, the antioxidant effect of the polysaccharide composition obtained in Examples 1-3 is better than that of the polysaccharide composition obtained in Comparative Examples 1-5, mainly because the difference in the extraction process of the materials leads to better function; the antioxidant effect of the polysaccharide composition obtained in Examples 1-3 is better than that of the polysaccharide composition obtained in Comparative Examples 6-9, mainly because the difference in material combination leads to better function.

[0117] 3. Comparison of anti-inflammatory functions

[0118] The T / GDCDC 030-2023 Cosmetic Soothing Efficacy Test - In Vitro Macrophage Nitric Oxide (NO) Release Inhibition was used to determine the inhibitory ability of the polysaccharide composition on NO. The T / SHRH 034-2021 Cosmetic Soothing Efficacy Test - In Vitro TNF-α Inflammatory Factor Content Determination in Lipopolysaccharide-Induced Macrophages RAW264.7 Test Method was used to determine the inhibitory ability of the polysaccharide composition on TNF-α.

[0119] 1. Toxicity detection based on macrophage RAW264.7

[0120] This experiment uses MTT to detect cell viability and screen the maximum safe concentration of cell sample administration. A blank control (culture medium) and a zero-adjustment well (PBS) were set up. Sample concentrations were set to have eight gradient concentrations per sample, with six replicate wells for each concentration.

[0121] (1) Prepare different concentrations of test substances using complete culture medium. Set 8 concentrations for each test substance: 20%, 9.3%, 4.32%, 2.01%, 0.93%, 0.432%, 0.201% and 0.093% (all percentages by mass);

[0122] (2) Inoculation: Take cells in the logarithmic growth phase, digest them and inoculate them into 96-well plates. Place the culture plates in a 37°C, 5% CO2 incubator and incubate for 20-48 hours.

[0123] (3) Sample addition: discard the supernatant from the culture plate after the above incubation, add the culture medium containing different concentrations of the test substance prepared in step (1), and place the culture plate in a 37°C, 5% CO2 incubator for further incubation for 24-48 hours;

[0124] (4) Detection: Discard the supernatant of the culture plate after the above incubation, add 50 μl of prepared and filtered MTT, continue to culture in a CO2 incubator for 2-6 hours, discard the culture medium, add 150 μl of DMSO to each well, shake, use 630 nm as the reference wavelength, and read the OD570 nm value with a microplate reader.

[0125] (5) The formula for calculating relative cell viability is as follows:

[0126] Cell viability (%) = sample OD value / blank OD value × 100%

[0127] (6) Based on the results of relative cell survival rate, the concentration of the test substance with no cytotoxicity was screened out to be 4.32% (0.0432% when converted into the concentration of the polysaccharide composition), and the LPS induction experiment was carried out based on this test substance concentration.

[0128] 2. Determination of relative NO content based on macrophage RAW264.7 cells

[0129] (1) Cell plating: Add 200 μL of cell suspension to each well of a 96-well plate, place in a CO2 incubator, and culture at 37°C and 5% CO2 for 20-24 hours.

[0130] (2) Solution preparation: Essences 1-12 were prepared into 4.32% solutions with complete culture medium, and 1 μg / mL of LPS was added as sample solution; 200 μg / mL of dexamethasone and 1 μg / mL of LPS were added to the complete culture medium as positive control; 1 μg / mL of LPS was added to the complete culture medium as negative control, and a blank control (complete culture medium) was also set up.

[0131] (3) Administration: Discard the culture medium in the 96-well plate and proceed with the induction and administration procedures. Add the sample solution to the test wells, the negative control solution to the negative control wells, the positive control solution to the positive control wells, and complete culture medium to the blank control wells, 200 μL per well. After administration, place the 96-well plate in a CO2 incubator and incubate for 24 h ± 2 h.

[0132] (4) Collection of cell supernatant: After the incubation, collect 200 μL of cell culture supernatant from each well into a 1.5 mL sterile centrifuge tube and store it in a -80°C ultra-low temperature freezer.

[0133] (5) Griess test: Griess test should be performed according to the operating instructions of the nitric oxide detection kit.

[0134] (6) The calculation formula of NO inhibition rate is as follows:

[0135] Inhibition rate (%) = (1-T / C) × 100%

[0136] Where: T is the average relative content of NO in the sample solution;

[0137] C is the average relative content of NO in the negative control.

[0138] (7) Results of inhibition assay of nitric oxide (NO) release in macrophage RAW264.7 cells:

[0139] The results of the relative content of NO in macrophage RAW264.7 are shown in Table 7, the results of the relative inhibition rate of NO in macrophage RAW264.7 are shown in Table 8, and the bar graph of the relative inhibition rate of NO in macrophage RAW264.7 is shown in Table 8. Figure 5 shown.

[0140] Table 7 Detection results of relative NO content in macrophage RAW264.7 cells

[0141]

[0142] Table 8 Relative inhibition rate of NO in macrophage RAW264.7 cells

[0143]

[0144] 3. TNF-a content determination based on macrophage RAW264.7

[0145] (1) Cell plating: Add 200 μL of cell suspension to each well of a 96-well plate, place the plate in a CO2 incubator, and culture at 37°C and 5% CO2 for 20-24 hours.

[0146] (2) Solution preparation: Essences 1-12 were prepared into 4.32% mass percent solutions in complete culture medium, and 1 μg / mL of LPS was added as sample solution; 100 μg / mL of dexamethasone and 1 μg / mL of LPS were added to complete culture medium as positive control; 1 μg / mL of LPS was added to complete culture medium as negative control, and a blank control (complete culture medium) was also set up.

[0147] (3) Administration: Discard the culture medium in the 96-well plate and proceed with the induction and administration procedures. Add the sample solution to the test wells, the negative control solution to the negative control wells, the positive control solution to the positive control wells, and complete culture medium to the blank control wells, 200 μL per well. After administration, place the 96-well plate in a CO2 incubator and incubate for 24 h ± 2 h.

[0148] (4) Cell supernatant collection: After the incubation culture is completed, 200 μL of cell culture supernatant is collected in each well in a 1.5 mL sterile centrifuge tube and stored in a -80 °C ultra-low temperature freezer.

[0149] (5) ELISA detection: ELISA detection needs to be detected according to the operating instructions of the detection kit. Because the expression amount of TNF-a after LPS induction is high, before formal detection, the dilution ratio should be set, pre-experimented, and the dilution ratio of the ELISA detection experimental group should be determined to ensure that the ELISA detection value falls within the standard curve range.

[0150] (6) The calculation formula of the inhibition rate of inflammatory factors is as follows:

[0151] Inhibition rate (%) = (1-T / C) x 100%

[0152] In the formula: T is the average value of the inflammatory factor content of the sample solution;

[0153] C is the average value of the inflammatory factor content of the negative control.

[0154] (7) The determination results of the inflammatory factor TNF-a content of macrophage RAW264.7:

[0155] The determination results of the inflammatory factor TNF-a content of macrophage RAW264.7 are shown in Table 9, the inhibition rate of the inflammatory factor TNF-a of macrophage RAW264.7 is shown in Table 10, and the columnar chart of the inhibition rate of the inflammatory factor TNF-a of macrophage RAW264.7 is shown in Figure 6 .

[0156] Table 9 Determination results of the inflammatory factor TNF-a content (ng / mL) of macrophage RAW264.7

[0157]

[0158] Table 10 Inhibition rate of the inflammatory factor TNF-a of macrophage RAW264.7

[0159]

[0160] It can be concluded from the above data that the polysaccharide composition of the present invention has significant anti-inflammatory efficacy, and the function of the composition is significantly enhanced after the combination and process of the present invention. In addition to the functional enhancement caused by the key process of the raw materials themselves, the re-combination of the raw materials under a specific production process also achieves the effect of 1+1>2. For example, the anti-inflammatory effect of the polysaccharide composition obtained in Examples 1-3 is better than that of the polysaccharide composition obtained in Comparative Examples 1-5, mainly because the difference in the extraction process of the materials leads to better functions; the anti-inflammatory effect of the polysaccharide composition obtained in Examples 1-3 is better than that of the polysaccharide composition obtained in Comparative Examples 6-9, mainly because the difference in the material combination leads to better functions.

[0161] The experiment was conducted using the "QB / T 4256-2011 Guidelines for the Evaluation of Moisturizing Efficacy of Cosmetics" as a method guide. 35 healthy subjects aged 20 to 55 years with no history of skin diseases or allergies were selected. The inner forearms of each subject were divided into product application areas and marked on the corresponding application areas. Essences 1-13 were applied to different areas for 28 consecutive days. Subsequently, the skin transepidermal water loss test and keratin moisture content test were performed. The data after 0 and 28 days were recorded and compared. The average values ​​are shown in Table 11 below, and the transepidermal water loss bar graph is shown below. Figure 7 As shown, the histogram of stratum corneum water content is as follows Figure 8 shown.

[0162] Table 11 Transepidermal water loss and stratum corneum water content

[0163]

[0164] The above data demonstrate that the polysaccharide composition of the present invention is more effective in repairing the skin barrier (improving the skin barrier and minimizing transepidermal water loss), and significantly helps the skin retain moisture. The composition's functionality is enhanced after treatment according to the present invention's combination and specific process. In addition to the enhanced functionality resulting from the key processes involved in the raw materials themselves, the recombination of the raw materials under specific production processes also achieves a 1+1>2 effect. For example, the polysaccharide compositions obtained in Examples 1-3 are more effective at repairing the skin barrier than the polysaccharide compositions obtained in Comparative Examples 1-5, primarily due to differences in the material extraction processes. The polysaccharide compositions obtained in Examples 1-3 are more effective at repairing the skin barrier than the polysaccharide compositions obtained in Comparative Examples 6-9, primarily due to differences in the material combinations.

[0165] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A natural polysaccharide composition, characterized in that: The natural polysaccharide composition is composed of the following components in percentage by mass: Pseudoalteromonas fermentation product extract 50-70% Locust bean gum 10-15% Cassia gum 5-10% Plantain seed gum 15-25%; The Pseudoalteromonas fermentation product extract refers to the exopolysaccharide produced by fermentation of Pseudoalteromonas maritima DSM15203, and is specifically prepared according to the following steps: adding Pseudoalteromonas maritima DSM15203 to a culture medium, placing the culture medium in a constant temperature shaker, and culturing the culture medium at a temperature of 10-15°C and a rotation speed of 200-230 r / min for 36-48 hours; then sequentially performing inactivation, coarse filtration, centrifugal separation of impurities, protease deproteinization, inactivation, decolorization, passing through a UF ultrafiltration membrane, concentration, separation, drying, and pulverization to obtain the Pseudoalteromonas fermentation product extract whose main active ingredient is the exopolysaccharide; the culture medium comprises the following components: 150g-200g / L of mixed algae powder consisting of brown algae, red algae, and copper algae, 2.0-3.0g / L of ammonium sulfate, 25.2-37.3g / L of seawater, and the balance being water, and the pH value of the culture medium is 7.0-7.5; The locust bean gum is extracted from the seeds of the locust tree and is prepared by the following steps: selecting dried locust beans, and sequentially subjecting them to peeling, degerming, powdering, screening, decoloring, hydration, filtration, centrifugation, deproteinization, inactivation, mannanase hydrolysis, UF ultrafiltration, concentration, separation, drying and pulverization to obtain locust bean gum whose main active ingredient is a low-molecular-weight polysaccharide of 50,000-100,000D; The cassia gum is extracted from the seed endosperm of the cassia plant and is prepared by the following steps: selecting dried cassia seeds, and sequentially subjecting them to peeling, removing the embryo, powdering, screening, decoloring, hydrating, filtering, centrifuging, deproteinizing, inactivating, hydrolyzing with mannanase, passing through a UF ultrafiltration membrane, concentrating, separating, drying, and pulverizing to obtain cassia gum whose main active ingredient is a low-molecular-weight polysaccharide of 50,000-100,000D; The psyllium seed gum is extracted from the seed coat of plantain and is prepared specifically according to the following steps: selecting dried plantain seeds, and sequentially subjecting them to core removal and shelling, powdering, sieving, decolorization, hydration, filtration, centrifugation, deproteinization, inactivation, xylanase hydrolysis, UF ultrafiltration membrane, concentration, separation, drying and pulverization to obtain psyllium seed gum whose main active ingredients are low and medium molecular weight polysaccharides of 50,000-100,000D.

2. The method for preparing a natural polysaccharide composition according to claim 1, characterized in that The following steps are included: Pseudoalteromonas fermentation product extract, locust bean gum, cassia gum and psyllium seed gum are weighed and dissolved in water to obtain aqueous solutions with a mass percentage concentration of 2% respectively. The obtained aqueous solutions of Pseudoalteromonas fermentation product extract, locust bean gum, cassia gum and psyllium seed gum are mixed and stirred uniformly to obtain a polysaccharide composition solution. The polysaccharide composition solution is vacuum freeze-dried and pulverized to obtain a natural-source polysaccharide composition.

3. Use of a natural polysaccharide composition according to claim 1 in the preparation of cosmetics having antioxidant, anti-inflammatory and skin barrier repair effects.

Images