Anti-allergy soothing polysaccharide composition as well as preparation method and application thereof

Abstract

The invention discloses an anti-allergy soothing polysaccharide composition as well as a preparation method and application thereof. The anti-allergy soothing polysaccharide composition comprises the following components in parts by weight: 3-5 parts of peach gum extracting solution; 3-5 parts of rhizoma bletillae polysaccharide; 3-5 parts of polygonatum polysaccharide; 3-5 parts of a white poria extract; and 5-10 parts of betaine supramolecular crystal powder. The invention aims to provide a polysaccharide composition with remarkable anti-allergy and soothing effects by optimizing extraction and purification processes of polysaccharides and combining with compounding effects of different polysaccharides, and provides a new choice for the fields of cosmetics and medicines.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and in particular to an anti-allergic and soothing polysaccharide composition, a preparation method and application thereof. Background Art

[0002] With the acceleration of the pace of modern life, people's skin is more and more susceptible to external stimuli, leading to discomfort such as sensitivity, redness, swelling, itching, etc. Therefore, the development of cosmetics and drugs with anti-allergic and soothing effects has become a hot topic in current research. As a class of natural polymer compounds, polysaccharides have been widely used in the fields of cosmetics and drugs due to their good biocompatibility, moisturizing and antioxidant properties.

[0003] Traditionally, polysaccharides are mainly derived from plants, microorganisms and animals, such as hyaluronic acid, chitosan, dextran, etc. They have been proven to have moisturizing, repairing and anti-inflammatory effects on the skin. However, these polysaccharides have limited effects in anti-allergic and soothing, and some polysaccharides have limited sources, high costs or complex extraction processes, which limit their wide application in cosmetics and medicines.

[0004] In recent years, researchers have begun to explore new sources and extraction methods of polysaccharides in order to obtain more efficient and economical anti-allergic and soothing ingredients. For example, polysaccharides in traditional Chinese medicines such as peach gum, white radix, polygonatum, and white tuckahoe have attracted much attention due to their unique biological activity and low toxicity. These polysaccharides not only have good moisturizing and antioxidant properties, but also show significant anti-allergic and soothing effects, which can effectively reduce the skin's sensitive response to external stimuli and promote skin health.

[0005] However, there are still some problems with the extraction and purification technology of these Chinese herbal medicine polysaccharides, such as low extraction efficiency, low purity, and complex process, which makes it difficult to ensure the quality and stability of the products. In addition, the research on the compounding effect and mechanism of action of different polysaccharides is not in-depth enough, which limits their further application in cosmetics and medicines.

[0006] Therefore, the development of an efficient, economical, and stable anti-allergic and soothing polysaccharide composition and its preparation method is of great significance for meeting market demand and promoting the development of the cosmetics and pharmaceutical industries. The present invention aims to provide a polysaccharide composition with significant anti-allergic and soothing effects by optimizing the extraction and purification process of polysaccharides and combining the compounding effects of different polysaccharides, thereby providing a new option for the cosmetics and pharmaceutical fields. Summary of the invention

[0007] In view of this, the purpose of the present invention is to provide an anti-allergic and soothing polysaccharide composition and its preparation method and application. The present invention aims to provide a polysaccharide composition with significant anti-allergic and soothing effects by optimizing the extraction and purification process of polysaccharides and combining the compounding effects of different polysaccharides, thereby providing a new option for the cosmetics and pharmaceutical fields.

[0008] The objective of the present invention is achieved through the following technical solutions: <First aspect> An anti-allergic soothing composition comprises the following components in parts by weight: 3-5 parts of peach gum extract; 3-5 parts of Bletilla striata polysaccharide; 3-5 parts of Polygonatum sibiricum polysaccharide; 3-5 parts of white Poria cocos extract; Betaine supramolecular powder 5-10 parts.

[0009] The peach gum extract is prepared by a method comprising the following steps: Take dried peach gum powder, add deionized water; extract at 50-60°C for 4-6 hours, centrifuge at 4000-5000rpm for 10-20min, collect the extract, add 85-95% ethanol to a final concentration of 40 wt%-80 wt%, add 0-600mL saturated sodium chloride solution, let stand for precipitation, centrifuge at 4000-5000rpm for 10-20min, collect the precipitate to obtain crude polysaccharide, and dissolve in water to obtain peach gum extract.

[0010] The solid-liquid ratio is preferably 1:10-50; the concentration of the peach gum extract is 5 - 15 mg / mL.

[0011] The preparation method of the betaine supramolecular crystal powder comprises the following steps: Step 1, taking betaine extract powder, adding water, dissolving at 30-40°C, and preparing a betaine extract solution with a concentration of 15-20% (w / w); Step 2, placing the betaine extract solution in an ultrasonic oscillator for 20-30 minutes at a power of 200-250W to self-assemble into a supramolecular structure; Step 3: The betaine extract solution treated in step 2 is filtered through a microporous filter membrane to obtain a supramolecular solution, which is then dried into a powder to obtain the betaine supramolecular crystal powder.

[0012] The betaine extract is prepared by a method comprising the following steps: Fresh wolfberry leaves are washed, frozen at -18°C to -20°C for 20-24 hours, dried, crushed, sieved, mixed with the extract, added with activated carbon, extracted, centrifuged, adsorbed by macroporous adsorption resin, eluted, vacuum concentrated and dried to obtain betaine extract.

[0013] In the extraction step, the extract is ethanol with a mass concentration of 50-60%, the amount of activated carbon added is 0.5%-1% of the mass of the wolfberry leaf powder, the extraction temperature is 20-40°C, the extraction time is 1-2h, the centrifugal conditions are 4000-5000r / min, and the centrifugal time is 5-8min.

[0014] In the eluting step, the eluent includes a polyol and / or water; the polyol includes butanediol.

[0015] The weight average molecular weight Mw of the Bletilla striata polysaccharide is 30kDa-50kDa, and the purity is greater than 99%.

[0016] The weight average molecular weight Mw of the polygonatum sibiricum polysaccharide is 3kDa-6kDa, and the purity is greater than 99%.

[0017] The CAS number of the white Poria cocos extract is 168456-53-9.

[0018] The preparation method of the anti-allergic soothing polysaccharide composition comprises the following steps: S1. Prepare the following components in parts by weight: 3-5 parts of peach gum extract; 3-5 parts of Bletilla striata polysaccharide; 3-5 parts of Polygonatum sibiricum polysaccharide; 3-5 parts of Poria cocos extract; 5-10 parts of betaine supramolecular crystal powder; S2. Evenly mix the above components according to the above parts by weight to obtain an anti-allergic and soothing polysaccharide composition.

[0019] The use of the anti-allergic and soothing polysaccharide composition in the preparation of cosmetics or medicines with soothing effects also falls within the protection scope of the present invention.

[0020] Compared with the prior art, the anti-allergic and soothing polysaccharide composition provided by the present invention and its preparation method and application have significant beneficial effects, which are specifically manifested in the following aspects: 1. Significant anti-allergic and soothing effects: The present invention prepares a polysaccharide composition with significant anti-allergic and soothing effects by carefully screening and optimizing the proportion of multiple natural polysaccharide components (peach gum extract, Bletilla striata polysaccharide, Polygonatum sibiricum polysaccharide, white Poria cocos extract and betaine supramolecular crystal powder). The performance test results in the embodiment show that the composition can significantly improve the skin sensitivity problem of the subjects, reduce skin acne and oiliness, and reduce skin sensitivity by up to 40%.

[0021] 2. Highly effective hyaluronidase inhibition: Hyaluronidase is an important factor causing allergic reactions. The anti-allergic soothing polysaccharide composition of the present invention showed a highly effective inhibitory effect on hyaluronidase in in vitro tests. The experimental results showed that at different concentrations, the inhibition rate of the composition on hyaluronidase reached a significant level, especially at a lower concentration (such as 5 mg / mL), the inhibition rate could reach 53.82%, effectively preventing the occurrence of allergic reactions.

[0022] 3. Significant anti-inflammatory effect: In the in vitro TNF-α inflammatory factor content determination experiment, the anti-allergic soothing polysaccharide composition of the present invention can significantly reduce the increase in TNF-α content caused by LPS stimulation, and the inhibition rate can reach 41.35%. This result shows that the composition has a significant effect in inhibiting skin inflammatory reactions and helps maintain skin health.

[0023] 4. Natural ingredients and high safety: The anti-allergic and soothing polysaccharide composition of the present invention uses natural plant polysaccharide ingredients, has low toxicity and good biocompatibility, and avoids the side effects that may be caused by chemical synthetic ingredients. This makes the application of the composition in cosmetics and medicines safer and more reliable.

[0024] 5. The preparation process is simple and easy to scale up: The preparation method provided by the present invention has clear steps, is easy to operate, and the required equipment and raw materials are easy to obtain, which makes it possible to mass-produce the composition, reduces production costs, and is conducive to its market popularization and promotion.

[0025] The anti-allergic and soothing polysaccharide composition of the present invention is not only applicable to the cosmetics field, as an effective ingredient for soothing the skin and improving the condition of sensitive skin; it can also be expanded to the pharmaceutical field, providing a new option for treating skin sensitivity, inflammation and other related diseases. In addition, the composition can also play an important role in the fields of health care products and meet the needs of different consumer groups. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings: Figure 1 This is a picture of the soothing effect test results; Figure 2 It is the reference curve of hyaluronidase inhibition by vitamin C (VC); Figure 3 is the standard curve of TNF-α; Figure 4 This is a diagram of cell migration in the blank group of the scratch experiment; Figure 5This is a diagram of cell migration in the scratch experiment group. DETAILED DESCRIPTION

[0027] The present invention is described in detail below in conjunction with embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, some adjustments and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0028] In the following embodiments and comparative examples: Preparation Example 1 Preparation of Betaine Supramolecular Crystalline Powder 1. Preparation of Lycium barbarum leaf betaine extract Step (1), washing fresh wolfberry leaves, freezing at -18°C for 24 hours; drying at 60°C for 12 hours, crushing, and passing through a 200-mesh sieve to obtain wolfberry leaf powder; Step (2), 10 g of wolfberry leaf powder was mixed with an extract (100 mL of 50% ethanol by mass concentration), 0.5 g of activated carbon was added, and the mixture was extracted at 20-40° C. for 2 h to obtain an extract; the extract was centrifuged at 4000 r / min for 5 min, and the supernatant was collected; Step (3), after the filtrate is adsorbed by a medium-polarity macroporous adsorption resin, it is first eluted with deionized water until it is sugar-free, and then eluted with 25 times the bed volume of a methanol-water-acetic acid solution from the wolfberry leaf betaine adsorbed on the macroporous resin to obtain an eluate containing wolfberry leaf betaine; Wherein: the volume ratio of methanol, water and acetic acid in the methanol-water-acetic acid solution is 5:5:0.01; Step (4), the eluate of step (3) is concentrated under vacuum at a vacuum degree of 0.05 MPa and a temperature of 20° C. to obtain a wolfberry leaf betaine concentrate; the concentrate is dried to obtain a wolfberry leaf betaine extract powder.

[0029] 2. Preparation of betaine supramolecular crystal powder Step (1), taking an appropriate amount of betaine extract powder, adding deionized water, stirring and dissolving at 40° C., and preparing a betaine extract solution with a concentration of 15% (w / w).

[0030] Step (2), placing the betaine extract solution in an ultrasonic oscillator for 30 minutes at a power of 200 W to promote intermolecular interaction, self-assembly, and formation of a supramolecular structure.

[0031] The betaine extract solution after the treatment in step (3) is filtered through a microporous filter membrane (0.22 μm) to obtain a clarified supramolecular solution, which is then vacuum dried (drying temperature is 45-55° C., vacuum degree is less than 5 Pa) into a powder to obtain betaine supramolecular crystal powder.

[0032] Preparation Example 2 Preparation of Betaine Supramolecular Crystalline Powder 1. Preparation of Lycium barbarum leaf betaine extract Step (1), washing fresh wolfberry leaves, freezing at -20°C for 20 hours; drying at 70°C for 10 hours, crushing, and passing through a 200-mesh sieve to obtain wolfberry leaf powder; Step (2), 10 g of wolfberry leaf powder was mixed with an extract (100 mL of 50% ethanol by mass concentration), 1 g of activated carbon was added, and the mixture was extracted at 40° C. for 1 h to obtain an extract; the extract was centrifuged at 5000 r / min for 8 min, and the supernatant was collected; Step (3), after the filtrate is adsorbed by a medium-polarity macroporous adsorption resin, it is first eluted with deionized water until it is sugar-free, and then eluted with 25 times the bed volume of a methanol-water-acetic acid solution from the wolfberry leaf betaine adsorbed on the macroporous resin to obtain an eluate containing wolfberry leaf betaine; Wherein: the volume ratio of methanol, water and acetic acid in the methanol-water-acetic acid solution is 5:5:0.01; Step (4), the eluate of step (3) is concentrated under vacuum at a vacuum degree of 0.05 MPa and a temperature of 20° C. to obtain a wolfberry leaf betaine concentrate; the concentrate is dried to obtain a wolfberry leaf betaine extract powder.

[0033] 2. Preparation of betaine supramolecular crystal powder Same as Preparation Example 1.

[0034] Comparative Preparation Example 1 The difference between this comparative preparation example and preparation example 1 is that in step (1), no freezing step is performed. Step (1) is specifically as follows: Step (1), washing the wolfberry leaves, drying at 60-80° C. for 12 h, crushing, and passing through a 200-mesh sieve to obtain wolfberry leaf powder; The remaining steps are the same as in Preparation Example 1.

[0035] Comparative Preparation Example 2 The difference between this comparative preparation example and preparation example 2 is that in step (1), no freezing step is performed. Step (1) is specifically as follows: Step (1), washing fresh wolfberry leaves, freezing at -18°C for 24 hours, and grinding to obtain wolfberry slurry; The remaining steps are the same as in Preparation Example 1.

[0036] Comparative Preparation Example 3 The difference between this comparative preparation example and preparation example 1 is that in step (1), the freezing temperature condition is -10°C; the remaining steps are the same as those in preparation example 1.

[0037] Preparation Example 3 Take dried peach gum powder, add deionized water (solid-liquid ratio 1:10-50), extract at 50℃ for 4 hours, centrifuge at (4000rpm×20min), collect the extract, add 95% ethanol to a final concentration of 50%, add 600mL of saturated sodium chloride solution, let stand and precipitate, centrifuge (4000rpm×15min), collect the precipitate to obtain crude polysaccharide, add appropriate amount of water to dissolve to obtain peach gum extract (10mg / mL).

[0038] Example 1-2 Embodiment 1-2 relates to an anti-allergic soothing polysaccharide composition and a preparation method thereof. The components and dosages of the composition of Embodiment 1-2 are shown in Table 1, where “-” in Table 1 means no addition.

[0039] Table 1 (Unit: parts by weight)

[0040] in: Bletilla striata polysaccharide: Mw 41.3kDa (Mw, weight average molecular weight, detected by HPGPC method); purity>99% (detected by HPGPC method, calculated by area normalization method); Polygonatum sibiricum polysaccharide: Mw 4.3kDa (Mw, weight average molecular weight, detected by HPGPC method); purity>99% (detected by HPGPC method, calculated by area normalization method); Poria cocos extract: CAS:168456-53-9.

[0041] The preparation method of embodiment 1: 1. Prepare the ingredients: Peach gum extract: prepared according to the method described in Preparation Example 3.

[0042] Bletilla striata polysaccharide: ensure that the weight average molecular weight Mw is 41.3kDa and the purity is greater than 99%.

[0043] Polygonatum sibiricum polysaccharide: ensure that the weight average molecular weight Mw is 4.3kDa and the purity is greater than 99%.

[0044] White Poria cocos extract: purchased or prepared under the designated CAS number 168456-53-9.

[0045] Betaine supramolecular crystal powder: prepared according to the method described in Preparation Example 1.

[0046] 2. Weigh each component: Accurately weigh each component according to the component ratio of Example 1 (3 parts of peach gum extract, 5 parts of Bletilla striata polysaccharide, 3 parts of Polygonatum sibiricum polysaccharide, 5 parts of White Poria cocos extract, and 5 parts of betaine supramolecular crystal powder).

[0047] 3. Mixing: Pour the weighed components into a clean container.

[0048] Use a blender or stir manually to evenly mix the components to form an anti-allergic and soothing polysaccharide composition.

[0049] The preparation method of embodiment 2: 1. Prepare the ingredients: Peach gum extract: prepared according to the method described in Preparation Example 3.

[0050] Bletilla striata polysaccharide: ensure that the weight average molecular weight Mw is 41.3kDa and the purity is greater than 99%.

[0051] Polygonatum sibiricum polysaccharide: ensure that the weight average molecular weight Mw is 4.3kDa and the purity is greater than 99%.

[0052] White Poria cocos extract: purchased or prepared under the designated CAS number 168456-53-9.

[0053] Betaine supramolecular crystal powder: prepared according to the method described in Preparation Example 2.

[0054] 2. Weigh each component: Accurately weigh each component according to the component ratio of Example 2 (5 parts of peach gum extract, 3 parts of Bletilla striata polysaccharide, 5 parts of Polygonatum sibiricum polysaccharide, 3 parts of white Poria cocos extract, 10 parts).

[0055] 3. Mixing: Pour the weighed components into a clean container.

[0056] A stirrer is used to mix the components evenly to form an anti-allergic soothing polysaccharide composition.

[0057] Comparative Example 1 The difference between this comparative example and Example 1 is that the betaine supramolecular crystal powder prepared in Comparative Preparation Example 1 is used.

[0058] Comparative Example 2 The difference between this comparative example and Example 1 is that the betaine supramolecular crystal powder is the betaine supramolecular crystal powder prepared in Comparative Preparation Example 2.

[0059] Comparative Example 3 The difference between this comparative example and Example 1 is that the betaine supramolecular crystal powder is the betaine supramolecular crystal powder prepared in Comparative Preparation Example 3.

[0060] Comparative Example 4 The difference between this comparative example and Example 1 is that the betaine supramolecular crystal powder is the betaine extract powder prepared in step 1 of Preparation Example 1.

[0061] Performance Test Example 1 Soothing Efficacy Test Test method: The compositions prepared in Example 1 and Comparative Examples 1-4 were prepared into 0.5 mg / mL samples and applied to the face for 30 min; instrumental tests (AISIA) were performed on the face of the subjects.

[0062] 2. Test results The results are as follows Figure 1 The subjects of Example 1 had acne and blackheads reduced by 13.6%, oiliness of the skin was also improved, and sensitivity was significantly improved, reduced by 40%.

[0063] Table 2

[0064] Performance Test Example 2 Soothing Efficacy Test-Hyaluronidase Inhibition Rate Test Hyaluronidase is the main factor causing allergic reactions. When allergens invade the body for the first time, the body will produce antibodies IgE. When allergens invade the body again, they will bind to the IgE antibodies on the surface of mast cells, thereby initiating activation signals and releasing histamine. Histamine produces allergic reactions under the activation of hyaluronidase. Therefore, inhibiting the activity of hyaluronidase can effectively prevent the occurrence of allergic reactions. Hyaluronidase has a strong correlation with inflammatory reactions and allergic reactions. After years of testing and research, the industry has also found that the in vitro hyaluronidase inhibition test method is consistent with the conclusion of the human soothing effect evaluation.

[0065] 1. Test on hyaluronidase inhibition rate of the composition of Example 1 1.1. Draw the reference curve of hyaluronidase inhibition of the system reference product Vitamin C (VC) was used as the system reference and diluted to the concentration in Table 3 using phosphate buffer (pH 7.0). The concentrations shown are tested and calculated according to the test method in step 1.2, with the reference concentration as the x-axis. The hyaluronidase inhibition rate is on the y-axis, and a reference curve is drawn.

[0066]

[0067] 1.2. In vitro hyaluronidase inhibition test Sample solution preparation: The sample of Example 1 was prepared with sterile water into sample solutions with concentrations of 30 mg / mL, 10 mg / mL, 5 mg / mL, 3 mg / mL, and 1 mg / mL, respectively.

[0068] Prepare the reaction system according to the amount and order of each reagent in Table 4. After mixing, react according to the corresponding reaction time and temperature. Set 3 replicate wells and 1 background control well for each concentration.

[0069] Table 4 In vitro hyaluronidase inhibition test reaction system

[0070] After the reaction is completed, 200 μL of the reaction solution is added to a 96-well plate, the absorbance OD value is read at 600 nm, and the inhibition rate of the sample on hyaluronidase is calculated according to the following formula.

[0071] Inhibition rate of sample on hyaluronidase (%) = ×100% Where: A——Absorbance of sodium hyaluronate system, i.e. absorbance value of solution without hyaluronidase B——Sodium hyaluronate system solvent background absorbance C——Sample absorbance value, i.e., the absorbance value of the solution after the sample reacts with the hyaluronidase solution D——Sample background absorbance 1.3、Reference curve of hyaluronidase inhibition of system reference (see Figure 2 ) Table 5 Analysis of system reference results (Mean)

[0072] Fitting equation: y = (AD) / [1+(x / C)^B]+D A=86.92, B= -16.69, C= 44.79, D=8.09, r2=0.9994 1.4. Results of in vitro hyaluronidase inhibition test (see Table 6) Table 6 Analysis of hyaluronidase inhibition test results (Mean±SD)

[0073] The results showed that the inhibition rate of the composition prepared in Example 1 on hyaluronidase activity was 19.55% at a concentration of 30 mg / mL; the inhibition rate on hyaluronidase activity was 24.09% at a concentration of 10 mg / mL; the inhibition rate on hyaluronidase activity was 53.82% at a concentration of 5 mg / mL; the inhibition rate on hyaluronidase activity was 45.38% at a concentration of 3 mg / mL; and the inhibition rate on hyaluronidase activity was 43.30% at a concentration of 1 mg / mL.

[0074] 2. Test on hyaluronidase inhibition rate of the compositions of Example 2 and Comparative Examples 1-4 The compositions of Comparative Examples 1 to 4 were respectively prepared with sterile water into sample solutions with a concentration of 30 mg / mL.

[0075] The test method is the same as step 1.

[0076] The test results are shown in Table 7. Table 7

[0077] The test results show that: Comparative Example 1: The betaine extract that was not frozen had a reduced effect; Comparative Example 2 uses frozen but not dried wolfberry slurry to extract betaine, which is slightly higher than that of Comparative Example 1; In Comparative Example 3, chitinase was replaced by chitosanase to prepare peach gum extract, which affected the effect of the overall composition.

[0078] Performance Test Example 3 Cosmetic Soothing Efficacy Test - In Vitro TNF-α Inflammatory Factor Content Determination In this test example: the sample of Example 1 was used to determine the content of TNF-α inflammatory factor in vitro 1. Experimental Materials Cells used: Macrophage Raw264.7 (purchased from the Cell Bank of the Chinese Academy of Sciences, TCM13) Culture medium: High glucose DMEM (Gibco) Serum: Fetal bovine serum (ExCell) Buffer: PBS (Gibco) Antibiotics: Double antibiotics (Soleba, penicillin + streptomycin) Culture plate: 96-well plate (Nice) Centrifuge tube: 50mL sterile centrifuge tube (Labselect) Culture dish: cell culture dish (Nice, 60mm) Kit: NO kit (Shanghai Biotech Biotechnology Co., Ltd.) Stimulant: LPS (Sigma, L2880) Positive control: dexamethasone (Sigma, D4902) 2. Experimental Methods 1. Cell seeding: Macrophages Raw264.7 were seeded into 24-well plates at a seeding density of 2×10^5 cells / well.

[0079] Culture overnight in a 37°C, 5% CO2 incubator to allow the cells to adhere to the wall and reach a suitable growth state.

[0080] 2. Liquid preparation: According to the experimental design, the sample of Example 1 was prepared into a working solution with a mass concentration of 0.3% for standby use.

[0081] Meanwhile, prepare blank control (BC, no additives), negative control (NC, medium + cells only), and positive control (PC, dexamethasone 100 μg / mL + LPS 1 μg / mL).

[0082] 3. Drug administration: When the cell confluence reaches 40%-60%, the drugs are administered in groups according to the experimental design.

[0083] Add 990 μL of the corresponding working solution to each well, and set up three replicate wells in each group.

[0084] The culture plate was placed in a 37°C, 5% CO2 incubator for further 2 hours.

[0085] 4. LPS stimulation: Add 10 μL of LPS working solution (final concentration of 1 μg / mL) to each well of the drug-treated plate to allow the drug to act together with LPS. Gently shake the plate to mix the drugs evenly and continue to culture in the incubator for 22 hours.

[0086] 5. Collection of cell supernatant: After the culture was completed, 800 μL of cell culture supernatant from each well was collected in a 1.5 mL sterile centrifuge tube and the supernatant was frozen in a -80°C ultra-low temperature refrigerator for future use.

[0087] 6. TNF-α content detection: The TNF-α content in the supernatant was detected according to the operating instructions of the NO kit.

[0088] The standard curve of TNF-α is as follows: Figure 3 .

[0089] Equation: y = a + bx a= 0.03072, b= 0.00362, r 2= 0.99257 The experimental results are shown in Table 8.

[0090] Table 8

[0091] Result analysis: (1) Under the stimulation of 1 μg / mL LPS, the TNF-α content in the negative group increased significantly (P < 0.001), indicating that the modeling was successful. (2) Compared with the blank control, the TNF-α content in the negative control was increased by 240.72 times; compared with the negative control, the TNF-α content inhibition rate in the positive control was 56.73%, which met the standard requirements of TNF-α content increase ≥5 times and TNF-α content inhibition rate ≥25%, proving that the experimental system is effective. (3) Compared with the negative control, the sample group of Example 1 had the effect of inhibiting the TNF-α content at a concentration of 0.3%, and the inhibition rate was 41.35%.

[0092] Performance Test Example 4 Scratch Test In this test example: the sample of Example 1 was used to carry out a scratch test.

[0093] The scratch test was performed to simulate the repair process of cells after injury, and to observe whether the sample of Example 1 could promote cell migration, thereby exploring its potential efficacy in cell repair.

[0094] Experimental materials and reagents: Cell line: 3T3 NIH cell line (from the Institute of Cell Biology, Chinese Academy of Sciences, Shanghai).

[0095] Culture medium and serum: DMEM culture medium (Gibco), double antibodies (penicillin, streptomycin), FBS fetal bovine serum (ExCell), PBS buffer (Gibco).

[0096] Experimental consumables: 24-well plate, 0.22μl filter membrane (Biosharp).

[0097] Control substance: Commercially available EGF protein (Pepro Tech, 315-09-100UG).

[0098] Experimental methods: Draw positioning lines: Use a marker pen to draw horizontal lines evenly on the back of the 24-well plate and align them with a ruler. Draw three parallel lines across each well to position and observe the subsequent scratching operation.

[0099] Preparation of monolayer cells: 3T3 NIH cell suspension was inoculated into a 24-well plate with 1 mL per well, and then placed in a 5% 37°C incubator for 12 hours. After that, the medium was replaced with basal medium containing 0.5% serum and cultured for another 12 hours to obtain a confluent monolayer of 3T3 cells.

[0100] Scratch operation: On the second day, after observing that the cells in the 24-well plate have grown into a monolayer, aspirate the original culture medium. Use a 100μl / 1mL pipette tip to scratch perpendicular to the horizontal line on the back, making one scratch per well. Then add PBS to the plate and wash three times to remove the suspended cells scratched and ensure the consistency of the cell state at the edge of the scratch.

[0101] Grouping and sampling Blank group: Add 1 mL of serum-free culture medium.

[0102] Experimental group: Add 1 mL of 1% FBS culture medium containing 0.5 wt % of the sample prepared in Example 1. After adding the sample, place the 24-well plate in a constant temperature incubator for culture.

[0103] Cell migration record: Take photos at 0 hours after scratching to record the position of each well. Then observe and take photos of the fixed positions at 12h, 24h, and 36h of culture to analyze cell migration. The results are shown in Figure 4 (blank group) and Figure 5 .

[0104] Statistical method: Image J software was used to measure the scratch area. Cell migration rate = (0h scratch area - scratch area after culture) / 0h scratch area × 100% The experimental results are shown in Tables 9-10, as well as: Table 9 Cell migration rate in blank group (mean)

[0105] Table 10 Cell migration rate in experimental groups (mean)

[0106] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. An anti-allergic soothing polysaccharide composition, characterized in that: The composition comprises the following components in parts by weight: 3-5 parts of peach gum extract; 3-5 parts of Bletilla striata polysaccharide; 3-5 parts of Polygonatum sibiricum polysaccharide; 3-5 parts of white Poria cocos extract; Betaine supramolecular crystal powder 5-10 parts.

2. The anti-allergic soothing polysaccharide composition according to claim 1, characterized in that: The peach gum extract is prepared by a method comprising the following steps: Take dried peach gum powder, add deionized water; extract at 50-60°C for 4-6 hours, centrifuge at 4000-5000rpm for 10-20min, collect the extract, add 85-95% ethanol to a final concentration of 40 wt%-80 wt%, add 0-600mL saturated sodium chloride solution, let stand for precipitation, centrifuge at 4000-5000rpm for 10-20min, collect the precipitate to obtain crude polysaccharide, and dissolve in water to obtain peach gum extract.

3. The anti-allergic soothing polysaccharide composition according to claim 2, characterized in that: The solid-liquid ratio is preferably 1:10-50; the concentration of the peach gum extract is 5 - 15 mg / mL.

4. The anti-allergic soothing polysaccharide composition according to claim 1, characterized in that: The preparation method of the betaine supramolecular crystal powder comprises the following steps: Step 1, taking betaine extract powder, adding water, dissolving at 30-40°C, and preparing a betaine extract solution with a concentration of 15-20% (w / w); Step 2, subjecting the betaine extract solution to ultrasonic oscillation for 20-30 minutes at an ultrasonic power of 200-250W to self-assemble to form a supramolecular structure; Step 3: The betaine extract solution treated in step 2 is filtered through a microporous filter membrane to obtain a supramolecular solution, which is then dried into a powder to obtain the betaine supramolecular crystal powder.

5. The anti-allergic soothing polysaccharide composition according to claim 4, characterized in that: The preparation method of the betaine extract powder comprises the following steps: After washing the fresh wolfberry leaves, freezing them at -18°C to -20°C for 20-24h, drying, crushing, sieving, mixing with the extract, adding activated carbon, extracting, centrifuging, adsorbing with macroporous adsorption resin, eluting, vacuum decompression concentration and drying, to obtain betaine extract; In the extraction step, the extract is ethanol with a mass concentration of 50-60%, the amount of activated carbon added is 0.5%-1% of the mass of the wolfberry leaf powder, the extraction temperature is 20-40°C, the extraction time is 1-2h, the centrifugal conditions are 4000-5000r / min, and the centrifugal time is 5-8min.

6. The anti-allergic soothing polysaccharide composition according to claim 5, characterized in that: In the eluting step, the eluent includes a polyol and / or water; the polyol includes butanediol.

7. The anti-allergic soothing polysaccharide composition according to claim 1, characterized in that: The weight average molecular weight Mw of the Bletilla striata polysaccharide is between 30 kDa and 50 kDa, and the purity is greater than 95%.

8. The anti-allergic soothing polysaccharide composition according to claim 1, characterized in that: The weight average molecular weight Mw of the polygonatum sibiricum polysaccharide is between 3kDa and 6kDa, and the purity is greater than 95%.

9. A method for preparing the anti-allergic soothing polysaccharide composition according to any one of claims 1 to 8, characterized in that: The components are mixed evenly to obtain the anti-allergic and soothing polysaccharide composition.

10. Use of the anti-allergic and soothing polysaccharide composition according to any one of claims 1 to 8 in the preparation of cosmetics or medicines with soothing effects.

Images