Amphiphilic chitosan oligosaccharide and its preparation method and application

Abstract

The present invention belongs to the technical field of chitosan oligosaccharide modification, specifically relating to an amphiphilic chitosan oligosaccharide, its preparation method, and application. The amphiphilic chitosan oligosaccharide has the structural formula shown in Formula I: #imgabs0#, wherein m and n are both positive integers, and m+n is between 3 and 30; and -N=R represents a Schiff base structure formed by the reaction of an amino group with an aldehyde, wherein the aldehyde is citral. By grafting the aldehyde onto the chitosan oligosaccharide, the chitosan oligosaccharide possesses both amphiphilic and aldehyde properties. This amphiphilicity can be utilized to prepare skin care products in various dosage forms, while also imparting the skin care products with the fragrance and efficacy of the aldehyde.

Description

Technical Field

[0001] The invention belongs to the technical field of chitosan oligosaccharide modification, and particularly relates to amphiphilic chitosan oligosaccharide and a preparation method and application thereof. Background Art

[0002] The statements herein merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] The skin is the largest organ in the human body and performs crucial physiological functions. From a physiological perspective, it serves as a barrier against physical, chemical, and microbial intrusion; it also regulates body temperature, prevents excessive water loss, and maintains a stable internal environment. From a psychological perspective, healthy, smooth, and elastic skin can significantly enhance a person's self-confidence and image, enabling them to project a more positive outlook in social interactions and daily life.

[0004] As we age, our skin inevitably develops a range of problems. On the surface, collagen and elastin fiber synthesis decreases and degradation increases, leading to decreased skin elasticity and the appearance and deepening of wrinkles. Subcutaneous fat is lost or its distribution changes, causing sagging skin. The skin's ability to retain moisture weakens, leading to dry skin problems such as roughness and scaling. Furthermore, the skin's immune function gradually declines, increasing sensitivity to external stimuli and making it more susceptible to allergies and inflammation. These skin problems can seriously impact a person's self-confidence and quality of life.

[0005] To overcome the negative effects of skin aging, researchers are continuously developing skincare ingredients with specialized benefits. Chitosan oligosaccharides, a degradation product of chitosan, offer superior water solubility, antibacterial properties, antioxidant properties, biocompatibility, and the ability to promote the growth of skin fibroblasts. Their use in skincare products can impart moisturizing, antibacterial, anti-inflammatory, and anti-aging properties. However, their excellent water solubility limits their application in a wider range of skincare formulations. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the present invention aims to provide an amphiphilic chitosan oligosaccharide and a preparation method and application thereof.

[0007] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0008] In a first aspect, the present invention provides an amphiphilic chitosan oligosaccharide, the structural formula of which is shown in Formula I:

[0009]

[0010] Formula I;

[0011] Wherein, m and n are both positive integers, and m+n is between 3 and 30; -N=R is a Schiff base structure formed by the reaction of an amino group and an aldehyde, and the aldehyde is citral.

[0012] Citral, also known as geranial, has a strong lemon scent that can add a fresh and pleasant aroma to skin care products and enhance the sensory experience of the product; it also gives skin care products antibacterial, antioxidant, soothing and anti-inflammatory functions.

[0013] By grafting aldehyde onto chitosan oligosaccharide, chitosan oligosaccharide has both amphiphilic and aldehyde properties. Its amphiphilicity can be used to prepare skin care products in various dosage forms, and at the same time, the skin care products are given the fragrance and efficacy of aldehyde.

[0014] In some embodiments, the citral is trans-citral and / or cis-citral.

[0015] Preferably, the content of trans-citral in the amphiphilic chitosan oligosaccharide is greater than 50%. Trans-citral has a lemon scent, while cis-citral has no scent. The content of trans-citral is greater than 50% to highlight its scent.

[0016] In some embodiments, the molecular weight of the amphiphilic chitosan oligosaccharide before modification is less than 5000, and the degree of deacetylation is greater than 80%.

[0017] In some embodiments, the degree of substitution of the amphiphilic chitosan oligosaccharide is 10%-80%, preferably 20%-60%.

[0018] Preferably, the aggregate particle size of the amphiphilic chitosan oligosaccharide is 200-700 nm.

[0019] The aggregate particle size here refers to the particle size after 21 days of standing. The particle size before 21 days is unstable.

[0020] Amphiphilic chitosan oligosaccharides exhibit self-aggregation behavior, and the aggregate size is affected by the degree of substitution, hydrophobic chain length, and standing time. When the chain lengths are equal, the greater the degree of substitution, the smaller the aggregate size. When the degree of substitution is equal, the longer the chain length, the larger the aggregate size. When the amphiphilic chitosan oligosaccharide solution is left to stand for 0-21 days, the particle size gradually decreases with increasing standing time, and after 21 days, the particle size no longer changes with time. Smaller amphiphilic chitosan oligosaccharide particles are more likely to penetrate the skin, allowing them to fully demonstrate their advantages.

[0021] In a second aspect, the present invention provides a method for preparing the amphiphilic chitosan oligosaccharide, comprising the following steps: preparing a chitosan oligosaccharide aqueous solution and an ethanol solution of aldehyde, and heating them to 30-60° C.;

[0022] Gradually add the ethanol solution of aldehyde to the chitosan oligosaccharide aqueous solution, stirring continuously during the addition process. After the addition is completed, continue stirring for a set time to allow for full reaction;

[0023] After the reaction is completed, the reaction system is cooled and ethanol is removed by rotary evaporation to obtain a modified product;

[0024] After the modified product is freeze-dried, sponge-like amphiphilic chitosan oligosaccharide is obtained.

[0025] In some embodiments, the reaction time is 1-6 hours.

[0026] In some embodiments, the molar ratio of the aldehyde group of the aldehyde to the primary amino group in the chitosan oligosaccharide is 0.01-1:1, preferably 0.1-1:1.

[0027] In some embodiments, the freeze-drying temperature is -60 to -85°C, preferably -70 to -80°C.

[0028] In some embodiments, the vacuum degree of the freeze-drying is in the range of 5-10 Pa, preferably 5-10 Pa.

[0029] In a third aspect, the present invention provides a method for preparing the amphiphilic chitosan oligosaccharide, comprising the following steps: mixing and grinding the aldehyde group of aldehyde and the primary amino group of chitosan oligosaccharide in a molar ratio of 0.01-1:1, applying a force of 5-20N during grinding, and grinding for 2-15min, and freeze-drying the modified product at -85°C to obtain amphiphilic chitosan oligosaccharide.

[0030] This preparation method is a solid-phase method. By changing the type of aldehyde, the molar ratio of aldehyde to chitosan oligosaccharide (calculated as -NH2), the grinding force, and the grinding time, amphiphilic chitosan oligosaccharides with a degree of substitution between 20% and 80% can be prepared.

[0031] The liquid-phase method for preparing amphiphilic chitosan oligosaccharides is relatively cumbersome and time-consuming, and produces waste liquid during the preparation process. The solid-phase method only requires sample pre-drying, is simple and rapid to operate, and does not produce waste liquid. If put into industrial production, it has obvious advantages over the liquid-phase method.

[0032] During the preparation of amphiphilic chitosan oligosaccharides, if a small amount of aldehyde is not completely reacted and separated, it will remain in the amphiphilic chitosan oligosaccharide as a fragrance.

[0033] In a fourth aspect, the present invention provides the use of the amphiphilic chitosan oligosaccharide in the preparation of skin care cosmetics.

[0034] In some embodiments, the skin care cosmetics include but are not limited to lotion, moisturizer, facial mask, toner, lipstick, sunscreen, powder, makeup remover, cleanser, shower gel or shampoo.

[0035] The beneficial effects achieved by one or more embodiments of the present invention are as follows:

[0036] The amphiphilic chitosan oligosaccharide provided by the present invention has the characteristics of simple preparation process, excellent performance, and high safety. The modified amphiphilic chitosan oligosaccharide of the present invention has the ability to self-aggregate in aqueous solution, and the particle size of the aggregates can be adjusted by the degree of substitution and the length of the hydrophobic segment to achieve good transdermal absorption. The modified amphiphilic chitosan oligosaccharide is non-toxic to the fibroblast HFF-1 cell line, has antibacterial properties (Staphylococcus aureus, Escherichia coli, and Propionibacterium acnes), significantly inhibits the activity of inflammatory factors (TNF-2 and IL-6) and hyaluronidase, promotes fibroblast growth, has antioxidant properties (DPPH, hydroxyl radicals, and superoxide radicals), promotes transdermal absorption, and can encapsulate / sustained-release oil-soluble active ingredients. Furthermore, the amphiphilic chitosan oligosaccharide self-aggregates have the properties of stabilizing Pickering emulsions. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0038] Figure 1 is the infrared spectrum of the amphiphilic chitosan oligosaccharide prepared in Example 1;

[0039] Figure 2 1 is the NMR spectrum of the amphiphilic chitosan oligosaccharide prepared in Example 1, wherein the embedded figure is a schematic diagram of the molecular structure of the amphiphilic chitosan oligosaccharide;

[0040] Figure 3 2 are the thermogravimetric curves of amphiphilic chitosan oligosaccharides with different degrees of substitution prepared in Examples 1, 2 and 4;

[0041] Figure 4 This is the particle size distribution diagram of the aggregates of the amphiphilic chitosan oligosaccharide with a substitution degree of 19.9% ​​prepared in Example 2;

[0042] Figure 5 This is the particle size distribution diagram of the aggregates of the amphiphilic chitosan oligosaccharide with a substitution degree of 24.3% prepared in Example 3;

[0043] Figure 6 This is the particle size distribution diagram of the amphiphilic chitosan oligosaccharide with a substitution degree of 31.4% prepared in Example 1;

[0044] Figure 7 1 is a particle size distribution diagram of the aggregates of amphiphilic chitosan oligosaccharides with substitution degrees of 19.9%, 24.3% and 31.4% prepared in Examples 1-3 after standing for 21 days;

[0045] Figure 8 This is a transmission electron micrograph of the aggregate of the amphiphilic chitosan oligosaccharide having a degree of substitution of 38.0% prepared in Example 5 after standing for 21 days;

[0046] Figure 9 This is a comparison chart of the cytotoxicity of different concentrations of amphiphilic chitosan oligosaccharides with substitution degrees of 19.9%, 24.3% and 31.4% prepared in Examples 1-3 to fibroblast HFF-1, wherein (0) is a chitosan oligosaccharide system with a concentration of 0.05 g / L;

[0047] 1-a is an amphiphilic chitosan oligosaccharide with a substitution degree of 19.9% ​​and a concentration of 0.05 g / L; 1-b is an amphiphilic chitosan oligosaccharide with a substitution degree of 19.9% ​​and a concentration of 0.1 g / L; 1-c is an amphiphilic chitosan oligosaccharide with a substitution degree of 19.9% ​​and a concentration of 0.2 g / L;

[0048] 2-a is an amphiphilic chitosan oligosaccharide with a substitution degree of 24.3% and a concentration of 0.05 g / L; 2-b is an amphiphilic chitosan oligosaccharide with a substitution degree of 24.3% and a concentration of 0.1 g / L; 2-c is an amphiphilic chitosan oligosaccharide with a substitution degree of 24.3% and a concentration of 0.2 g / L;

[0049] 3-a is an amphiphilic chitosan oligosaccharide with a substitution degree of 31.4% and a concentration of 0.05 g / L; 3-b is an amphiphilic chitosan oligosaccharide with a substitution degree of 31.4% and a concentration of 0.1 g / L; 3-c is an amphiphilic chitosan oligosaccharide with a substitution degree of 31.4% and a concentration of 0.2 g / L;

[0050] Figure 10 This is a comparative graph of the antibacterial effects of amphiphilic chitosan oligosaccharides with different degrees of substitution prepared in Examples 1-3 on Escherichia coli A, Staphylococcus aureus B, and Propionibacterium acnes C, wherein A1, B1, and C1 are amphiphilic chitosan oligosaccharides with a degree of substitution of 19.9%; A2, B2, and C2 are amphiphilic chitosan oligosaccharides with a degree of substitution of 24.3%; and A3, B3, and C3 are amphiphilic chitosan oligosaccharides with a degree of substitution of 31.4%.

[0051] Figure 11 This is a comparative chart of the inhibition of TNF-α and IL-6 inflammatory factors by amphiphilic chitosan oligosaccharides at different concentrations in Example 1, wherein A represents TNF-α; B represents IL-6;

[0052] Figure 12 This is a graph showing the growth-promoting effect of the amphiphilic chitosan oligosaccharide (100 μg / mL) prepared in Example 1 on fibroblasts, wherein A is the image after 0 h, B is the image after 24 h, and C is the image after 36 h (the colors of the images are to distinguish the time);

[0053] Figure 13 This is a graph showing the inhibitory effect of the amphiphilic chitosan oligosaccharide with a degree of substitution of 31.4% prepared in Example 1 on hyaluronidase;

[0054] Figure 14This is a comparison of the transdermal absorption effects of chitosan oligosaccharide and the amphiphilic chitosan oligosaccharide prepared in Example 1, wherein A is chitosan oligosaccharide and B is amphiphilic chitosan oligosaccharide with a degree of substitution of 31.4%. (The skin used in the experiment is the abdominal skin of 5-week-old mice, in vivo experiment), and the scale bar is 100 μm.

[0055] Figure 15 The figures are the effect of the amphiphilic chitosan oligosaccharide self-aggregate stabilizing the Pickering emulsion prepared in Example 1, wherein A is a water phase fluorescence confocal microscopy image, B is an oil phase fluorescence confocal microscopy image, C is an oil-water coexistence fluorescence microscopy image, and D is a polarizing microscopy image. The scale bar is 10 μm.

[0056] Figure 16 This is a Pickering emulsion diagram stabilized by amphiphilic chitosan oligosaccharide with a substitution degree of 31.4%, where a The oil content is 20%, b Oil content 30%, c Oil content 40%, d Oil content 50%, e Oil content 60%;

[0057] Figure 17 The relationship between the particle size of the Pickering emulsion stabilized by amphiphilic chitosan oligosaccharide with a substitution degree of 31.4% and time;

[0058] Figure 18 A comparison of the sustained release of resveratrol from Pickering emulsions stabilized by different amphiphilic chitosan oligosaccharides;

[0059] Figure 19 The antioxidant effect of the resveratrol-loaded Pickering emulsion stabilized by amphiphilic chitosan oligosaccharides, where (A) is the antioxidant rate against DPPH, (B) is the antioxidant rate against hydroxyl radicals, and (C) is the antioxidant rate against superoxide radicals.

[0060] Figure 20 A comparison chart of the photostability of resveratrol-loaded Pickering emulsions stabilized by different amphiphilic chitosan oligosaccharides. DETAILED DESCRIPTION

[0061] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0062] The following is an example of preparing amphiphilic chitosan oligosaccharide by modifying chitosan oligosaccharide with citral, and the present invention is further described in conjunction with the examples.

[0063] Example 1

[0064] The molar ratio of the aldehyde group of citral to the primary amino group -NH2 in chitosan oligosaccharide is 1:1. The corresponding masses of citral and chitosan oligosaccharide are weighed respectively. The reaction temperature is 60℃ and the reaction time is 5h to obtain citral-modified amphiphilic chitosan oligosaccharide with a degree of substitution of 31.4%.

[0065] The specific steps are as follows:

[0066] First, prepare a chitosan oligosaccharide aqueous solution with a concentration of 20 mg / mL and heat it to 60°C;

[0067] In another beaker, prepare a 20 mg / mL citral solution in ethanol and heat the solution to 60°C.

[0068] The ethanol solution of citral was added dropwise to the chitosan oligosaccharide aqueous solution while stirring continuously at a speed of 300 rpm. After the addition was completed, stirring was continued at the same temperature for 5 h to allow the reaction to proceed fully.

[0069] After the reaction was completed, the reaction system was cooled to room temperature, and ethanol was removed by reduced pressure distillation using a rotary evaporator to obtain a modified product.

[0070] The modified product was freeze-dried at -85°C and 5Pa vacuum to obtain spongy citral-modified amphiphilic chitosan oligosaccharide.

[0071] Example 2

[0072] The molar ratio of the aldehyde group of citral to the primary amino group -NH2 in chitosan oligosaccharide is 1:5, the reaction temperature is 40℃, and the reaction time is 2h, and citral-modified amphiphilic chitosan oligosaccharide with a degree of substitution of 19.9% ​​is obtained.

[0073] The specific steps are the same as those in Example 1.

[0074] Example 3

[0075] The molar ratio of the aldehyde group of citral to the primary amino group -NH2 in chitosan oligosaccharide was 1:2, the reaction temperature was 50℃, and the reaction time was 4h, and citral-modified amphiphilic chitosan oligosaccharide with a degree of substitution of 24.3% was obtained.

[0076] The specific steps are the same as those in Example 1.

[0077] Example 4

[0078] The molar ratio of the aldehyde group of citral to the primary amino group -NH2 in chitosan oligosaccharide was 1:1, the reaction temperature was 50℃, and the reaction time was 6h, and citral-modified amphiphilic chitosan oligosaccharide with a substitution degree of 40.0% was obtained.

[0079] The specific steps are the same as those in Example 1.

[0080] Example 5

[0081] At 25°C, the molar ratio of the aldehyde group of citral to the primary amino group -NH2 in chitosan oligosaccharide was 1:1. The mixture of citral and chitosan oligosaccharide was uniformly ground with a force of 5N for 10 minutes to obtain citral-modified amphiphilic chitosan oligosaccharide with a degree of substitution of 38.0%.

[0082] The specific steps are as follows: according to the molar ratio of the aldehyde group of citral to the -NH2 of chitosan oligosaccharide being 1:1, dry chitosan oligosaccharide is weighed and placed in a mortar, citral is added dropwise into the mortar, and at the same time, it is ground with a force of 5N. After all the citral is added, the grinding is continued with the same force for 10 minutes. The product is freeze-dried at -85°C and a vacuum degree of 10Pa to obtain amphiphilic chitosan oligosaccharide.

[0083] The performance test of the amphiphilic chitosan oligosaccharide prepared in the embodiment was carried out:

[0084] Infrared spectrum test:

[0085] Test method: Weigh approximately 5 mg of amphiphilic chitosan oligosaccharide, grind it and mix it evenly with dry potassium bromide. Press the mixture into a transparent sheet and measure the wavelength of the product using a Nicolet iS10 infrared spectrometer (Tensor27, Shimadzu, Japan) at room temperature in the wavenumber range of 4000 cm -1 -400cm -1 .

[0086] Infrared spectrum such as Figure 1 As shown in the figure, compared with the infrared spectrum curve of chitosan oligosaccharide, the spectrum curve of amphiphilic chitosan oligosaccharide at 1625cm -1 、1515cm -1 and 1381cm -1 There are new peaks, which belong to the C=C double bond, C=N and C=C conjugated double bonds, and CH vibration absorption peaks on the terminal methyl group in the citral molecule; at the same time, the infrared spectrum of chitosan oligosaccharide is located at 1257cm -1 The peak intensity of the chitosan oligosaccharide was significantly weakened. This is because the nitrogen atom in the chitosan oligosaccharide molecule is linked to the hydrophobic segment of citral, increasing the energy required for the vibration of the C-N bond. The above peak changes prove that citral is successfully incorporated into the chitosan oligosaccharide molecule.

[0087] MRI test:

[0088] Test method:

[0089] About 2 mg of amphiphilic chitosan oligosaccharide was weighed, dissolved in 1 mL of deionized water, and transferred to an NMR tube. The spectra were measured on a Bruker Advance II 400 spectrometer (Bruker, Switzerland) at room temperature.

[0090] NMR spectra such as Figure 2As shown in the figure, new peaks appeared at 1.21ppm, 1.44ppm, 1.33ppm, 2.03ppm and 2.19ppm. These peaks came from H on the carbon chain of citral, proving that citral was successfully connected to the chitosan oligosaccharide molecule.

[0091] Calculate the degree of substitution based on the peak area of ​​the nuclear magnetic resonance spectrum: Use the software that comes with the nuclear magnetic resonance spectrometer to integrate the characteristic peaks, and then substitute the integrated area into the calculation formula below to obtain the degree of substitution.

[0092] Taking the amphiphilic chitosan oligosaccharide prepared by citral modification as an example, the calculation formula of the degree of substitution DS is as follows:

[0093] ;

[0094] Among them, CH3 is Figure 2 The two -CH3 H, H b-f,f’ It is the H linked to the corresponding C on the sugar ring.

[0095] Thermodynamic stability test:

[0096] About 6 mg of sample was weighed and placed in a crucible. The thermodynamic stability of the sample in the temperature range of 25-500°C was measured using a synchronous thermal analyzer (SDT Q600, TA Instruments, USA). During the test, the heating rate was 5°C / min and the N2 flow rate was 100 mL / min.

[0097] like Figure 3 As shown, the thermogravimetric curves of the three amphiphilic chitosan oligosaccharides with different substitution degrees prepared in Examples 1-3 are almost identical to that of chitosan oligosaccharide, which proves that the thermodynamic stability of the amphiphilic chitosan oligosaccharides is consistent with that of chitosan oligosaccharide.

[0098] Self-aggregation behavior (critical self-aggregation concentration):

[0099] A series of amphiphilic chitosan oligosaccharide solutions with different concentrations were prepared at 25°C. Acetone was used as the solvent to prepare solutions with a concentration of 1×10 -3 Take another test tube with the same number of amphiphilic chitosan oligosaccharide solutions and add 1 mL of pyrene solution to each test tube. Then blow dry with nitrogen gas to keep the pyrene molecules on the inner wall of the test tube.

[0100] The prepared amphiphilic chitosan oligosaccharide solutions of different concentrations were poured into the test tubes containing pyrene to make the final concentration of pyrene 10 -5 The test tube was placed in an ultrasonic chamber at room temperature and 30 W power for 30 minutes to dissolve the pyrene molecules in the hydrophobic core of the self-aggregate, and then allowed to stand for 60 minutes.

[0101] Fluorescence changes in amphiphilic chitosan oligosaccharide solutions of varying concentrations were measured using a fluorescence spectrophotometer (Hitachi F4600, Japan), recording the peak intensities of five emission peaks (373 nm, 379 nm, 383 nm, 390 nm, and 397 nm). The peak intensities at 373 nm and 383 nm were plotted on the y-axis (with the peak intensities designated as I1 and I3, respectively) against the concentration of amphiphilic chitosan oligosaccharide. The critical self-aggregation concentration (CAC) was determined by plotting the peak intensity ratio at 373 nm and 383 nm as the ordinate (with the peak intensities designated as I1 and I3, respectively).

[0102] Aggregate Particle Size and Potential: Amphiphilic chitosan oligosaccharide solutions were prepared at concentrations above the critical aggregation concentration (CAC). Aggregate size and distribution were measured using a Zetasizer Nano ZS90 particle size analyzer (Malvern, England). The aggregate potential was also determined.

[0103] Transmission electron microscopy: The prepared amphiphilic chitosan oligosaccharide solution was dripped onto a copper grid, and the aggregates were stained with phosphotungstic acid. The grid was then dried. The morphology of the self-aggregates was observed using a transmission electron microscope (TEM, Tecnai-12, Philip Apparatus Co, Netherlands).

[0104] Figure 4 : This is a particle size distribution diagram of the aggregates of amphiphilic chitosan oligosaccharide with a degree of substitution of 19.9% ​​prepared in Example 2; it can be seen from the figure that in the newly configured solution, the particle size of the aggregates is closely related to the concentration of amphiphilic chitosan oligosaccharide, and the higher the concentration, the larger the particle size; as the standing time increases, the effect of concentration on particle size gradually weakens. After 21 days, the particle size of the amphiphilic chitosan oligosaccharide aggregates at the three concentrations is significantly smaller than the initial particle size, proving that the aggregate particle size is closely related to the standing time;

[0105] Figure 5 The particle size distribution diagram of the amphiphilic chitosan oligosaccharide with a substitution degree of 24.3% prepared in Example 3 is obtained through the figure. Figure 4 Same conclusion;

[0106] Figure 6 The particle size distribution diagram of the amphiphilic chitosan oligosaccharide with a substitution degree of 31.4% prepared in Example 1 is obtained through the figure. Figure 4 Same conclusion;

[0107] Figure 7 The particle size distribution diagram of the aggregates of amphiphilic chitosan oligosaccharides prepared in Examples 1-3 with substitution degrees of 31.4%, 19.9% ​​and 24.3% respectively after standing for 21 days; it can be seen that the greater the substitution degree, the larger the aggregate particle size of the stable system;

[0108] Figure 8This is a transmission electron micrograph of the aggregates of the amphiphilic chitosan oligosaccharide with a substitution degree of 38.0% prepared in Example 5 after standing for 21 days; closely adjacent amphiphilic chitosan oligosaccharide aggregates can be seen, proving the formation of self-aggregates.

[0109] Antibacterial activity: tested using the agar inhibition zone diffusion method.

[0110] The antimicrobial properties of amphiphilic chitosan oligosaccharides were tested using Staphylococcus aureus, Escherichia coli, and Propionibacterium acnes as bacterial models. All experiments were conducted in a cleanroom, free of other bacteria. The bacterial culture medium consisted of peptone (1.0%, w / v), yeast extract (0.5%, w / v), and sodium chloride (1.0%, w / v). Agar powder (1.5%, w / v) was added to the culture medium to create a solid culture medium. The solid culture medium was then sterilized in a high-temperature, high-pressure autoclave.

[0111] The strains were added to the culture medium at a volume ratio of 1:100 and incubated in a shaker at 37°C for 12 hours. 80 mL of the culture medium was placed in a cell culture dish. After solidification, 100 μL of Staphylococcus aureus, Escherichia coli, and Propionibacterium acnes were smeared on the surface. 6 mm diameter amphiphilic chitosan oligosaccharide discs with degrees of substitution of 19.9%, 24.3%, and 31.4% were placed on the culture medium containing the bacteria. The culture medium was placed in a 37°C incubator and the size of the inhibition zone was observed and recorded for 12 hours.

[0112] Figure 10 This is a comparative diagram of the antibacterial effects of the amphiphilic chitosan oligosaccharides with different substitution degrees prepared in Examples 1-3 on Escherichia coli (A), Staphylococcus aureus (B) and Propionibacterium acnes (C). Figure 10 Amphiphilic chitosan oligosaccharides have strong inhibitory effects on Escherichia coli, Staphylococcus aureus, and Propionibacterium acnes, indicating that they help regulate skin microbiota, prevent or improve oily skin and acne-prone skin, and protect skin health. They also reduce reliance on traditional chemical preservatives and the potential irritation of skincare cosmetics, satisfying consumer demand for natural, gentle products. This provides theoretical support for the use of amphiphilic chitosan oligosaccharides as a core antibacterial ingredient in cleansers and acne serums, and for their potential use in soothing and repairing skincare products to reduce skin inflammation.

[0113] In vitro cytotoxicity:

[0114] The cytotoxicity of amphiphilic chitosan oligosaccharides was detected by cell viability assay kit using fibroblasts as a model.

[0115] Approximately 10,000 fibroblasts were cultured in a medium containing glucose, 10% fetal bovine serum, and 1% (v / v) penicillin-streptomycin (500 U / mL). Approximately 10,000 fibroblasts were seeded in a 96-well plate, and the plate was placed in a biochemical incubator at 37°C and incubated for 24 h.

[0116] The amphiphilic chitosan oligosaccharide prepared in Examples 1-3 was added to the wells containing cells to a final concentration of 0.05 g / L, 0.1 g / L, and 0.5 g / L in each well, and the cells were incubated in a 37° C. biochemical incubator for 24 h.

[0117] Then, 100 μL of 5 mg / mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution was added to each well, and the plate was incubated in a 37°C incubator for 4 h. After that, it was rinsed with phosphate buffer solution and 100 μL of dimethyl sulfoxide solution was added to fully dissolve it for 20 min.

[0118] The absorbance of the final sample was read at 490 nm using a microplate reader. The absorbance of the different samples was compared with the absorbance of the blank sample to determine the cytotoxicity of the amphiphilic chitosan oligosaccharide on fibroblasts.

[0119] Figure 9 The cytotoxicity of different concentrations of amphiphilic chitosan oligosaccharides with substitution degrees of 19.9%, 24.3% and 31.4% prepared in Examples 1-3 to fibroblast HFF-1 is compared. Figure 9 It can be seen that amphiphilic chitosan oligosaccharide has no cytotoxicity to fibroblast HFF-1, and has a significant growth-promoting effect on fibroblasts, such as Figure 12 As shown in A and B, it is proven to be safe for skin cells and will not cause adverse reactions such as allergies and inflammation, thereby improving the safety and applicability of the product and increasing consumers' trust in the product. When used in skin care cosmetics, it helps maintain the integrity of the skin, stimulates fibroblasts to synthesize collagen and elastic fibers, increases the elasticity and toughness of the skin, makes the skin firm and smooth, and exerts an anti-aging effect. It can be used as an effective ingredient in anti-aging skin care products and sensitive skin repair products. It can also be used in medical cosmetics after-surgery products to accelerate skin regeneration.

[0120] Significantly inhibits TNF-α and IL-6 inflammatory factors:

[0121] Mouse mononuclear phagocyte leukemia cells were used as the research subjects. A cellular inflammation model was established by stimulating the cells with lipopolysaccharide bacterial endotoxin. Different test substances and positive controls were added and cultured for 24 hours. Then the cell supernatant was collected and the enzyme-linked immunosorbent assay kit was used to analyze the release of inflammatory factors in mouse mononuclear phagocyte leukemia cells, and the levels of proinflammatory cytokines IL-6 and TNF-α were analyzed.

[0122] Figure 11This is a comparative graph of the inhibition of TNF-α and IL-6 inflammatory factors by amphiphilic chitosan oligosaccharides at different concentrations in Example 1, wherein A is TNF-α; B is IL-6; and the reference group is naturally grown mouse mononuclear macrophages in a system without the addition of lipopolysaccharide and chitosan oligosaccharide.

[0123] Depend on Figure 11 It can be seen that amphiphilic chitosan oligosaccharides have significant inhibitory effects on TNF-α and IL-6 inflammatory factors. When used in repair creams to shield damaged skin, they can soothe the skin and relieve skin inflammation caused by external stimuli such as ultraviolet rays and pollutants; when used in anti-aging essences, they can protect collagen and elastic fibers in the skin and maintain skin elasticity and toughness; when used in sunscreens or after-sun repair products, they can reduce ultraviolet-induced oxidative stress and inflammatory responses, and enhance photoaging protection and inflammation control effects.

[0124] Inhibitory effect of hyaluronidase:

[0125] The inhibition rate of amphiphilic chitosan oligosaccharides on hyaluronidase was determined by the modified Elson-Morgan method.

[0126] Figure 13 is a graph showing the inhibitory effect of the amphiphilic chitosan oligosaccharide with a substitution degree of 31.4% on hyaluronidase prepared in Example 1. Figure 13 It can be seen that amphiphilic chitosan oligosaccharides have a significantly better inhibitory effect on hyaluronidase than chitosan oligosaccharides, and the inhibition rate is positively correlated with the concentration of amphiphilic chitosan oligosaccharides. Amphiphilic chitosan oligosaccharides are used in anti-aging serums, anti-allergic serums, creams, and facial masks to reduce redness, swelling, and itching caused by allergies, while also prolonging the moisturizing and anti-wrinkle effects of the products, making them suitable for sensitive skin. They can also be used in sunscreen and after-sun repair products to create a dual "antioxidant + hydrating" protection mechanism. In the field of medical aesthetics, they can prolong the filling effect of hyaluronic acid, reduce inflammation and edema reactions, and can also be used in post-operative repair dressings or scar care products.

[0127] It has a significant growth-promoting effect on fibroblasts:

[0128] The effect of amphiphilic chitosan oligosaccharides on fibroblast proliferation was investigated using a wound wound assay. Three horizontal lines were marked on the back of a 6-well plate with a marker before the experiment. The experiment began when the fibroblasts reached adherent monolayers. The experiment consisted of a blank control (Ctr) containing culture medium, a negative control (NC), and a sample group (Sample).

[0129] Before timing, use a 200 μL sterile pipette tip to evenly scratch the center of the cell plate perpendicular to the baseline. Rinse the scratch three times with phosphate buffered saline. Then add fresh serum-free medium and take a photo to time the wound. Observe and photograph every 24 hours and measure the scratch width. Calculate the scratch healing rate according to the following formula:

[0130] Scratch healing rate (%) = (L0-L 24 ) / L0×100%;

[0131] Preparation of Pickering emulsion (taking amphiphilic chitosan oligosaccharide modified with citral as an example):

[0132] At room temperature, a 1% aqueous solution of amphiphilic chitosan oligosaccharide and a 1% aqueous solution of sodium tripolyphosphate were mixed at a mass ratio of 9:1 and stirred at 800 rpm for 30 minutes to prepare the aqueous phase of the emulsion. Aqueous phases with varying chitosan oligosaccharide concentrations were prepared by varying the concentration of the amphiphilic chitosan oligosaccharide.

[0133] At room temperature, olive oil and resveratrol are fully mixed and used as the oil phase.

[0134] At room temperature, the aqueous phase and the oil phase were mixed in a mass ratio of 1:1 and homogenized at a speed of 8000 rpm for 3 min to obtain a Pickering emulsion.

[0135] Appearance of Pickering emulsions: There are two main methods for observing emulsions. The first is to take a photograph of the emulsion at rest to assess its static stability. The second is to observe the emulsion more specifically using an optical microscope to assess the size and structure of the emulsion droplets.

[0136] Pickering emulsion droplet diameter and droplet stability: The droplet diameter of the emulsion was measured using a Zetasizer Nano ZS90 (Malvern, England) laser particle size analyzer. Emulsion stability was evaluated by storage and centrifugation. Storage stability was primarily assessed by changes in emulsification rate and droplet diameter.

[0137] To further evaluate the emulsion's performance, the effects of environmental factors (ionic strength (sodium chloride concentration, calcium chloride concentration), temperature (storage for 24 hours at -40°C, 0°C, 25°C, and 50°C), and pH (buffer solution, pH values ​​of 4.00, 6.86, and 9.18, respectively)) on the emulsion's stability were investigated. All samples were tested after 24 hours of storage at room temperature.

[0138] Pickering emulsion showed good stability in the range of -40-50°C for 24 hours.

[0139] Figure 15 The effect diagram of the amphiphilic chitosan oligosaccharide self-aggregate stabilizing Pickering emulsion prepared in Example 1 is shown, wherein A is a water phase fluorescence confocal microscopy image, B is an oil phase fluorescence confocal microscopy image, C is an oil-water coexistence fluorescence microscopy image, and D is a polarizing microscopy image. The scale bars are all 10 μm. Figure 15The outer layer of the emulsion is adsorbed with amphiphilic chitosan oligosaccharide aggregates, stabilizing the emulsion and demonstrating the formation of a Pickering emulsion. The polydisperse nature of the emulsion droplets facilitates the prolonged release of the encapsulated active ingredient, reducing skin irritation. This Pickering emulsion structure is expected to exert a long-lasting effect.

[0140] Figure 16 This is a Pickering emulsion diagram stabilized by amphiphilic chitosan oligosaccharide with a substitution degree of 31.4%, where a The oil content is 20%, b Oil content 30%, c Oil content 40%, d Oil content 50%, e Oil content 60%. Figure 16 Particle size and potential measurements of Pickering emulsions revealed particle sizes ranging from 200-2000 nm and a positive charge of 18-50 mV. These Pickering emulsions can efficiently encapsulate oil-soluble active ingredients or photosensitizers, which can be encapsulated in the hydrophobic core of the amphiphilic chitosan oligosaccharide self-aggregates or dissolved in the oil phase of the Pickering emulsion. The hydrophobic core of the self-aggregates and the oil phase of the Pickering emulsion can each encapsulate / dissolve different oil-soluble active ingredients, enabling the preparation of multifunctional Pickering emulsions. The positive charge facilitates adsorption and uniform distribution of the emulsion on the skin surface. Emulsions with particle sizes less than 500 nm can penetrate the skin, where the positive charge enhances the binding of droplets to the sebum layer of hair follicles, increasing the efficacy of the active ingredients released within the skin. Emulsions with particle sizes greater than 500 nm exert their skincare benefits by regulating the skin's surface microbiome and releasing active ingredients. In addition, the emulsion with a particle size of 200-2000nm has a delicate texture, which keeps the skin fresh and comfortable, and helps to enhance the quality of skin care cosmetics.

[0141] Laser confocal scanning microscopy:

[0142] A Pickering emulsion was prepared using olive oil as the oil phase and an aqueous phase composed of amphiphilic chitosan oligosaccharides / sodium tripolyphosphate (same as above). The dyes Nile Red and Nile Blue were added to the two phases for staining. After 12 hours, the dyed oil and aqueous phases were blended and emulsified to create the Pickering emulsion. The excitation wavelengths for Nile Red were 488 nm, and for Nile Blue were 633 nm. Fluorescence confocal images of the aqueous phase, oil phase, and Pickering emulsion were captured using a laser confocal scanning microscope (CLSM-TCSSP8CARS, Germany) at 64x magnification at both wavelengths.

[0143] Figure 17 The relationship between the particle size of the Pickering emulsion stabilized by amphiphilic chitosan oligosaccharide with a substitution degree of 31.4% and time is shown in the figure. Figure 17 It can be seen that the particle size of the emulsion is closely related to the oil-water mass ratio. The higher the oil content, the larger the particle size of the Pickering emulsion. The particle size of the stable emulsion basically does not change with the standing time. Amphiphilic chitosan oligosaccharide-stabilized Pickering emulsions with different oil-water ratios can be prepared as needed.

[0144] Entrapment and release of resveratrol encapsulated in Pickering emulsion: First, the UV-visible absorption spectrum of resveratrol dissolved in ethanol solution was measured, and the absorbance of each concentration gradient was recorded at 306 nm to establish a standard curve for resveratrol.

[0145] Resveratrol was encapsulated in a Pickering emulsion to investigate its encapsulation and release behavior. First, 0.1 g of resveratrol was dissolved in 10 mL of olive oil and stirred at 300 rpm for 1 hour at 40°C to fully dissolve the resveratrol. Using the resveratrol / olive oil solution as the oil phase, a Pickering emulsion was prepared according to the previously described method.

[0146] Dilute 1 mL of the Pickering emulsion with 10 mL of methanol and sonicate for 10 minutes to break the Pickering emulsion and release resveratrol. Filter the sample through a 0.2 μm filter and measure the absorbance of the solution using a UV-visible spectrophotometer to calculate the resveratrol concentration.

[0147] Separately, 1 mL of the Pickering emulsion was centrifuged at 10,000 rpm for 15 minutes. The resulting supernatant was collected, filtered, and quantitatively analyzed for resveratrol (i.e., resveratrol not entrapped in the emulsion). The resveratrol entrapment efficiency (EE%) was calculated using the following formula:

[0148] EE%=(amount of added resveratrol-amount of unentrapped resveratrol) / (amount of added resveratrol)×100%.

[0149] The release behavior of resveratrol from Pickering emulsion was studied using a Franz diffusion cell with an effective diffusion area of ​​0.64 cm 2(The interface between the donor and receptor chambers and between the donor chambers). The receptor chamber used an ethanol / water solution (ethanol to water mass ratio of 1:1) containing 5 g / L Tween 20. The receptor medium was magnetically stirred at 600 rpm and maintained at a constant temperature in a water bath at 37°C. 300 μL of Pickering or resveratrol control solution (water / alcohol solution (20% v / v ethanol)) was filled into the donor chamber. Both samples contained an equal amount of resveratrol (1 mg / mL). At regular time intervals, 2 mL of the receptor solution was removed and immediately replaced with 2 mL of fresh medium to ensure that the receptor chamber volume was 5 mL. A lead acetate cellulose membrane with a pore size of 0.2 μm was used. The collected samples were used for quantitative identification of resveratrol by UV-visible absorption spectroscopy. The cumulative release (%) of resveratrol was calculated as follows:

[0150] Resveratrol release rate (%) = (cumulative release amount at a certain time) / (total amount encapsulated) × 100%.

[0151] Figure 18 Comparison of the sustained release of resveratrol from Pickering emulsions stabilized by different amphiphilic chitosan oligosaccharides. Figure 18 It can be seen that Pickering emulsion can encapsulate and slowly release oily active ingredients (such as resveratrol), with rapid release in the first two hours, reaching a release rate of 27%, and sustained release over 24 hours, with a maximum release rate of approximately 45%. Based on the Pickering emulsion's immediate efficacy and long-lasting protection, it is possible to design enhanced and daytime protection and nighttime repair products for anti-wrinkle, antioxidant, whitening, spot-lightening, sunscreen, and repair skincare.

[0152] Determination of free radical scavenging ability of Pickering emulsion:

[0153] (1) 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) free radical scavenging ability

[0154] This experiment was conducted in a 96-well plate with a blank control group, a solvent control group, and a sample group. Three replicate wells were set for each group, with a volume of 270 μL per well.

[0155] Control group: The same volume of solvent was used instead of the test substance, and 90 μL of 0.1 mmol / L 1,1-diphenyl-2-trinitrophenylhydrazine solution was added to each well.

[0156] Sample group: Dissolve an appropriate amount of the test substance in 180 μL of deionized water, and add 90 μL of 0.1 mmol / L 1,1-diphenyl-2-trinitrophenylhydrazine solution to the reaction system so that the final concentrations of the test substance in the reaction system are 1 μg / mL, 5 μg / mL, 10 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL, respectively.

[0157] After mixing according to the proportion, place on a shaker in the dark for 15 minutes, and measure the absorbance at 520 nm with a microplate reader. The calculation formula for the scavenging rate of 1,1-diphenyl-2-trinitrophenylhydrazine free radical is: scavenging rate = (A0-A X ) / A0×100%;

[0158] A0 represents the absorbance of the control group, A x Represents the absorbance of the test substance group (OD 520 ).

[0159] (2) Hydroxyl radical scavenging ability

[0160] This experiment uses the salicylic acid method, in which the hydroxyl radicals produced by the Foton reaction react with salicylic acid. The product has a special absorption peak at 510nm. The absorbance intensity (OD value) of the substance is detected to evaluate the hydroxyl radical scavenging ability of the test substance.

[0161] The experimental groups were blank control group, background group, and test substance groups with different concentration gradients. The concentrations of the test substances were set to 1 μg / mL, 5 μg / mL, 10 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL, and the reaction system was 1.5 mL.

[0162] The calculation formula for hydroxyl radical scavenging rate is:

[0163] Clearance rate = (A0-(A x -A x0 )) / A0×100%;

[0164] A0 represents the absorbance of the blank control, A x Indicates the absorbance of the test substance group, A x0 Indicates the absorbance of the background group of the sample to be tested (OD 510 ).

[0165] (3) Superoxide radical scavenging ability

[0166] Under weak alkaline conditions, pyrogallol readily autoxidizes, producing a colored intermediate with a distinct absorption peak at 320 nm. By measuring changes in absorbance, the effect of the test substance on pyrogallol autoxidation can be assessed. If the test substance possesses antioxidant activity, the production of the colored intermediate decreases, causing a change in the solution's absorbance. This principle allows the antioxidant capacity of the test substance to be evaluated.

[0167] Zero adjustment solution: Place 600 μL of 0.1 M Tris hydrochloride buffer (pH = 8.2) and 600 μL of deionized water in a new centrifuge tube, mix well, and place in a 25°C water bath for 20 min.

[0168] Solution I: Take 600 μL of 0.1 M Tris hydrochloride buffer (pH = 8.2) solution and 580 μL of deionized water and mix them evenly. Add the sample and set up a blank control group (solvent group). The concentrations of the test substance in the experimental groups are 1 μg / mL, 5 μg / mL, 10 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL.

[0169] Solution II: 20 μL of 5 mM pyrogallol solution. Rapidly mix Solution I and Solution II at 37°C in a water bath for 10 minutes. Measure the absorbance (OD) of the solution at 325 nm. Record the OD values ​​five times, with the first measurement being marked as 0 minutes. Each measurement is recorded with a 1-minute interval. The formula for free radical scavenging rate is:

[0170] Clearance = ((A0-A x )) / (A0)×100%;

[0171] A0 represents the absorbance of the blank control, A x Indicates the absorbance of the test substance group.

[0172] Figure 19 The antioxidant effect diagram of the Pickering emulsion loaded with resveratrol stabilized by amphiphilic chitosan oligosaccharide, where (A) is the antioxidant rate to DPPH, (B) is the antioxidant rate to hydroxyl radicals, and (C) is the antioxidant rate to superoxide radicals. Figure 19 It can be seen that compared with the single resveratrol system, the Pickering emulsion loaded with resveratrol showed better antioxidant properties; the greater the amount of amphiphilic chitosan oligosaccharide in the Pickering emulsion, the better the antioxidant effect of the system; the scavenging effect on DPPH and hydroxyl free radicals was better than the scavenging effect on superoxide free radicals, with the maximum scavenging rate of DPPH and hydroxyl free radicals being 80%, and it can be used in anti-aging essences, creams, lotions, eye creams, facial masks and other products developed for photoaging and oxidative damage.

[0173] Photostability of Pickering emulsion after encapsulation of resveratrol:

[0174] The photostability of resveratrol was studied by encapsulating resveratrol in Pickering emulsions stabilized with an ethanolic solution of resveratrol and 1.5% or 1% amphiphilic chitosan oligosaccharide self-aggregates. The samples containing resveratrol were irradiated with 365 nm UV light for 4 hours. 15 mL of each sample was placed in a glass circular plate (6 cm diameter) 20 cm from the lamp. Samples were collected after 0.5, 1, 2, 3, and 4 hours of irradiation, and the trans-resveratrol content was quantitatively determined by UV-visible spectrophotometry.

[0175] Figure 20 Comparison of the photostability of Pickering emulsions loaded with resveratrol stabilized by different amphiphilic chitosan oligosaccharides. Figure 20 It can be seen that Pickering emulsion has good light stability and can effectively protect resveratrol for a long time.

[0176] Skin retention assay using confocal fluorescence scanning microscopy (CLSM):

[0177] The retention of samples in the abdominal skin of 5-week-old mice was assessed using Nile Red as a fluorescent probe to observe their retention and distribution within the skin layers. This technique has become a well-established method for monitoring the distribution of hydrophobic drugs across different skin layers. A Pickering emulsion formulation was prepared using 0.01% w / v Nile Red according to the above-described procedures. A control sample was prepared by dissolving an equal amount of Nile Red in olive oil. 500 μL of Pickering emulsion (or control sample) was applied to the abdomen of the mouse. Six hours later, the skin samples were removed and thoroughly washed with deionized water and phosphate buffered saline to remove any excess, undiffused Nile Red. The skin samples were placed in tissue freezing medium and frozen at -20°C overnight. The next day, the samples were cryosectioned using a cryostat at 80 μm thickness. The sections were covered with a small amount of mounting medium and a cover glass and observed using a confocal microscope with an excitation wavelength of 561 nm and emission wavelengths of 570 nm and 691 nm.

[0178] Depend on Figure 14As can be seen from Figures A and B, a large amount of red dye in the Pickering emulsion stabilized by amphiphilic chitosan oligosaccharide penetrates into the skin, and the red area is much larger than that of the chitosan oligosaccharide / ethanol solution system. Moreover, the red color is more evenly distributed, indicating that the Pickering emulsion stabilized by amphiphilic chitosan oligosaccharide has a stronger transdermal effect. The software of the fluorescence confocal scanning microscope calculated that the depth of the dye in the Pickering emulsion in the sliced ​​skin is 55 μm, which is deeper than the depth of the dye in the chitosan oligosaccharide / ethanol system penetrating into the skin, further proving that amphiphilic chitosan oligosaccharide has the ability to promote penetration.

[0179] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. An application of an amphiphilic chitosan oligosaccharide in the preparation of skin care cosmetics, characterized in that: The structural formula of the amphiphilic chitosan oligosaccharide is shown in Formula I: Formula I; wherein m and n are both positive integers, and m+n is between 3 and 30; -N=R is a Schiff base structure formed by the reaction of an amino group and an aldehyde, and the aldehyde is citral; The aggregate particle size of the amphiphilic chitosan oligosaccharide is 200-700 nm; The degree of substitution of the amphiphilic chitosan oligosaccharide is 19.9-80%.

2. The use according to claim 1, characterized in that: The skin care cosmetics include lotion, moisturizing cream, facial mask, toner, lipstick, sunscreen, pressed powder, makeup remover oil, cleanser, shower gel or shampoo.

3. The use according to claim 1, characterized in that: The citral is trans-citral and / or cis-citral, and the content of trans-citral in the amphiphilic chitosan oligosaccharide is greater than 50%.

4. The use according to claim 1, characterized in that: The molecular weight of the amphiphilic chitosan oligosaccharide before modification is less than 5000, and the degree of deacetylation is greater than 80%.

5. The amphiphilic chitosan oligosaccharide according to any one of claims 1 to 4, characterized in that: The structural formula of the amphiphilic chitosan oligosaccharide is shown in Formula I: Formula I; wherein m and n are both positive integers, and m+n is between 3 and 30; -N=R is a Schiff base structure formed by the reaction of an amino group and an aldehyde, and the aldehyde is citral; The aggregate particle size of the amphiphilic chitosan oligosaccharide is 200-700 nm; The degree of substitution of the amphiphilic chitosan oligosaccharide is 19.9-80%.

6. The amphiphilic chitosan oligosaccharide according to claim 5, wherein: The citral is trans-citral and / or cis-citral, and the content of trans-citral in the amphiphilic chitosan oligosaccharide is greater than 50%.

7. The amphiphilic chitosan oligosaccharide according to claim 5, wherein: The molecular weight of the amphiphilic chitosan oligosaccharide before modification is less than 5000, and the degree of deacetylation is greater than 80%.

8. The method for preparing the amphiphilic chitosan oligosaccharide according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: preparing a chitosan oligosaccharide aqueous solution and an aldehyde ethanol solution, and heating the solution to 30-60° C.; Gradually add the ethanol solution of aldehyde to the chitosan oligosaccharide aqueous solution, stirring continuously during the addition process. After the addition is complete, continue stirring for 1-6 hours to allow for sufficient reaction; After the reaction is completed, the reaction system is cooled and ethanol is removed by rotary evaporation to obtain a modified product; After the modified product is freeze-dried, sponge-like amphiphilic chitosan oligosaccharide is obtained.

9. The method for preparing amphiphilic chitosan oligosaccharide according to claim 8, wherein: The molar ratio of the aldehyde group of the aldehyde to the primary amino group in the chitosan oligosaccharide is 0.01-1:

1.

10. The method for preparing the amphiphilic chitosan oligosaccharide according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: mixing and grinding the aldehyde group of aldehyde and the primary amino group of chitosan oligosaccharide at a molar ratio of 0.01-1:1, applying a force of 5-20N during grinding for 2-15 minutes, and freeze-drying the modified product at -85°C to obtain amphiphilic chitosan oligosaccharide.

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