Tremella polysaccharide-chitosan oligosaccharide-collagen peptide nanocomposite as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of nano materials, and particularly relates to a tremella polysaccharide-chitosan oligosaccharide-collagen peptide nano compound as well as a preparation method and application thereof. The tremella polysaccharide-chitosan oligosaccharide-collagen peptide nano compound comprises a shell layer formed by tremella polysaccharide and chitosan oligosaccharide, and collagen peptide embedded by the shell layer, the mass ratio of the tremella polysaccharide to the chitosan oligosaccharide is (0.5-4): 1; the mass ratio of the collagen peptide to the chitosan oligosaccharide is (2-10): 1. The nano-composite disclosed by the invention is high in water solubility, good in dispersity, high in stability and easy to absorb and utilize by cells, so that the bioavailability of the tremella polysaccharide and the collagen peptide is remarkably improved, more biological functions are achieved, and the dual effects of a carrier and a medicine can be achieved when a medicine delivery carrier is prepared; meanwhile, the tremella polysaccharide-chitosan oligosaccharide-collagen peptide nano-composite has good repairing activity and UVB light damage resisting activity, and has wide application prospects in the aspect of development of natural skin care products.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials technology, specifically relating to a tremella polysaccharide-chitosan oligosaccharide-collagen peptide nanocomposite, its preparation method, and its application. Background Technology

[0002] Natural polysaccharides possess high biocompatibility, biodegradability, and excellent controlled-release properties, making them commonly used functional carriers in drug delivery systems. This provides a feasible approach for precise drug delivery and controlled release. This is primarily based on the following: polysaccharides are high-molecular-weight compounds that can bind to drugs through covalent or non-covalent bonds to form nanocomposites, thereby exerting nano-effects; and polysaccharides contain abundant active groups, making them easily chemically modified to improve their properties and better meet the requirements of constructing nano-drug delivery systems.

[0003] Tremella polysaccharide (TFP) is a type of macromolecular polysaccharide extracted from the fruiting body of Tremella fuciformis. It has effects such as damage repair, anti-aging, moisturizing, lowering blood sugar, enhancing human immunity, anti-cancer, and antioxidant properties, and has the potential to be developed into skin care products. However, Tremella polysaccharide is a macromolecular polysaccharide with poor water solubility, making it difficult to enter cells and limiting its biological activity.

[0004] Collagen peptides are a class of substances with a relative molecular mass of less than 10,000 Da, obtained from fresh animal tissues (including skin, bones, tendons, ligaments, and scales) rich in collagen through extraction and hydrolysis. Collagen peptides possess various biological activities, including antioxidant, anti-aging, damage repair, blood sugar lowering, blood pressure lowering, blood lipid lowering, and antifreeze activities. However, collagen peptides alone are highly photosensitized and easily oxidized upon exposure to ultraviolet light, leading to loss of activity and potentially causing discomfort such as skin redness, swelling, and itching. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a Tremella fuciformis polysaccharide-chitosan oligosaccharide-collagen peptide nanocomposite, its preparation method, and its application. This invention combines Tremella fuciformis polysaccharide, chitosan oligosaccharide, and collagen peptides to form a Tremella fuciformis polysaccharide-chitosan oligosaccharide-collagen peptide nanocomposite with small particle size, making it easy to enter cells and exert biological activity. The collagen peptides are embedded inside the composite, improving stability and avoiding discomfort symptoms such as skin redness and swelling.

[0006] This invention provides a tremella polysaccharide-chitosan oligosaccharide-collagen peptide nanocomposite, comprising a shell formed by tremella polysaccharide and chitosan oligosaccharide, and collagen peptides embedded in the shell; The mass ratio of the tremella polysaccharide to the chitosan oligosaccharide is 0.5~4:1; The mass ratio of collagen peptides to chitosan oligosaccharides is 2~10:1.

[0007] Preferably, the mass ratio of the tremella polysaccharide to the chitosan oligosaccharide is 1~3:1.

[0008] Preferably, the mass ratio of collagen peptides to chitosan oligosaccharides is 4-8:1.

[0009] Preferably, the collagen peptide is cod collagen peptide; the particle size of the tremella polysaccharide-chitosan oligosaccharide-collagen peptide nanocomposite is 400~600nm.

[0010] This invention also provides a method for preparing the above-described Tremella fuciformis polysaccharide-chitosan oligosaccharide-collagen peptide nanocomposite, comprising the following steps: A mixture of chitosan oligosaccharide and collagen peptides was added dropwise to a solution of Tremella fuciformis polysaccharide to obtain a suspension. The pH of the suspension was adjusted to 5.8-6.6, and then freeze-dried to obtain a tremella polysaccharide-chitosan oligosaccharide-collagen peptide nanocomplex.

[0011] Preferably, the method for preparing the mixture of chitosan oligosaccharide and collagen peptide includes the following steps: adding a collagen peptide solution dropwise to a chitosan oligosaccharide solution to obtain a mixture of chitosan oligosaccharide and collagen peptide.

[0012] Preferably, the concentration of the collagen peptide solution is 1~5 mg / mL.

[0013] Preferably, the dripping is carried out under stirring conditions, and the stirring rate is 300~700 r / min.

[0014] Preferably, the pH of the suspension is adjusted to 6.0~6.4.

[0015] The present invention also provides the application of the above-described Tremella polysaccharide-chitosan oligosaccharide-collagen peptide nanocomposite or the Tremella polysaccharide-chitosan oligosaccharide-collagen peptide nanocomposite obtained by the above preparation method in the preparation of cosmetics.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a tremella polysaccharide-chitosan oligosaccharide-collagen peptide nanocomposite, comprising a shell formed by tremella polysaccharide and chitosan oligosaccharide, and collagen peptides embedded in the shell; the mass ratio of tremella polysaccharide to chitosan oligosaccharide is 0.5~4:1; the mass ratio of collagen peptides to chitosan oligosaccharide is 2~10:1.

[0017] The amino groups in chitosan oligosaccharide molecules hydrolyze to form -NH3 + This leads to the chitosan oligosaccharide being electropositive in aqueous solution. The -NH2 and -OH groups on this chitosan oligosaccharide can bind to collagen peptides through grafting and chelation coordination reactions. At the same time, the carboxyl groups of the Tremella fuciformis polysaccharide molecule hydrolyze to form -COO.- It is electronegative. Therefore, chitosan oligosaccharide can be electrostatically attracted to collagen peptides and Tremella fuciformis polysaccharide. The cationic chitosan oligosaccharide and Tremella fuciformis polysaccharide are combined to form a nanocarrier via a polyelectrolyte assembly method, and collagen peptides are loaded within it, forming a polyelectrolyte nanocomposite (a composite system formed by weak interactions or chemical bonding of charged polymers through electrostatic interactions, hydrogen bonds, hydrophobic interactions, etc.). The nanocomposite of this invention has a small particle size, making it easy to enter cells and exert biological activity; the collagen peptides are embedded internally, improving stability and avoiding discomfort such as skin redness and swelling. The nanocomposite of this invention has high water solubility, good dispersibility, and high stability, making it easily absorbed and utilized by cells, thus significantly improving the bioavailability of Tremella fuciformis polysaccharide and collagen peptides, enabling them to exert more biological functions. In the preparation of drug delivery carriers, it can exert the dual effects of carrier and drug; at the same time, the Tremella fuciformis polysaccharide-chitosan oligosaccharide-collagen peptide nanocomposite has excellent repair activity and anti-UVB photodamage activity, showing broad application prospects in the development of natural skin care products. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The results show the effect of the initial pH of the COS solution on the particle size of TFP-COS NPs in Example 1. Figure 2 The results show the effect of the mass concentrations of TFP solution and COS solution on the particle size of TFP-COS NPs in Example 1. Figure 3 The results show the effect of the TFP to COS mass ratio on the particle size of TFP-COS NPs in Example 1. Figure 4 The results show the effect of stirring speed on the particle size of TFP-COS NPs in Example 1; Figure 5 This illustrates the effect of the CP to COS mass ratio on the encapsulation efficiency of TFP-COS-CP NPs in Example 2. Figure 6 The effect of CP solution mass concentration on the encapsulation efficiency of TFP-COS-CP NPs in Example 2; Figure 7 The effect of pH value of TFP-COS-CP NPs suspension on the encapsulation efficiency of TFP-COS-CP NPs in Example 2; Figure 8 FTIR images of different samples in Test Example 1; Figure 9 TEM images of TFP-COS NPs in Test Example 1; Figure 10 TEM images of TFP-COS-CP NPs in Test Example 1; Figure 11 The results show the UVB photodamage repair activity of TFP-COS-CP NPs in Test Example 1. Detailed Implementation

[0020] The present invention provides a tremella polysaccharide-chitosan oligosaccharide-collagen peptide nanocomposite (denoted as TFP-COS-CPNPs), comprising a shell formed by tremella polysaccharide and chitosan oligosaccharide, and collagen peptides embedded in the shell; The mass ratio of the tremella polysaccharide to the chitosan oligosaccharide is 0.5~4:1; The mass ratio of collagen peptides to chitosan oligosaccharides is 2~10:1.

[0021] In this invention, the collagen peptide is preferably cod collagen peptide.

[0022] In this invention, the preferred mass ratio of Tremella fuciformis polysaccharide (TFP) to chitosan oligosaccharide (COS) is 1 to 3:1, specifically 0.5:1, 1:1, 2:1, 3:1, or 4:1 in the embodiments of this invention. The TFP to COS mass ratio described in this invention results in a nanocomposite with a small particle size.

[0023] In this invention, the preferred mass ratio of collagen peptides (CP) to chitosan oligosaccharides (COS) is 4-8:1, specifically 2:1, 4:1, 6:1, 8:1, or 10:1 in the embodiments of this invention. The collagen peptide to chitosan oligosaccharide mass ratio described in this invention results in a nanocomposite with high encapsulation efficiency of the collagen peptides.

[0024] In this invention, the particle size of the Tremella polysaccharide-chitosan oligosaccharide-collagen peptide nanocomposite is preferably 400~600nm, specifically 437.5nm.

[0025] This invention provides a method for preparing the Tremella fuciformis polysaccharide-chitosan oligosaccharide-collagen peptide nanocomposite described in the above technical solution, comprising the following steps: A mixture of chitosan oligosaccharide and collagen peptides was added dropwise to a solution of Tremella fuciformis polysaccharide to obtain a suspension. The pH of the suspension was adjusted to 5.8-6.6, and then freeze-dried to obtain a tremella polysaccharide-chitosan oligosaccharide-collagen peptide nanocomplex.

[0026] Unless otherwise specified, all materials and equipment used in this invention are commercially available products in the field.

[0027] In this invention, a mixture of chitosan oligosaccharide and collagen peptides is added dropwise to a solution of Tremella fuciformis polysaccharide to obtain a suspension.

[0028] In this invention, the method for preparing the mixture of chitosan oligosaccharide and collagen peptide preferably includes the following steps: adding a collagen peptide solution dropwise to a chitosan oligosaccharide solution to obtain a mixture of chitosan oligosaccharide and collagen peptide.

[0029] In this invention, the concentration of the collagen peptide solution is preferably 1-5 mg / mL, specifically 1.0, 2.0, 3.0, 4.0, or 5.0 mg / mL. The concentration of the chitosan oligosaccharide solution is preferably 0.5-2.5 mg / mL, specifically 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, or 2.5 mg / mL. The volume ratio of the collagen peptide solution to the chitosan oligosaccharide solution is preferably 2-10:1, specifically 5:1. The dropping rate is preferably 60-120 drops / min, specifically 90 drops / min.

[0030] In this invention, the concentration of the solution of Tremella polysaccharide is preferably 0.5~2.5 mg / mL, specifically 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL or 2.5 mg / mL.

[0031] In this invention, the preparation method of the Tremella polysaccharide preferably includes the following steps: The fruiting body powder of Tremella fuciformis was mixed with water and extracted to obtain an extract; The extract was mixed with the first ethanol and subjected to first alcohol precipitation, followed by solid-liquid separation to obtain a purified solution. The purified liquid and the second ethanol were mixed for a second alcohol precipitation, followed by solid-liquid separation. The resulting solid was washed and dried to obtain the Tremella polysaccharide.

[0032] In this invention, the preferred ratio of the Tremella fuciformis fruiting body powder to water is 1g:70mL. The preferred extraction temperature is 100℃, the preferred extraction time is 2 hours, and the preferred number of extractions is 2. The extraction process preferably includes concentration, and the volume of the concentrated product is preferably half the volume before concentration.

[0033] In this invention, the volume fraction of ethanol in the system after mixing the extract and the first ethanol is preferably 50%. The temperature of the first ethanol precipitation is preferably 4°C, and the time is preferably 8 hours; the solid-liquid separation is preferably centrifugation, and the centrifugation speed is preferably 8000 r / min, and the time is preferably 20 minutes.

[0034] In this invention, the volume fraction of ethanol in the system after mixing the purified liquid and the second ethanol is preferably 70%. The temperature for the second alcohol precipitation is preferably 4°C, and the time is preferably 8 hours; the solid-liquid separation is preferably centrifugation, the centrifugation speed is preferably 8000 r / min, and the time is preferably 20 minutes. The washing reagent is preferably a 70% ethanol solution, and the washing is preferably performed twice; the drying is preferably freeze-drying, and the freeze-drying time is preferably 4 days.

[0035] In this invention, the dripping rate is preferably 70-100 drops / min, specifically 90 drops / min. The dripping is preferably carried out under stirring conditions, and the stirring rate is preferably 300-700 r / min, specifically 300 r / min, 400 r / min, 500 r / min, 600 r / min, or 700 r / min. After the dripping, it is preferable to continue stirring, and the continued stirring time is preferably 20-60 min, specifically 30 min.

[0036] After obtaining the suspension, the pH value of the suspension was adjusted to 5.8~6.6, and then freeze-dried to obtain a Tremella polysaccharide-chitosan oligosaccharide-collagen peptide nanocomplex.

[0037] In this invention, the pH value of the suspension is preferably adjusted to 6.0~6.4, and specifically 5.8, 6.0, 6.2, 6.4 or 6.6 in the embodiments of this invention.

[0038] In this invention, the freeze-drying is preferably vacuum freeze-drying, specifically: the material is pre-frozen at -20°C for 6 hours, then transferred to -80°C for overnight (24 hours); placed in a freeze dryer; vacuum degree 0.1~1 Pa, freeze-dried for more than 3 days until the adsorbed water inside the material is removed.

[0039] The present invention also provides the application of the above-described Tremella fuciformis polysaccharide-chitosan oligosaccharide-collagen peptide nanocomposite or the Tremella fuciformis polysaccharide-chitosan oligosaccharide-collagen peptide nanocomposite obtained by the above preparation method in the preparation of cosmetics.

[0040] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes the Tremella fuciformis polysaccharide-chitosan oligosaccharide-collagen peptide nanocomposite, its preparation method, and its application, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0041] In the embodiments or comparative examples of this invention, the raw materials used include: Tremella fruiting bodies (Fujian Shanhu Herbal Tremella Co., Ltd.), chitosan oligosaccharides (Shanghai Maclean Biochemical Technology Co., Ltd.), and cod collagen peptides (Beijing Jiuyan Technology Co., Ltd.).

[0042] Example 1 1. Extraction of Tremella polysaccharides: The dried Tremella fruiting bodies were pulverized into small particles. The pulverized fruiting bodies were then placed in hot water (100℃) at a ratio of 1:70 (g:mL) for 2 hours. The mixture was filtered to obtain a filtrate and a residue. The residue was then extracted again in hot water at a ratio of 1:70 (g:mL) for 2 hours. The filtrates from both filtrations were combined and concentrated to a ratio of 1:2 (v / v). Ethanol was added until the ethanol volume fraction reached 50%. The mixture was then allowed to stand at 4℃ for 8 hours for alcohol precipitation. Centrifuge at 000 r / min for 20 min, add ethanol to the supernatant until the ethanol volume fraction is 70%, place in a 4℃ refrigerator and let stand for ethanol precipitation for 8 h, centrifuge at 8000 r / min for 20 min, wash the precipitate twice with 70% ethanol solution, then dissolve it completely in distilled water, evaporate the alcohol in a water bath at 100℃ until there is no alcohol odor, freeze dry in a freeze dryer at -50℃ for 4 days to obtain Tremella polysaccharide (purity 72.07%, yield 14.27%).

[0043] The monosaccharide composition and molecular weight distribution of Tremella fuciformis polysaccharide were determined. The polysaccharide mainly consists of fucose, glucose, xylose, mannose, and glucuronic acid. The weight-average molecular weight is 8.098 × 10⁻⁶. 4 Da.

[0044] 2. Preparation of Tremella fuciformis polysaccharide-chitosan oligosaccharide nanocomposite (denoted as TFP-COS NPs) A certain mass of Tremella fuciformis polysaccharide and chitosan oligosaccharide were weighed and added to distilled water. To ensure complete dissolution, the mixture was placed under 200W ultrasonic treatment for 30 minutes, followed by high-pressure homogenization at 8000 rpm for 10 minutes and centrifugation at 12000 rpm for 15 minutes. The supernatant was collected to prepare Tremella fuciformis polysaccharide (TFP) and chitosan oligosaccharide (COS) solutions of a certain concentration. Under magnetic stirring, the COS solution was added dropwise to the TFP solution, and stirring was continued for 30 minutes to obtain a TFP-COS NPs suspension.

[0045] Using TFP-COS NPs particle size as the screening index, the effects of TFP to COS mass ratio, initial pH of COS solution, TFP mass concentration, and magnetic stirring speed on TFP-COS NPs particle size were investigated.

[0046] (1) Prepare chitosan oligosaccharide (COS) and tremella polysaccharide (TFP) solutions with a concentration of 1 mg / mL using distilled water. Adjust the pH of the COS solution to 5.8, 6.0, 6.2, 6.4, and 6.6 respectively using 0.5 mol / mL HCl or NaOH solution. Add the COS solution dropwise to the TFP solution at a mass ratio of 1:1 with tremella polysaccharide and chitosan oligosaccharide, and continue stirring for 30 min to obtain a TFP-COS NPs suspension.

[0047] Take 1.5 mL of TFP-COS NPs suspension and place it in a cuvette. Measure the particle size using a Malvern particle size analyzer. The test results are as follows: Figure 1 As shown in the figure and Table 1.

[0048] Table 1. Effect of initial pH of COS solution on particle size of TFP-COS NPs

[0049] Depend on Figure 1 As shown in Table 1, when the initial pH of the COS solution is between 5.8 and 6.6, the particle size of TFP-COS NPs first decreases and then increases. The particle size is smallest when the initial pH of the COS solution is 6.0.

[0050] (2) TFP-COS NP suspension was prepared according to method (1), the only difference being that the initial pH of the COS solution was 6.0, and the mass concentrations of TFP and COS solutions were 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, and 2.5 mg / mL, respectively (the mass ratio of Tremella fuciformis polysaccharide to chitosan oligosaccharide was 1:1). The particle size of TFP-COS NPs is as follows: Figure 2 As shown in Table 2.

[0051] Table 2. Effects of TFP and COS solution concentrations on TFP-COS NPs particle size.

[0052] Depend on Figure 2 As shown in Table 2, when the mass concentrations of TFP and COS solutions are between 0.5 and 2.5 mg / mL, the particle size of TFP-COS NPs first decreases and then increases with the concentration of TFP and COS solutions. At very low concentrations, the probability of intermolecular collisions decreases, primarily reflecting the particle size of TFP in the solution.

[0053] (3) TFP-COS NPs suspensions were prepared according to method (1). The concentrations of TFP and COS solutions were both 1 mg / mL, with the only difference being that the mass ratios of TFP to COS were 1:2, 1:1, 2:1, 3:1, and 4:1 (volume variation). The particle size of TFP-COS NPs is as follows: Figure 3 As shown in Table 3.

[0054] Table 3. Effect of TFP to COS mass ratio on TFP-COS NPs particle size.

[0055] Depend on Figure 3 As shown in Table 3, when the TFP to COS mass ratio is in the range of 1:2 to 4:1, the particle size of TFP-COS NPs first decreases and then increases. The particle size is smallest when the TFP to COS mass ratio is 2:1. Then, as the TFP to COS mass ratio increases, the particle size of TFP-COS NPs gradually increases.

[0056] (4) TFP-COS NPs suspension was prepared according to method (1), except that the mass ratio of TFP to COS was 2:1, and the magnetic stirring speeds were 300 r / min, 400 r / min, 500 r / min, 600 r / min, and 700 r / min, respectively. The particle size of TFP-COS NPs is as follows: Figure 4 As shown in Table 4.

[0057] Table 4. Effect of magnetic stirring speed on the particle size of TFP-COS NPs

[0058] Depend on Figure 4 As shown in Table 4, the particle size of TFP-COS NPs first decreases and then increases when the stirring speed is between 300 and 700 r / min. When the stirring speed is too low, the local concentration becomes too high, and TFP and COS are prone to aggregation, resulting in larger particle sizes. The particle size of TFP-COS NPs is smallest at 500 r / min.

[0059] Example 2 Preparation of Tremella fuciformis polysaccharide-chitosan oligosaccharide-cod collagen peptide nanocomplex (TFP-COS-CP NPs): A method for preparing Tremella fuciformis polysaccharide-chitosan oligosaccharide nanocomposites (initial pH of COS solution 6.0, mass concentrations of TFP and COS solutions 1.0 mg / mL, and mass ratio of TFP to COS 2:1) was used to prepare Tremella fuciformis polysaccharide-chitosan oligosaccharide-cod collagen peptide nanocomposites. The difference was that the collagen peptide solution was first gradually added dropwise to the chitosan oligosaccharide solution, and then the chitosan oligosaccharide-collagen peptide composite solution was gradually added dropwise to the Tremella fuciformis polysaccharide solution to obtain a TFP-COS-CP NPs suspension. The suspension was then freeze-dried to obtain the Tremella fuciformis polysaccharide-chitosan oligosaccharide-cod collagen peptide nanocomposites.

[0060] (1) Prepare solutions of Tremella fuciformis polysaccharide (TFP), chitosan oligosaccharide (COS), and collagen peptide (CP) with a concentration of 1 mg / mL using distilled water. Adjust the pH of the COS solution to 6.0 using 0.5 mol / mL HCl and NaOH solutions. Under magnetic stirring at 500 r / min, add the CP solution dropwise to the COS solution at mass ratios of 2:1, 4:1, 6:1, 8:1, and 10:1 (CP:COS), and continue stirring for 30 min. Then add the COS-CP solution dropwise to the TFP solution at a mass ratio of 2:1 (TFP:COS) to obtain a TFP-COS-CP NPs suspension. Adjust the pH of the TFP-COS-CP NPs suspension to 6.4 using 0.5 mol / mL HCl or NaOH solutions.

[0061] The encapsulation efficiency of TFP-COS-CP NPs was determined by high performance liquid chromatography, and the results are as follows: Figure 5 As shown in Table 5.

[0062] Table 5. Effect of CP to COS mass ratio on encapsulation efficiency of TFP-COS-CP NPs

[0063] Depend on Figure 5 As shown in Table 5, when the TFP to CP mass ratio is 2:1 to 10:1, the CP encapsulation efficiency first increases and then decreases, and the CP encapsulation efficiency reaches its maximum when the TFP to CP mass ratio is 6:1.

[0064] (2) TFP-COS-CP NPs suspension was prepared according to method (1), except that the mass ratio of TFP to CP was 6:1, the concentrations of the CP solution were 1.0, 2.0, 3.0, 4.0, and 5.0 mg / mL, and the concentrations of the other solutions were kept at 1.0 mg / mL. The volume was changed to make the mass ratio of TFP to CP 6:1. The encapsulation efficiency of TFP-COS-CP NPs is as follows: Figure 6 As shown in Table 6.

[0065] Table 6. Effect of CP solution concentration on the encapsulation efficiency of TFP-COS-CP NPs

[0066] Depend on Figure 6 As shown in Table 6, the CP encapsulation efficiency first increases and then decreases when the CP solution concentration is between 1.0 and 5.0 mg / mL. The encapsulation efficiency is the highest at 3.0 mg / mL.

[0067] (3) Prepare TFP-COS-CP NPs suspension according to method (2), the only difference being that the concentration of CP solution is 3.0 mg / mL. Adjust the pH of TFP-COS-CP NPs suspension to 5.8, 6.0, 6.2, 6.4, and 6.6 respectively. The encapsulation efficiency of TFP-COS-CP NPs is as follows: Figure 7 As shown in Table 7.

[0068] Table 7. Effect of pH value of TFP-COS-CP NPs suspension on encapsulation efficiency of TFP-COS-CP NPs

[0069] Depend on Figure 7 As shown in Table 7, the CP encapsulation efficiency first increases and then decreases when the solution pH is between 5.8 and 6.6. The CP encapsulation efficiency is the highest at pH 6.0.

[0070] Test Example 1: Testing and Characterization of the TFP-COS-CP NPs Complex The following test data are all TFP-COS NPs and TFP-COS-CP NPs under optimal conditions.

[0071] 1. Table 8 shows the particle size of different samples.

[0072] Table 8 Particle size of different samples

[0073] It can be seen that the particle size of the TFP-COS-CP NPs complex is much smaller than that of Tremella polysaccharide.

[0074] 2. Figure 8 FTIR images of different samples.

[0075] Depend on Figure 8 It can be seen that at 3200 cm -1 ~3500 cm -1 The relatively broad absorption peaks nearby correspond to the stretching vibrations of OH and NH, reflecting the presence of numerous hydroxyl and amino groups in TFP, COS, and TFP-COS NPs molecules; at 2800 cm⁻¹...-1 ~3000 cm -1 The nearby absorption peak is related to the stretching vibration of CH; 1000 cm⁻¹ -1 ~1200 cm -1 The absorption peak in this region is typically due to the stretching vibration of COC, reflecting the pyranose ring structure of the polysaccharide. In the TFP infrared spectrum, the peak is at 1602.98 cm⁻¹. -1 The absorption peaks correspond to the carboxylic ester (C=O) and carboxylate ion (COO) of glucuronic acid. - The vibration of 1408.21 cm -1 The absorption peak at 798.50 cm⁻¹ is related to the symmetric stretching vibration of the carboxyl group. -1 The nearby peaks indicate the presence of α-D-mannopyranose. The absorption peak positions vary slightly among different substances, which may be due to changes in the vibrational environment of CH caused by intermolecular interactions during the formation of the nanocomposite. Furthermore, the shapes of some peaks also differ; for example, the peaks of TFP and CP are relatively sharp in certain wavelength bands, while the peaks of TFP-COS-CP NPs are relatively broad, indicating that intermolecular interactions are enhanced and the molecular structure's order and spatial conformation change during the formation of the nanocomposite.

[0076] 3. Figure 9 and Figure 10 TEM images of TFP-COS NPs and TFP-COS-CP NPs, respectively.

[0077] Depend on Figure 9 and Figure 10 As can be seen, TFP exhibits a typical chain-like structure, while COS is spherical. After binding with the TFP polysaccharide chains, it forms spherical or subspherical shapes with relatively uniform size. TFP-COS-CP NPs are elliptical with a dense internal structure, indicating an effective interaction between TFP, COS, and CP. This tight binding helps stabilize the structure of the nanocomposite and better maintain its properties in practical applications. Simultaneously, the successful encapsulation of white spherical CP particles within the nanocomposite demonstrates the successful encapsulation of CP during the preparation of TFP-COS-CP NPs, ensuring its functionality in subsequent applications and preventing premature release or degradation when used as a carrier to deliver collagen peptides.

[0078] 4. Bioactivity (UVB photodamage repair activity) of the TFP-COS-CP NPs complex Add 180 μL of HSF cells in logarithmic growth phase to each well of a 96-well plate. Each well contains approximately 10 cells. 596-well plates seeded with cells were placed in a 37°C incubator containing 5% CO2 for 24 h. 20 μL of TFP-COS-CP NPs at different concentrations (200, 400, and 800 μg / mL) were added to the sample group, while 20 μL of PBS buffer was added to both the control and model groups. Except for the control group, the sample and model groups were subjected to a 48 mJ / cm² temperature range. 2 Cells were irradiated with UVB and then cultured in complete culture medium for 24 h. Cell viability was calculated using the MTT assay.

[0079] UVB (medium-wave ultraviolet) photodamage can significantly reduce the proliferative capacity of skin fibroblasts. DNA damage and oxidative stress can activate intracellular stress signaling pathways, leading to cell cycle arrest, preventing cells from entering the normal proliferation phase, and delaying the repair and renewal of skin tissue.

[0080] Results of the TFP-COS-CP NPs' activity in repairing UVB photodamage are as follows: Figure 11 As shown in Table 9.

[0081] Table 9. Results of UVB photodamage repair activity in each group (cell survival rate, %)

[0082] Depend on Figure 11 As shown in Table 9, the samples exhibited protective effects against UVB photodamage to human skin fibroblasts at all concentrations. The protective effect was particularly significant at medium to high concentrations (800 μg / mL). This is likely due to the nanoscale structure of TFP-COS-CP NPs, which, at high concentrations, increases the contact area with cells, allowing more active ingredients to bind to cell surface receptors and promoting cellular uptake of the protective components. Simultaneously, the nanocomposite provides more stable encapsulation of the components, preventing rapid degradation in the complex cellular environment.

[0083] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on the present invention without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A tremella polysaccharide-chitooligosaccharide-collagen peptide nanocomplex, characterized in that, A shell layer formed by tremella polysaccharide and chitooligosaccharide, and collagen peptide embedded by the shell layer; The mass ratio of the tremella polysaccharide and the chitooligosaccharide is 0.5-4:

1. The mass ratio of the collagen peptide and the chitooligosaccharide is 2-10:

1.

2. The tremella polysaccharide-chitosan oligosaccharide-collagen peptide nanocomplex of claim 1, characterized in that, The mass ratio of the tremella polysaccharide and the chitooligosaccharide is 1-3:

1.

3. The tremella polysaccharide-chitosan oligosaccharide-collagen peptide nanocomplex of claim 1 or 2, characterized in that, The mass ratio of the collagen peptide and the chitooligosaccharide is 4-8:

1. 4.The tremella polysaccharide-chitosan oligosaccharide-collagen peptide nanocomplex of claim 1, characterized in that, The collagen peptide is cod collagen peptide; and the particle size of the tremella polysaccharide-chitooligosaccharide-collagen peptide nanocomposite is 400-600 nm.

5. The method for preparing the silver ear polysaccharide-chitooligosaccharide-collagen peptide nanocomplex according to any one of claims 1-4, characterized in that, The method comprises the following steps: The mixture of chitooligosaccharide and collagen peptide is added dropwise into a solution of tremella polysaccharide to obtain a suspension; The pH value of the suspension is adjusted to 5.8-6.6, and freeze-drying is performed to obtain the tremella polysaccharide-chitooligosaccharide-collagen peptide nanocomposite.

6. The production method according to claim 5, wherein The preparation method of the mixture of chitooligosaccharide and collagen peptide comprises the following steps: collagen peptide solution is added dropwise into a chitooligosaccharide solution to obtain the mixture of chitooligosaccharide and collagen peptide.

7. The production method according to claim 6, wherein The concentration of the collagen peptide solution is 1-5 mg / mL.

8. The preparation method according to claim 5, characterized in that, The dropwise adding is performed under stirring, and the stirring rate is 300-700 r / min.

9. The production method according to claim 5 or 8, characterized by, The pH value of the suspension is adjusted to 6.0-6.

4.

10. The application of the tremella polysaccharide-chitooligosaccharide-collagen peptide nanocomposite of any one of claims 1-4 or the tremella polysaccharide-chitooligosaccharide-collagen peptide nanocomposite obtained by the preparation method of any one of claims 5-9 in the preparation of cosmetics.

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