Preparation method of anti-photoaging peach gum polysaccharide

Abstract

The invention discloses a preparation method of anti-photoaging peach gum polysaccharide, and belongs to the technical field of food. The peach gum is treated by two steps of hydrogen peroxide, the low-molecular-weight peach gum polysaccharide with good solubility is prepared, and the prepared peach gum polysaccharide has good UVB damage resistance, cell apoptosis inhibition, intracellular oxidation resistance and anti-aging effects. The preparation method of the low-molecular-weight peach gum polysaccharide provided by the invention provides support for further application of peach gum.

Description

Technical Field

[0001] The invention relates to a method for preparing anti-photoaging peach gum polysaccharide, belonging to the technical field of food. Background Art

[0002] Peach gum is a kind of gum that peach trees secrete to protect themselves after being damaged and subjected to environmental stress. The Compendium of Materia Medica states that "blackened acne can be cured with Bisheng ointment". Modern research has confirmed that it has antibacterial and skin wound healing effects. my country produces 220,000 tons of peach gum every year, and the resources are abundant. The polysaccharide content in peach gum is as high as 80%, and after treatment, its emulsification and stability are better than those of gum arabic and guar gum. Peach gum polysaccharide can help wounds of patients with burns and scalds heal, with a moisture absorption rate of 81.5%, and a moisturizing effect better than sodium alginate, glycerol, etc.

[0003] Raw peach gum is a crystal-like substance that is very hard and translucent, with a brownish-yellow color. At room temperature, raw peach gum has a high molecular weight and low solubility, which severely limits its application in industry. In order to promote the utilization of peach gum resources, it is necessary to hydrolyze the crude peach gum to obtain water-soluble peach gum products. So far, several hydrolysis technologies have been developed, including thermal hydrolysis, acid hydrolysis, alkaline hydrolysis, H 2 O 2 Hydrolysis, enzymatic hydrolysis and combined hydrolysis. Thermal hydrolysis is the use of high temperature hydrolysis to hydrolyze peach gum into peach gum polysaccharides. The technology is simple and does not require the addition of other substances; however, if the water temperature is too high, peach gum polysaccharides will be further degraded into furfural and 5-hydroxymethylfurfural. In addition, thermal hydrolysis also has disadvantages such as time-consuming, energy-consuming, poor product reproducibility and relatively low yield. Acid or alkaline hydrolysis refers to the hydrolysis of peach gum with the assistance of acid or alkali. Compared with thermal hydrolysis, acid or alkaline hydrolysis has the advantages of high hydrolysis efficiency, low energy consumption and stable product reproducibility; however, the acid or alkaline hydrolysis conditions are harsh, which not only increases the difficulty of process control, but also the purification and separation costs are high. Enzymatic hydrolysis has the advantages of fast hydrolysis rate, mild hydrolysis conditions and high yield, but also has some limitations. For example, the suitable temperature range and pH range of enzymatic hydrolysis are usually very narrow. In order to keep the enzyme activity at the optimal level during the enzymatic hydrolysis process, the temperature and pH value must be strictly controlled. Compared with other hydrolysis technologies, H 2 O 2 Hydrolysis has the advantages of high hydrolysis efficiency, good product appearance and low hydrolysis temperature.

[0004] However, the peach gum polysaccharide prepared by the above method is generally a high molecular weight peach gum polysaccharide. Although the high molecular weight peach gum polysaccharide also has high antioxidant activity, it has the problems of high viscosity and low water solubility, and is more difficult to absorb in the body than the low molecular weight peach gum polysaccharide. The present invention provides a preparation method of low molecular weight peach gum polysaccharide to provide support for the further application of peach gum. Summary of the invention

[0005] In order to solve at least one of the above problems, the present invention provides a method for preparing anti-photoaging peach gum polysaccharide, which adopts two steps of H 2 O 2 Low molecular weight peach gum polysaccharide was prepared by hydrolysis method.

[0006] The first object of the present invention is to provide a method for preparing anti-photoaging peach gum polysaccharide, comprising the following steps:

[0007] S1, soaking peach gum for 10-30 hours and then stirring and homogenizing to obtain peach gum suspension;

[0008] S2, adjust the pH of peach gum suspension to 9.0-10.0, add 4%-6% H 2 O 2 , stirring and reacting for 7 to 9 hours to obtain a crude peach gum polysaccharide solution;

[0009] S3, collect the intermediate products in the peach gum crude polysaccharide solution, dissolve it in water, adjust the pH to 9.5-10.5, add 5%-7% H 2 O 2 , stirring and reacting for 7 to 9 hours to obtain a low molecular weight peach gum polysaccharide solution, dialyzing and alcohol precipitation, and freeze-drying the precipitate to obtain the peach gum polysaccharide.

[0010] In one embodiment of the present invention, in step S1, the soaking is done in water at 50-60°C.

[0011] In one embodiment of the present invention, the ratio of peach gum to water is 1g:80-120mL.

[0012] In one embodiment of the present invention, in step S2 or step S3, the stirring reaction temperature is 50-60°C.

[0013] In one embodiment of the present invention, in step S2 or step S3, the rotation speed of the stirring reaction is 400-600 rpm.

[0014] In one embodiment of the present invention, in step S3, the intermediate product in the peach gum crude polysaccharide solution is collected by concentrating the peach gum crude polysaccharide solution to 15-25% of the original volume, then centrifuging at 3000-5000 rpm for 10-30 min, collecting the supernatant, and freeze-drying it after alcohol precipitation.

[0015] In one embodiment of the present invention, when the intermediate product is dissolved in water, the mass ratio of the intermediate product to water is 1:80-120.

[0016] In one embodiment of the present invention, in step S3, the dialysis is performed at 20-30° C. for 40-50 h.

[0017] In one embodiment of the present invention, in step S3, the alcohol precipitation is performed by adding 3 to 5 times of anhydrous ethanol and standing at 0 to 10° C. for 8 to 12 hours to obtain a precipitate.

[0018] The second object of the present invention is to provide peach gum polysaccharide prepared by the method.

[0019] The third object of the present invention is to provide the application of the peach gum polysaccharide in the preparation of food, cosmetics and health products.

[0020] Beneficial Effects

[0021] The invention adopts two-step hydrogen peroxide treatment to peach gum to prepare low-molecular-weight peach gum polysaccharide with good solubility. The peach gum polysaccharide prepared by the invention has good effects of resisting UVB damage, inhibiting cell apoptosis, intracellular anti-oxidation and anti-aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a diagram showing the effect of peach gum polysaccharide on cells' resistance to UVB damage;

[0023] Figure 2 This is a diagram showing the effect of peach gum polysaccharide in inhibiting cell apoptosis;

[0024] Figure 3 This is the effect diagram of peach gum polysaccharide in reducing intracellular ROS;

[0025] Figure 4 This is the effect diagram of peach gum polysaccharide retaining intracellular SOD;

[0026] Figure 5 This is a diagram showing the effect of peach gum polysaccharide in inhibiting cell aging. DETAILED DESCRIPTION

[0027] The detection methods involved in the following embodiments are as follows:

[0028] 1. Solubility

[0029] Dissolve 5 g of peach gum polysaccharide sample in 10 mL of pure water, transfer to a glass centrifuge tube with a known constant weight Wb, centrifuge at 3000 r / min for 20 min, and dry the supernatant at 105°C to a constant weight Wh.

[0030] Solubility = (Wh-Wb) / 5

[0031] 2. Molecular weight

[0032] Laser light scattering can accurately determine the molecular weight and conformation of peach gum. The SEC-MALLS-RI-VISC system includes a high performance liquid chromatograph (LC-20, SHIMADZU, Japan), a guard column (OHpak SB-G, Shodex, Japan), a size exclusion chromatography column (SB-806HQ and SB-804HQ column, 7.8mm×300mm, Shodex, Japan), a multi-angle light scattering instrument (DAWNHELEOS II, λ0=660nm, Wyatt Technology, USA), a viscosity detector (ViscoStar III, Wyatt Technology, USA), and a differential detector (RID-20A, SHIMADIU, Japan).

[0033] 3.Chicken embryo chorioallantoic membrane test

[0034] The experimental method was carried out in accordance with the "Chicken Embryo Chorioallantoic Membrane Test for Eye Irritation / Corrosion of Cosmetics (SN / T 2329-2009)". CAM was prepared from 9-day-old chicken embryos. Incubation conditions: temperature (37.5±0.5)℃, relative humidity 45%RH~70%RH. First, use an egg candling device to check the eggs, observe the starting position, and mark it with a marker; use serrated curved tweezers to knock the eggshell at the air chamber position, and gently peel it off according to the mark to expose the white egg membrane. Careful operation should be performed throughout the process to ensure that the integrity of the egg membrane is not damaged. Use a pipette to drop the negative control (0.9% NaCl) solution and the peach gum polysaccharide solution of each group with a concentration of 1.2mg / mL to soak the egg membrane, and then carefully pour out the solution. Use tweezers to peel off the white egg membrane to ensure that the blood vessels are not damaged. Observe the growth of blood vessels, select chicken embryos with good blood vessel growth for the experiment, and discard those that cannot be used for the experiment.

[0035] 4. UVB-damaged HaCaT model

[0036] Human epidermal keratinocytes (HaCaT) were cultured in DMEM (GIBCO) supplemented with 10% (v / v) fetal bovine serum (FBS; GIBCO) and 1% penicillin-streptomycin. The cells were incubated at 37°C and 5% CO in a humidified atmosphere (HF 90, Heal Force). 2 To evaluate cell viability, HaCaT cells were cultured at 1 × 10 4Cells / well were seeded in a 96-well plate and incubated for 12 h. UVB light source (30 W, UVB313-EL, Guanhongya Optoelectronics Technology Co., Ltd., Shenzhen, China) with an emission wavelength of 313 nm was irradiated at a distance of 30 cm for 20 s, 50 s, 100 s, and 200 s. PBS was replaced with fresh medium and incubated overnight. Cell viability was detected by CCK-8 method to determine the experimental conditions for subsequent studies.

[0037] 5. Apoptosis

[0038] The apoptosis of UVB-damaged HaCaT cells was analyzed using the Annexin V-FITC apoptosis kit (C1062M, Beyotime). The cells were incubated in the dark for 20 minutes, and apoptosis was analyzed by flow cytometry (FACSCalibur, BD). After pretreatment and UVB irradiation, the cell cycle distribution was determined by propidium iodide staining, and red fluorescence and light scattering were detected at 488 nm.

[0039] 6. Intracellular Antioxidant

[0040] SOD activity and ROS levels in UVB-exposed HaCaT keratinocytes were measured using detection kits (S0101, S0052, S0033, Beyotime) and the relevant manufacturer's instructions. The absorbance of each solution was measured at 450 nm and 412 nm using a microplate reader to detect SOD activity. The flow cytometer was set to an excitation wavelength of 495 nm and an emission wavelength of 530 nm to detect the CM-DCF signal intensity indicating ROS levels.

[0041] 7. Immunoblotting

[0042] For in vitro immunoblotting, after incubation of HaCaT cells, the cells were washed three times with PBS and lysed. After centrifugation at 11000×g for 10 min at 4°C, the crude protein solution was obtained. The protein concentration was detected using the BCA protein assay kit (P0010, Beyotime). The harvested proteins were separated by electrophoresis on SDS-PAGE gel and transferred to NC membrane (HATF00010, Millipore). The membrane was blocked in TBST buffer containing 5% skim milk for 1 h at room temperature. Then, specific primary antibodies (P21, P16, P53, β-actin) were incubated with the membrane overnight at 4°C. The membrane was washed three times in TBST buffer and incubated with the secondary antibody dilution solution at room temperature for 1 h. Then, the washed membrane was treated with electrochemiluminescence (ECL) solution. The protein bands were visualized and quantified using the “Image J” software.

[0043] Embodiment 1:

[0044] Take 50g peach gum and soak it in 5L distilled water at 55℃ for 24h, then suspend it in a blender. Adjust the pH of the peach gum suspension to 9.5 with 2M NaOH, add 5% H 2 O 2 (v / v). Stir at 55°C and 500 rpm for 8 h. Concentrate the peach gum polysaccharide solution to 20% of the original volume, centrifuge at 4000 rpm for 20 min, collect the supernatant and lyophilize it to obtain the intermediate product powder. Dissolve the obtained powder in distilled water at a mass ratio of 1:100, adjust the pH to 10 with NaOH, add 6% H 2 O 2 (v / v), stirred at 55°C and 500 rpm for 8 h, dialyzed at room temperature for 48 h, added 4 times the volume of anhydrous ethanol, allowed to stand at 4°C for 10 h, concentrated and freeze-dried to obtain peach gum polysaccharide PGP-1.

[0045] Embodiment 2:

[0046] Take 50g of peach gum and soak it in 5L of distilled water at 55℃ for 24h, then suspend it in a blender. Adjust the pH of the peach gum suspension to 9.5 with 2M NaOH, add 4% H 2 O 2 (v / v). Stir at 55°C and 500 rpm for 9 h. Concentrate the peach gum polysaccharide solution to 20% of the original volume, centrifuge at 4000 rpm for 20 min, collect the supernatant and lyophilize it to obtain the intermediate product powder. Dissolve the obtained powder in distilled water at a mass ratio of 1:100, adjust the pH to 10 with NaOH, add 6% H 2 O 2 (v / v), stirred at 55°C and 500 rpm for 8 h, dialyzed at room temperature for 48 h, added 4 times the volume of anhydrous ethanol, stood at 4°C for 10 h, concentrated and freeze-dried to obtain peach gum polysaccharide PGP-1-2.

[0047] Embodiment 3:

[0048] Take 50g peach gum and soak it in 5L distilled water at 55℃ for 24h, then suspend it in a blender. Adjust the pH of the peach gum suspension to 9.5 with 2M NaOH, add 5% H 2 O 2 (v / v). Stir at 55°C and 500 rpm for 8 h. Concentrate the peach gum polysaccharide solution to 20% of the original volume, centrifuge at 4000 rpm for 20 min, collect the supernatant and lyophilize it to obtain the intermediate product powder. Dissolve the obtained powder in distilled water at a mass ratio of 1:100, adjust the pH to 10 with NaOH, add 7% H 2 O 2(v / v), stirred at 55°C and 500 rpm for 7 h, dialyzed at room temperature for 48 h, added 4 times the volume of anhydrous ethanol, stood at 4°C for 10 h, concentrated and freeze-dried to obtain peach gum polysaccharide PGP-1-3.

[0049] Comparative Example 1:

[0050] Take 50g of peach gum and soak it in 5L of distilled water at 55℃ for 24h, then suspend it in a blender. Adjust the pH of the peach gum suspension to 9.5 with 2M NaOH, add 3% H 2 O 2 (v / v). Stir at 55°C and 500 rpm for 8 h. Concentrate the peach gum polysaccharide solution to 20% of the original volume, centrifuge at 4000 rpm for 20 min, collect the supernatant and lyophilize it to obtain the intermediate product powder. Dissolve the obtained powder in distilled water at a mass ratio of 1:100, adjust the pH to 10 with NaOH, add 6% H 2 O 2 (v / v), stirred at 55°C and 500 rpm for 8 h, dialyzed at room temperature for 48 h. Anhydrous ethanol was added at a ratio of 4:1, allowed to stand at 4°C for 10 h, concentrated and freeze-dried to obtain peach gum polysaccharide PGP-2.

[0051] Comparative Example 2:

[0052] Take 50g peach gum and soak it in 5L distilled water at 55℃ for 24h, then suspend it in a blender. Adjust the pH of the peach gum suspension to 9.5 with 2M NaOH, add 5% H 2 O 2 (v / v). Stir at 55°C and 500 rpm for 5 h. Concentrate the peach gum polysaccharide solution to 20% of the original volume, centrifuge at 4000 rpm for 20 min, collect the supernatant and lyophilize it to obtain the intermediate product powder. Dissolve the obtained powder in distilled water at a mass ratio of 1:100, adjust the pH to 10 with NaOH, add 6% H 2 O 2 (v / v), stirred at 55°C and 500 rpm for 8 h, dialyzed at room temperature for 48 h, added 4 times the volume of anhydrous ethanol, allowed to stand at 4°C for 10 h, concentrated and freeze-dried to obtain peach gum polysaccharide PGP-3.

[0053] Comparative Example 3:

[0054] Take 50g of peach gum and soak it in 5L of distilled water at 55℃ for 24h, then suspend it in a blender. Adjust the pH of the peach gum suspension to 8 with 2M NaOH, add 5% H 2 O 2(v / v). Stir at 55°C and 500 rpm for 8 h. Concentrate the peach gum polysaccharide solution to 20% of the original volume, centrifuge at 4000 rpm for 20 min, collect the supernatant and lyophilize it to obtain the intermediate product powder. Dissolve the obtained powder in distilled water at a ratio of 1:100, adjust the NaOH to 10, add 6% H 2 O 2 (v / v), stirred at 55°C and 500 rpm for 8 h, dialyzed at room temperature for 48 h, added 4 times the volume of anhydrous ethanol, allowed to stand at 4°C for 10 h, concentrated and freeze-dried to obtain peach gum polysaccharide PGP-4.

[0055] Comparative Example 4:

[0056] Take 50g peach gum and soak it in 5L distilled water at 55℃ for 24h, then suspend it in a blender. Adjust the pH of the peach gum suspension to 13 with 2M NaOH, add 8% H 2 O 2 (v / v). Stir at 55°C and 500 rpm for 10 h. Concentrate the peach gum polysaccharide solution to 20% of the original volume, dialyze at room temperature for 48 h. Add 4 times the volume of anhydrous ethanol, let stand at 4°C for 10 h, concentrate and freeze-dry to obtain peach gum polysaccharide PGP-5.

[0057] Comparative Example 5:

[0058] Take 50g peach gum and soak it in 5L distilled water at 55℃ for 24h, then suspend it in a blender. Adjust the pH of the peach gum suspension to 9.5 with 2M NaOH, add 5% H 2 O 2 (v / v). Stir at 55°C and 500 rpm for 8 h. Concentrate the peach gum polysaccharide solution to 20% of the original volume, centrifuge at 4000 rpm for 20 min, collect the supernatant and lyophilize it to obtain the intermediate product powder. Dissolve the obtained powder in distilled water at a mass ratio of 1:100, adjust the pH to 10 with NaOH, add 8% H 2 O 2 (v / v), stirred at 55°C and 500 rpm for 8 h, dialyzed at room temperature for 48 h, added 4 times the volume of anhydrous ethanol, allowed to stand at 4°C for 10 h, concentrated and freeze-dried to obtain peach gum polysaccharide PGP-6.

[0059] Comparative Example 6:

[0060] Take 50g peach gum and soak it in 5L distilled water at 55℃ for 24h, then suspend it in a blender. Stir it at 95℃ and 500rpm for 2h. Centrifuge it at 4000rpm for 20min, collect the supernatant and repeat twice. Add 4 times the volume of anhydrous ethanol, let it stand at 4℃ for 10h, then concentrate and freeze-dry to obtain water-extracted peach gum polysaccharide PGP-7.

[0061] Comparative Example 7:

[0062] Take 50g peach gum and soak it in 5L distilled water at 55℃ for 24h, then suspend it in a blender. Adjust the pH of the peach gum suspension to 13 with 2M NaOH, add 5% H 2 O 2 (v / v). Stir at 55°C and 500 rpm for 8 hours. Concentrate the peach gum polysaccharide solution to 20% of the original volume, centrifuge at 4000 rpm for 20 minutes, collect the supernatant and lyophilize it to obtain peach gum polysaccharide PGP-8.

[0063] Result test example:

[0064] 1. Solubility

[0065] The solubility of the peach gum polysaccharides of Examples 1 to 3 and Comparative Examples 1 to 7 was tested, and the results are shown in Table 1.

[0066] Table 1. Solubility of peach gum polysaccharide

[0067]

[0068] The results in Table 1 show that the peach gum polysaccharides prepared in Examples 1 to 3 of the present invention have good water solubility. In addition, the peach gum polysaccharide prepared in Comparative Example 5 has the highest water solubility.

[0069] 2. Molecular weight

[0070] The molecular weight of the peach gum polysaccharides of Examples 1 to 3 and Comparative Examples 1 to 7 was measured, and the results are shown in Table 2.

[0071] Table 2. Molecular weight of peach gum polysaccharide

[0072]

[0073] The results in Table 2 show that the peach gum polysaccharides prepared in Examples 1 to 3 of the present invention have a relatively low molecular weight. In addition, the peach gum polysaccharide prepared in Comparative Example 5 has the lowest molecular weight. The molecular weight result corresponds to the solubility result. In the present invention, the free radicals formed by hydrogen peroxide are used to attack the glycosidic bond, resulting in the cleavage of the glycosidic bond. The present invention adopts secondary hydrogen peroxide treatment to more effectively reduce the molecular weight. It can be seen from the results of Example 1 and Comparative Examples 1 to 3 that during the hydrogen peroxide treatment process, the hydrogen peroxide concentration, reaction time and the pH value of the system during the reaction have a great influence on the effect of the reaction.

[0074] 3.Chicken embryo chorioallantoic membrane test

[0075] The peach gum polysaccharides prepared in Example 1 and Comparative Examples 1 to 7 were tested on chicken embryo chorioallantoic membrane. The results are shown in Table 3.

[0076] Table 3. Evaluation of the irritation of peach gum polysaccharide

[0077]

[0078] According to the solubility of peach gum polysaccharide and the daily usage concentration in the formula, a relatively high concentration solution was prepared, and the peach gum polysaccharide prepared in the examples of the present invention and the comparative examples was tested for safety at high concentrations, and the results showed that they were non-irritating.

[0079] 4. UVB-damaged HaCaT model

[0080] The peach gum polysaccharides prepared in Example 1 and Comparative Examples 1 to 7 were tested for anti-UVB damage, and CCK-8 was used to evaluate the effects of the eight peach gum polysaccharides on the proliferation rate of HaCaT cells. The initial cell seeding density was 1×10 4 Cells were maintained at 37°C and 5% CO 2 The cells were cultured well under the following conditions. The control group (CG) had no UVB irradiation or any treatment. The model group (MG) had the culture medium removed, washed 3 times with PBS, and then irradiated with UVB for 100s at a distance of 30cm after adding 100μL PBS. The PGP-1, 2, 3, 4, 5, 6, 7, and 8 groups were cultured for 24h after adding 1.5mg / ml peach gum polysaccharide to the culture medium before UVB irradiation, then the culture medium was removed, washed 3 times with PBS, and then irradiated with UVB for 100s at a distance of 30cm after adding 100μL PBS to each well. CCK-8 was used to detect cell proliferation, and the absorbance at 450nm was measured using a microplate reader (Multiskan GO, Thermo). The results are shown in Figure 1 It can be seen that the peach gum polysaccharide prepared in Example 1 can effectively inhibit the damage of cells under UVB irradiation.

[0081] 5. Apoptosis

[0082] The peach gum polysaccharide prepared in Example 1 and Comparative Examples 1 to 7 was subjected to a cell apoptosis experiment, wherein the control group (CG, no UVB irradiation or any treatment), the model group (MG, the culture medium was removed, PBS was washed three times, and 100 μL PBS was added before UVB irradiation for 100 s at a distance of 30 cm) and 8 experimental groups (PGP-1, 2, 3, 4, 5, 6, 7, 8, 1.5 mg / ml peach gum polysaccharide was added to the culture medium before UVB irradiation for 24 hours, then the culture medium was removed, PBS was washed three times, 100 μL PBS was added to each well, and UVB irradiation was performed at a distance of 30 cm for 100 s). After the irradiation, PBS was removed, and fresh complete culture medium was added to each well. After continuing to culture overnight, the relevant indicators were measured. The results are shown in FIG. Figure 2 As shown in the results, it can be seen that the peach gum polysaccharide prepared in Example 1 can effectively inhibit cell apoptosis.

[0083] 6. Intracellular Antioxidant

[0084] The peach gum polysaccharides prepared in Example 1 and Comparative Examples 1 to 7 were subjected to intracellular antioxidant tests. Human epidermal keratinocytes (HaCaT) were cultured in DMEM (GIBCO) containing 10% (volume ratio) fetal bovine serum (FBS; GIBCO) and 1% penicillin-streptomycin. The culture conditions followed the protocol described by Amal D. Premarathna et al. (2024). The cell line was maintained at 37°C and 5% CO 2 The cells were cultured in a humidified environment. The control group (CG, no UVB irradiation or any treatment), the model group (MG, the culture medium was aspirated, the cells were washed 3 times with PBS, and then 100 μL PBS was added and UVB irradiated for 100 s at a distance of 30 cm) and 8 experimental groups (PGP-1, 2, 3, 4, 5, 6, 7, 8, 1.5 mg / ml peach gum polysaccharide was added to the culture medium before UVB irradiation and cultured for 24 hours, then the culture medium was aspirated, the cells were washed 3 times with PBS, and 100 μL PBS was added to each well and UVB irradiated for 100 s at a distance of 30 cm) were added. After the irradiation, the PBS was aspirated, and fresh complete culture medium was added to each well. The cells were cultured overnight and the relevant indicators were measured. The results are shown in Figure 3 and Figure 4 shown.

[0085] Among them, ROS induces oxidative stress, resulting in redox imbalance and irreversible oxidative modification of macromolecules, leading to mitochondrial apoptosis. Figure 3 The results showed that PGP-1 could alleviate UVB-induced oxidative stress.

[0086] SOD has strong free radical scavenging ability and is a key enzyme in defending against UVB-induced oxidative stress. Figure 4 The results showed that PGP-1 could effectively retain SOD.

[0087] 7. Immunoblotting

[0088] Cell cycle arrest is a key feature of cellular senescence; cell cycle regulators such as p16, p21, and p53 are often used to detect senescent cells. p16 can inhibit the progression of the G1 / S cell cycle transition, thereby maintaining normal regulation of cell proliferation and homeostasis. p53 is a protein that regulates cellular senescence and also plays an important role in DNA damage and cellular stress. For example, p53 can promote cell cycle arrest, DNA repair, and apoptosis. P21 can be activated by p53 and plays a role in regulating the cell cycle and stopping or slowing cell proliferation. The present invention sets up a control group (CG, without UVB irradiation or any treatment), a model group (MG, the culture medium is aspirated, PBS is washed three times, and 100 μL PBS is added before UVB irradiation for 100 s at a distance of 30 cm) and 8 experimental groups (PGP-1, 2, 3, 4, 5, 6, 7, 8, 1.5 mg / ml peach gum polysaccharide is added to the culture medium before UVB irradiation for 24 hours, then the culture medium is aspirated, PBS is washed three times, 100 μL PBS is added to each well, and UVB irradiation is performed at a distance of 30 cm for 100 s). After the irradiation, PBS is aspirated, and fresh complete culture medium is added to each well. After continuing to culture overnight, relevant indicators are measured. In the UVB-induced HaCaT cell aging model, the expression of p16, p21 and p53 is significantly increased. Figure 5 As shown, the PGP-1 group can significantly reduce the expression of P16, 21, and 53, thereby inhibiting the senescence of HaCaT cells.

[0089] According to the comparison of the biological activity detection effects of the low molecular weight peach gum polysaccharides prepared in Example 1 of the present invention and Comparative Example 5, although they also have certain effects of anti-UVB damage, inhibition of cell apoptosis, intracellular antioxidant and anti-aging, they are not as good as the peach gum polysaccharide prepared in Example 1. It can be seen that further reducing the molecular weight does not further improve its biological activity, but rather decreases it to a certain extent.

[0090] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing anti-photoaging peach gum polysaccharide, characterized in that: The following steps are involved: S1, soaking peach gum for 10-30 hours and then stirring and homogenizing to obtain peach gum suspension; S2, adjusting the pH of the peach gum suspension to 9.0-10.0, adding 4%-6% H2O2 by volume, stirring and reacting for 7-9 hours to obtain a peach gum crude polysaccharide solution; S3, collecting the intermediate products in the crude peach gum polysaccharide solution, dissolving them in water, adjusting the pH to 9.5-10.5, adding 5%-7% H2O2 by volume, stirring and reacting for 7-9 hours to obtain a low molecular weight peach gum polysaccharide solution, dialyzing and precipitating with alcohol, and freeze-drying the precipitate to obtain the peach gum polysaccharide.

2. The preparation method according to claim 1, characterized in that: In step S1, the immersion is carried out in water at 50 to 60°C.

3. The preparation method according to claim 1, characterized in that: The ratio of peach gum to water is 1g:80~120mL.

4. The preparation method according to claim 1, characterized in that: In step S2 or step S3, the temperature of the stirring reaction is 50-60° C.; the rotation speed of the stirring reaction is 400-600 rpm.

5. The preparation method according to claim 1, characterized in that: In step S3, the intermediate product in the peach gum crude polysaccharide solution is collected by concentrating the peach gum crude polysaccharide solution to 15-25% of the original volume, then centrifuging at 3000-5000 rpm for 10-30 min, collecting the supernatant, and freeze-drying after alcohol precipitation.

6. The preparation method according to claim 1, characterized in that: When the intermediate product is dissolved in water, the mass ratio of the intermediate product to water is 1:80-120.

7. The preparation method according to claim 1, characterized in that: In step S3, dialysis is performed at 20 to 30°C for 40 to 50 hours.

8. The preparation method according to claim 1, characterized in that: In step S3, the alcohol precipitation is performed by adding 3 to 5 times of anhydrous ethanol and standing at 0 to 10° C. for 8 to 12 hours to obtain a precipitate.

9. Peach gum polysaccharide prepared by the method according to any one of claims 1 to 8.

10. Use of the peach gum polysaccharide according to claim 9 in the preparation of food, cosmetics and health products.

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