CN116803368B - Tremella aurantialba polysaccharide degradation product and application thereof in protecting skin cells from thermal injury - Google Patents

Abstract

The invention discloses a tremella aurantialba polysaccharide degradation product, which is prepared by degrading tremella aurantialba polysaccharide through a method of combining H ₂O₂ with vitamin C and selecting degradation products with molecular weight of more than 100k Da and 1k Da and molecular weight of less than 100k Da. The biological utilization rate of the tremella aurantialba polysaccharide can be greatly improved by effectively degrading the tremella aurantialba polysaccharide, and the tremella aurantialba polysaccharide degradation product I (E-TAP I) and the tremella aurantialba polysaccharide degradation product II (E-TAP II) have good application prospects in the aspect of repairing skin thermal injury.

Description

Tremella aurantialba polysaccharide degradation product and application thereof in protecting skin cells from thermal injury

Technical Field

The invention relates to a polysaccharide degradation product and application thereof, in particular to a tremella aurantialba polysaccharide degradation product and application thereof in protecting skin cells from thermal injury.

Background

With the progress of technology, there is an increasing demand for effective and safe facial rejuvenation, and a wide variety of medical cosmetic approaches are increasingly accepted and used by the public. Photothermal therapy is a widely used method in medical and beauty skin care, such as photon skin tendering, intense pulsed light, radio frequency, laser, heat-maskan and the like, and mainly causes certain changes of epidermis layers and dermis layers of skin by generating certain photothermal effects, promotes proliferation of collagen, improves skin elasticity, improves skin state, reduces pores, eliminates wrinkles and spots. However, the photo-thermal effect can easily cause damage to the skin barrier, so that keratinocytes are damaged, skin reddening, dryness, sensitivity and other adverse reactions are easily caused, dermis fibroblasts are easily damaged, collagen synthesis capacity is reduced, skin becomes loose, and skin depression is seriously caused. The medical cosmetology is performed in a correct mode, and meanwhile, care after medical cosmetology is also timely paid attention to, so that possible damage to skin of people can be greatly reduced.

At present, research and development of related products for relieving skin injury after operation by using photo-thermal effect are still lacking.

Natural skin care ingredients have been attracting attention from the skin care product market, and natural, green skin care ingredients are more favored by consumers. In recent years, the skin care effect of edible fungi is receiving more and more attention, such as beta-glucan from mushroom, schizophyllan, lentinan, ganoderic acid, ergothioneine and the like have important effects on anti-inflammation, ultraviolet resistance, antioxidation and the like. The edible fungi also have good health care effects of beautifying, resisting aging and the like, and the ganoderma lucidum which is one of the edible fungi is used as a health care product for beautifying and resisting aging in Asia for thousands of years. Edible fungi, especially mushrooms and mushroom extracts, have a strong skin care effect and are increasingly being used in the cosmetic field.

The tremella aurantialba (Tremella aurantialba) is rare and precious edible fungus, and the Chinese medicinal fungus records that the tremella aurantialba has the characteristics of Wen Zhongdai cold property, sweet taste, phlegm reduction, cough relieving, mainly treating diseases such as lung heat, excessive phlegm, asthma and the like, and has the effects of lubricating skin and the like due to fine and smooth colloid, white lubrication and delicate smell of tremella aurantialba. The tremella aurantialba polysaccharide is the main component of tremella aurantialba, and has certain anti-inflammatory, skin immunoregulatory, antioxidant and moisturizing effects.

Disclosure of Invention

The invention aims to provide a tremella aurantialba polysaccharide degradation product, which is degraded by a method of combining H ₂O₂ with vitamin C, and is prepared by an ultrafiltration fractionation method to obtain two tremella aurantialba polysaccharide degradation products I (E-TAP I) and tremella aurantialba polysaccharide degradation products II (E-TAP II) with different molecular weights. It is another object of the present invention to provide the use of the degradation products of auricular polysaccharides for protecting or repairing thermally damaged skin cells.

According to the technical scheme, the tremella aurantialba polysaccharide degradation product is prepared by degrading tremella aurantialba polysaccharide through a method of combining H ₂O₂ with vitamin C and selecting degradation products with molecular weights of more than 100k Da and 1k Da and molecular weight of less than 100k Da.

The application of the auricularia auricula polysaccharide degradation product in preparing a medicament for protecting or repairing skin thermal injury.

The application of the tremella aurantialba polysaccharide degradation products comprise tremella aurantialba polysaccharide degradation products I and tremella aurantialba polysaccharide degradation products II.

The application is that the grading of the auricularia auricula polysaccharide after degradation is carried out by an ultrafiltration grading method.

The molecular weight of the degradation product I of the auricularia auricula polysaccharide is more than 100k Da.

The molecular weight of the degradation product II of the auricularia auricula polysaccharide is more than 1k Da and less than 100k Da.

The application of the tremella aurantialba polysaccharide degradation product in preparing skin care products or cosmetics for protecting or repairing skin thermal injury.

The application of the tremella aurantialba polysaccharide degradation product I in the preparation of products for repairing keratinocyte injury caused by heat is provided, wherein the tremella aurantialba polysaccharide degradation product I is a degradation product of tremella aurantialba polysaccharide with molecular weight of more than 100k Da through a method of combining H ₂ O2 with vitamin C.

The application of the tremella aurantialba polysaccharide degradation product II in preparing a product for repairing fibroblast injury caused by heat is provided, wherein the tremella aurantialba polysaccharide degradation product II is a degradation product of tremella aurantialba polysaccharide by a method of combining H ₂O₂ with vitamin C, and 1k Da < molecular weight <100k Da is selected.

The preparation method of the auricularia auricula polysaccharide degradation product is characterized by comprising the following steps:

(1) Dissolving crude tremella aurantialba polysaccharide in water, fully swelling, and adding hydrogen peroxide and ascorbic acid;

(2) Dialyzing with dialysis membrane with molecular weight cut-off of 1000Da, and ultrafiltering and grading the cut-off part with ultrafiltration tube of 100kDa to obtain auris auriculata polysaccharide degradation product I with molecular weight greater than 100k Da and auris auriculata polysaccharide degradation product II with molecular weight less than 100k Da.

In a thermal injury skin cell model, auricularia auricula polysaccharide degradation products are used as active components, and auricularia auricula polysaccharide with different molecular weights obtained through degradation is applied to the protection of thermal injury of skin stratum corneum, dermis layer cells and tissues. The auricularia auricula polysaccharide degradation products I (E-TAP I) and II (E-TAP II) with different molecular weights are prepared by degrading auricularia auricula polysaccharide by a method of combining H ₂O₂ with vitamin C and performing ultrafiltration fractionation.

The in vitro antioxidant activity analysis is carried out on the two obtained degrading auricle polysaccharides and the undegraded auricle polysaccharide, and the auricle polysaccharides with different molecular weights obtained by degradation are found to have excellent reducing power, DPPH free radical scavenging ability and ABTS free radical scavenging ability, when the concentration of the polysaccharide is 2.0mg/ml, the E-TAP I reducing power, the DPPH free radical scavenging ability and the ABTS free radical scavenging ability are increased by 30-35%, 45-50% and 35-40% compared with the undegraded polysaccharide, and the E-TAP II reducing power, the DPPH free radical scavenging ability and the ABTS free radical scavenging ability are increased by 65-70%, 60-65% and 45-50% compared with the undegraded polysaccharide. The E-TAP II > E-TAP I > undegraded tremella polysaccharide obtained by degrading the total antioxidant capacity is far more than tremella polysaccharide sold in the market, which shows that both tremella polysaccharide with two molecular weights obtained by degrading have certain antioxidant capacity, and can reduce the degree of oxidative stress caused by heat to skin. The E-TAP I can remarkably enhance the proliferation and migration capacity of human keratinocytes, and can greatly increase the survival rate of the keratinocytes after heat shock compared with a non-administration group, and the experimental result shows that the E-TAP I obtained by degradation can relieve the damage of the human keratinocytes caused by heat. The E-TAP II can obviously enhance the survival rate of fibroblasts after the fibroblast heat shock is increased, and the experimental result shows that the E-TAP II obtained by degradation can relieve the fibroblast damage caused by heat.

The existing research on the auricularia auricula polysaccharide is generally focused on the extraction and research on the polysaccharide in the auricularia auricula fruiting body, but the auricularia auricula polysaccharide extracted from the fruiting body has high molecular weight, is difficult to enter the dermis layer through the skin stratum corneum to exert a relieving effect, the biological utilization rate of the auricularia auricula polysaccharide can be greatly improved by effectively degrading the auricularia auricula polysaccharide, and meanwhile, the obtained auricularia auricula polysaccharide degradation products with different molecular weights can exert different effects on different skin layers.

The key point of the invention is as follows:

1. Polysaccharide E-TAP I and E-TAP II with different molecular weights obtained by degrading auricularia auricula polysaccharide with hydrogen peroxide have antioxidant activity;

2. E-TAP I can repair keratinocyte injury caused by heat;

3. E-TAP II can repair the damage of fibroblast caused by heat.

Compared with the prior art, the invention has the following technical advantages that the tremella aurantialba polysaccharide with different molecular weights can be used as an active ingredient, is applied to photothermal post-medical repair products, exploits the new application of tremella aurantialba polysaccharide, and provides a new choice for post-medical repair products. Specifically, the application of the auricularia auricula polysaccharides with different molecular weights in medical and beauty skin care, the application of the auricularia auricula polysaccharides with different molecular weights in repairing and relieving products and the application of the auricularia auricula polysaccharides with different molecular weights in cosmetics.

Drawings

FIG. 1 is a graph showing the measurement of the reducing power of different polysaccharides;

FIG. 2 shows DPPH radical scavenging ability measurements for different polysaccharides;

FIG. 3 is a measurement of the ABTS radical scavenging ability of different polysaccharide species;

FIG. 4 shows the effect of E-TAP I on keratinocyte proliferation activity (12 h);

FIG. 5 shows the effect of E-TAP I on keratinocyte proliferation activity (24 h);

FIG. 6 shows the effect of E-TAP I on keratinocyte proliferation activity (48 h);

FIG. 7 shows the reverse thermal damage effect of E-TAP I on HaCaT;

FIG. 8 is a graph showing the migration ability impact of E-TAP I on HaCaT;

FIG. 9 shows the effect of E-TAP II on fibroblast proliferation activity (12 h);

FIG. 10 shows the effect of E-TAP II on fibroblast proliferation activity (24 h);

FIG. 11 shows the effect of E-TAP II on fibroblast proliferation activity (48 h);

FIG. 12 shows the reverse thermal damage effect of E-TAP II on NIH-3T 3.

Detailed Description

In the previous study, the inventor prepares tremella polysaccharide from tremella. The crude tremella aurantialba polysaccharide used in the experiment is extracted from tremella aurantialba, the total sugar content reaches 93.66%, and the tremella aurantialba polysaccharide is also called in the following example part. Drying and crushing tremella aurantialba fruiting bodies, adding 75-95% ethanol into L according to a feed liquid ratio of 1:10-1:20 g/mL or kg/L, stirring at normal temperature for 20-30 h, carrying out suction filtration, drying a filter cake to obtain defatted dry powder, adding water into the defatted dry powder, stirring until the defatted dry powder swells, adjusting pH to be 5.5-6.5, adding cellulase and pectase at 45-55 ℃, carrying out heat preservation and enzymolysis for 30-45 min at 45-55 ℃, extracting with water at 75-80 ℃ for 0.5-1.5 h, cooling, centrifuging, collecting supernatant, evaporating and concentrating, adding absolute ethanol to 75-80%, carrying out alcohol precipitation at 4-8 ℃ for 20-30 h, centrifuging, collecting precipitate, and drying to obtain tremella aurantialba crude polysaccharide. For specific extraction methods see cn202111271004.X.

Tremella polysaccharides are supplied by Shanghai Hui Biotechnology Co.

Example 1 preparation method of auricularia auricula polysaccharides with different molecular weights

The method for degrading the tremella aurantialba polysaccharide comprises the steps of dissolving tremella aurantialba crude polysaccharide 1g in 100ml of water, fully swelling, adding a proper amount of hydrogen peroxide and an equal proportion of ascorbic acid (7.5 mM), heating in a 50 ℃ water bath for 4 hours, cooling, adjusting pH to 7.0, dialyzing for 48 hours by using a dialysis membrane with a molecular weight cutoff of 1000, and carrying out ultrafiltration fractionation by using a 100kDa ultrafiltration tube to obtain tremella aurantialba polysaccharide degradation product I (E-TAPI) with a molecular weight cutoff of more than 100k Da and tremella aurantialba polysaccharide degradation product II (E-TAPII) with a molecular weight cutoff of less than 100k Da.

Example 2 measurement of reducing force of different molecular weight auricularia auricula polysaccharides

7.2G of dipotassium hydrogen phosphate dodecahydrate and 3.1g of potassium dihydrogen phosphate dihydrate were weighed, dissolved in 100mL of distilled water, respectively, and mixed in a ratio of 2:3 to prepare a 0.2mM PBS solution, and 1mL of each of E-TAPI sample, E-TAPII sample, tremella polysaccharide (TAP) sample and tremella polysaccharide (WSK) sample having concentrations of 0.25, 0.5, 1.0, 1.5 and 2.0 were subjected to water bath at 50 ℃ for 20 minutes in a dark place by mixing with 2.5mL of 0.2mM PBS solution and 2.5mL of 1% potassium ferricyanide solution. Then, 5mL of a 10% trichloroacetic acid solution was added, and after centrifugation for 10 minutes, 2.5mL of the supernatant was taken, 2.5mL of distilled water and 0.5mL of a 0.1% ferric trichloride solution were added, and the mixture was left to stand in a dark place for 10 minutes, and the absorbance was measured at a wavelength of 700 nm.

The experimental results are shown in FIG. 1.

As can be seen from the figure, the four polysaccharides have the reducing power of E-TAP II > E-TAP I > TAP > WSK. Compared with TAP, the reducing power of E-TAP I and E-TAP II is obviously improved (p is less than 0.01 and p is less than 0.001), and when the polysaccharide concentration is 2mg/ml, the reducing power (average value) of E-TAP II is 2.1 times of E-TAP I, 3.1 times of TAP and 4.3 times of tremella polysaccharide. The results showed that E-TAPII has excellent reducing power.

EXAMPLE 3 DPPH free radical scavenging ability measurement of different molecular weight auricularia auricula polysaccharides

A certain amount of DPPH was weighed and dissolved in absolute ethanol to prepare a solution of DPPH of 0.2 mM. E-TAPI sample, E-TAPII sample, tremella polysaccharide sample and tremella polysaccharide sample with the concentration of 0.25, 0.5, 1.0, 1.5 and 2.0mM are prepared respectively, 1mL of DPPH solution and 1mL of absolute ethyl alcohol are respectively added, a blank group uses absolute ethyl alcohol solvent to replace the sample, namely 1mL of absolute ethyl alcohol and 1mLDPPH solution, the blank group is vibrated and mixed uniformly, and the blank group reacts in a water bath at 37 ℃ for 30 minutes in a dark place, and the absorbance of the blank group is measured at the wavelength of 517 nm. The clearance capacity of the auricularia auricula polysaccharide and degradation products thereof to DPPH is calculated according to the following formula:

Absorbance of Ai-experimental group

A _i0 absorbance of control group

Absorbance of A ₀ -blank control group

The experimental results are shown in FIG. 2.

From the graph, the DPPH free radical scavenging ability of the four samples is E-TAP II > E-TAP I > TAP > WSK, and the E-TAP I and E-TAPIIDPPH free radical scavenging ability is obviously increased compared with TAP (p is less than 0.001). When the polysaccharide concentration was 2mg/ml, the clearance (average value) of E-TAP II was 1.5 times that of E-TAP I, 2.8 times that of TAP, and 5.6 times that of tremella polysaccharide. The results show that E-TAP II has excellent DPPH free radical scavenging ability.

Example 4 measurement of ABTS free radical scavenging Capacity of different molecular weight auricularia auricula polysaccharides

ABTS solution with concentration of 7mmol/L and potassium persulfate solution with concentration of 140mmol/L are accurately prepared. Mixing the two materials according to a certain proportion, standing in dark for 12 hours, and regulating the absorbance of the ABTS to 0.7 (734 nm) by using absolute ethyl alcohol. Taking 1 mL mM E-TAPI sample, E-TAPII sample, tremella polysaccharide sample and tremella polysaccharide sample with concentrations of 0.25, 0.5, 1.0, 1.5 and 2.0mM respectively, respectively 1 mL, mixing with 1 mLABTS and 1 mL distilled water respectively, replacing the sample with distilled water in a blank control group, shaking uniformly, standing for 1 hour in a dark environment, and measuring absorbance at 734nm wavelength. The cleaning capacity of the auricularia auricula polysaccharide and degradation products thereof to ABTS is calculated according to the following formula:

in A _i -absorbance of experimental group

A _i0 absorbance of control group

Absorbance of A ₀ -blank control group

The experimental results are shown in FIG. 3.

From the graph, the ABTS free radical scavenging ability of the four polysaccharides is E-TAP II > E-TAP I > TAP > WSK, and the E-TAP I and E-TAP II ABTS free radical scavenging ability is obviously increased compared with TAP (p <0.05, p < 0.001). When the polysaccharide concentration was 2mg/ml, the clearance (average value) of E-TAP II was 1.2 times that of E-TAP I, 1.9 times that of TAP, and 3.1 times that of tremella polysaccharide. The results show that E-TAPII has excellent ABTS free radical scavenging ability.

Example 5 cell proliferation assay of auris degradation product I (E-TAP I) on keratinocyte (HaCaT)

HaCaT cells were inoculated into 96-well plates, cultured in a 37℃5% CO2 incubator for 24 hours, the cell density and morphology were observed under a microscope, old medium was aspirated, and treated with 25, 50, 100, 150, 200. Mu.g/mL E-TAP I, respectively, cultured in a 37℃5% CO2 incubator for 12 hours, incubated with CCK-8 while a blank control group was set for zeroing, and absorbance was measured at 450nm using a microplate reader. And cultured for 24 and 48 hours in the same manner for measurement.

The experimental results of 12h, 24h and 48h cell survival rates are shown in FIGS. 4, 5 and 6.

From the graph, the effect on cell proliferation was very remarkable at the concentration of ETAP-I of 50. Mu.g/mL (p < 0.001) and at the concentrations of 100. Mu.g/rnL and 150. Mu.g/mL (p < 0.01) after 12h of culture. At 24h of culture, the concentration of 200 μg/mL had a significant effect on cell proliferation (< 0.05 p), none of the other concentrations were significant. At 48h incubation, the effect on cell proliferation was very pronounced at a concentration of 50 μg/mL for aurora degradation product I (< p < 0.01) and at a concentration of 25 μg/mL (< p < 0.05). In general, E-TAP I is non-toxic to keratinocytes in the range of 25-200. Mu.g/ml and has a pro-proliferative effect.

EXAMPLE 6 thermal injury reversing effect of auris degradation product I (E-TAP I) on HaCaT

Firstly, grouping cells inoculated to a 96-well plate, namely a control group (37 ℃ culture group), a dosing group (E-TAPI), a molding group (48 ℃ molding group), a molding and dosing group (48 ℃ plus E-TAPI), culturing for 24 hours in a 37 ℃ 5% CO2 constant temperature incubator, adding serum-free MEM culture medium for starvation for 24 hours, sealing a culture dish by using a sealing film, and adding the culture dishes of the corresponding groups into a water bath kettle of 37 ℃ and 48 ℃ for incubation for 30 minutes, wherein the bottoms of the culture dishes are completely contacted with the water surface. Serum-free MEM medium containing E-TAP I at the corresponding concentrations was added to the molded dishes, incubated for 24 hours in an incubator, incubated with CCK-8 while a blank control was set to zero, and absorbance was measured at 450nm using a microplate reader.

The experimental results are shown in FIG. 7.

As can be seen from the graph, the survival rate of HaCaT cells can be remarkably reduced by 48 ℃ (. P < 0.001), compared with a modeling module, the survival rate of HaCaT can be remarkably improved by adding 25-50 mug/ml of E-TAP I (#p < 0.05), and experimental results show that the E-TAP I can reverse keratinocyte injury caused by heat.

EXAMPLE 7 cell migration experiment of auris degradation product I (E-TAP I) on keratinocyte (HaCaT)

HaCaT cells were seeded at a concentration of 1.0X105 cells/mL in 6-well cell culture plates with a medium of complete MEM medium, 2mL per well, and cultured in a 5% CO2, 37℃incubator until the cell fusion rate became 100%, followed by cell streaking with a 10. Mu.L gun head. E-TAP I was added to a serum-free MEM basal medium to prepare 25, 50, 100. Mu.g/mL sample solutions (serum-free MEM basal medium as a blank) and incubated with the cells for 24h, respectively, and observed under a microscope and photographed at 0h and 24h (0 h corresponding to the area of the antigen, 24 corresponding to the area after scratch), and the mobility (%) was calculated according to the following formula:

The experimental results are shown in FIG. 8.

As can be seen from the graph, compared with the Control group, the concentrations of 12.5, 25, 50 and 100 μg/mL of E-TAP I have a significant promoting effect on keratinocyte migration (p < 0.001) and the migration promoting capacity concentration is increased in a dependent manner, which indicates that the tremella aurantiaca degradation product has the effect of promoting repair of the keratinocyte injury.

Example 8 cell proliferation assay of auris degradation product II (E-TAPII) on fibroblasts (NIH-3T 3)

NIH-3T3 cells were inoculated into 96-well plates, cultured in a 37℃5% CO2 incubator for 24 hours, the cell density and morphology were observed under a microscope, old medium was aspirated, E-TAP II was added at 25, 50, 100, 150, 200. Mu.g/mL, respectively, and treated, incubated in a 37℃5% CO2 incubator for 12 hours, incubated with CCK-8 while a blank control group was set for zeroing, and absorbance was measured at 450nm using a microplate reader. And cultured for 24 and 48 hours in the same manner for measurement.

The experimental results of 12h, 24h and 48h show the cell viability in FIGS. 9, 10 and 11.

E-TAP II at 200 μg/ml had pro-proliferative effects on NIH-3T3 cells 12, 48 hours after administration, with significant differences (< p < 0.05), and E-TAP II at 50, 100, 150 μg/ml had pro-proliferative effects on NIH-3T3 cells 24 hours after administration with significant differences (< p <0.05, < p < 0.001). In general, E-TAP II is non-toxic to keratinocytes in the range of 25-200. Mu.g/ml and has a pro-proliferative effect.

EXAMPLE 9 thermal injury reversing effect of auris degradation product II (E-TAPII) on fibroblasts (NIH-3T 3)

Firstly, grouping cells inoculated to a 96-well plate, namely a control group (37 ℃), a drug administration group (E-TAP II), a molding group (48 ℃) and a molding drug administration group (48 ℃) and E-TAP II), culturing for 24 hours in a 37 ℃ 5% CO2 constant temperature incubator, adding serum-free DMEM culture medium for starvation for 24 hours, sealing a culture dish by using a sealing film, adding the culture dishes of the corresponding groups into a water bath kettle of 37 ℃ and 48 ℃, and incubating for 10 minutes, wherein the bottoms of the culture dishes are completely contacted with the water surface. Serum-free DMEM medium containing E-TAP II at the corresponding concentration was added to the molded dishes, incubated in an incubator for 24 hours, incubated with CCK-8 while a blank control group was set to zero, and absorbance was measured at 450nm using a microplate reader.

The experimental results are shown in FIG. 12.

As can be seen from the graph, 48 ℃ can significantly reduce the survival rate of NIH-3T3 cells (p < 0.001), and compared with the 48 ℃ group without E-TAP II, the addition of 50, 100 μg/ml of E-TAP I can significantly improve the survival rate of NIH-3T3 (p < 0.001), and the experimental result shows that the E-TAP II can reverse the damage of fibroblasts caused by heat.

Claims (2)

1. Application of Auricularia auricula polysaccharide degradation product II in the preparation of products for repairing heat-induced fibroblast damage, wherein Auricularia auricula polysaccharide degradation product II is a degradation product of Auricularia auricula polysaccharide by _H2O2 combined with vitamin _C , and the degradation product with molecular weight < 1k Da < 100k Da is selected. 2. The application according to claim 1, characterized in that the preparation method of the degradation product II of *Auricularia auricula-judae* polysaccharide includes the following steps: (1) Dissolve crude auricularia polysaccharide in water, allow it to swell fully, and add hydrogen peroxide and ascorbic acid; (2) Dialysis was performed using a dialysis membrane with a molecular weight cutoff of 1000 Da. The cutoff portion was then subjected to ultrafiltration fractionation using a 100 kDa ultrafiltration tube to obtain degradation product I of Auricularia auricula polysaccharide with a molecular weight greater than 100 kDa and degradation product II of Auricularia auricula polysaccharide with a molecular weight less than 100 kDa.