Schizophyllan as well as preparation method and application thereof

Abstract

The invention discloses schizophyllan, which has two components with molecular weights of 1.54 * 10 < 7 > Da and 1.85 * 10 < 4 > Da respectively, and consists of glucose, mannose, galactose and fucose, and the molar ratio of glucose to mannose to galactose to fucose is 86: 8: 5: 1. The schizophyllan is a heterogeneous polysaccharide with glucose as a main component, and can promote proliferation of HaCaT cells and HUVEC cells. Meanwhile, the schizophyllan can remarkably promote the healing speed of a full-thickness skin wound model mouse wound and has excellent effects in the aspects of epithelium reformation, granulation tissue formation and tissue remodeling, and when 4 mg / mL of schizophyllan is used for treating the mouse wound, the healing rate reaches 96.65%.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to Schizophyllan and a preparation method and application thereof. Background Art

[0002] Skin wound healing is a complex and important biological process that is essential for maintaining human health and function. However, if complications such as infection or excessive inflammation occur in the wound, it will hinder or delay its normal healing. Therefore, in order to improve the wound healing effect, the development of efficient wound dressings has become an important direction of biomedical research in recent years.

[0003] Existing wound dressings are mainly made of natural or synthetic materials. Common protein materials include gelatin, collagen and fibrin, while polysaccharide materials include seaweed polysaccharides, chitosan and hyaluronic acid. These materials not only need to have good mechanical strength, appropriate porosity and sufficient flexibility, but also should provide a microenvironment that is conducive to cell migration and proliferation, and support wound functional repair and tissue regeneration.

[0004] Schizophyllan is a polysaccharide material extracted from the fermentation broth of Schizophyllan. Currently, most Schizophyllan are homopolymeric Schizophyllan, which have a complex structure and low functional stability. In addition, homopolymeric Schizophyllan is often difficult to dissolve due to its large molecular weight, which also causes Schizophyllan to often precipitate white flocs in water environments.

[0005] Therefore, the development of biomaterials that can biomimetically replicate the microenvironment of skin tissue regeneration has become an important research direction in the fields of materials science, bioengineering and clinical medicine. Summary of the invention

[0006] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art and to provide a schizophyllan and a preparation method and application thereof.

[0007] The first object of the present invention is to provide a Schizophyllan.

[0008] The second objective of the present invention is to provide the use of the above-mentioned Schizophyllan in the preparation of a product for promoting the proliferation of HaCaT cells and / or HUVEC cells.

[0009] The third object of the present invention is to provide the use of the above-mentioned Schizophyllan in the preparation of a product for promoting HUVEC cell migration.

[0010] The fourth object of the present invention is to provide the use of the above-mentioned Schizophyllan in the preparation of products that promote collagen fiber deposition.

[0011] The fifth object of the present invention is to provide the use of the above-mentioned Schizophyllan in the preparation of products promoting wound healing.

[0012] The sixth object of the present invention is to provide a product for promoting the proliferation of HaCaT cells and / or HUVEC cells.

[0013] A seventh object of the present invention is to provide a product for promoting wound healing.

[0014] In order to achieve the above object, the present invention is implemented by the following scheme:

[0015] A schizophyllan polysaccharide with two molecular weights of 1.54×10 7 Da and 1.85×10 4 The components of Da are composed of glucose, mannose, galactose and fucose, and the molar ratio of glucose, mannose, galactose and fucose is 86:8:5:1.

[0016] The present invention also claims protection for a Schizophyllan polysaccharide, wherein the preparation method of the Schizophyllan polysaccharide comprises the following steps:

[0017] S1. Fully ferment using Schizophyllum GDMCC NO.5.43, collect the supernatant after the fermentation to obtain Schizophyllum exopolysaccharide fermentation liquid;

[0018] S2. The Schizophyllum exopolysaccharide fermentation liquid obtained in step S1 is separated into solid and liquid, purified and protein removed to obtain Schizophyllum polysaccharide.

[0019] Among them, the schizophyllan GDMCC NO.5.43 comes from the Guangdong Provincial Microbial Culture Collection Center.

[0020] Preferably, the fermentation conditions in step S1 are: when fermenting for 0 to 84 hours, the dissolved oxygen is controlled at 20% to 40%; when fermenting for 84 to 108 hours, the dissolved oxygen is controlled at 25% to 50%; and the temperature is controlled at 25 to 30° C. during the fermentation process.

[0021] More preferably, the fermentation conditions are: when fermenting for 0 to 36 hours, the dissolved oxygen is controlled at 40%; when fermenting for 36 to 84 hours, the dissolved oxygen is controlled at 30%; when fermenting for 84 to 108 hours, the dissolved oxygen is controlled at 45%; and the temperature is controlled at 28°C during the fermentation process.

[0022] Further preferably, the fermentation in step S1 comprises the following steps:

[0023] S11. Solid culture of Schizophyllum: aerial hyphae of Schizophyllum are picked up with an inoculation loop and inoculated into a solid plate medium, the inoculated solid plate medium is placed in a constant temperature incubator, inverted and cultured at 28°C for 5 days, and activated Schizophyllum strains are obtained when the hyphae grow all over the plate, and the strains are preserved for later use; wherein the solid plate medium contains 30 g / L glucose, 3 g / L yeast powder, 1 g / L KH2PO4, 0.5 g / L MgSO4·H2O and 18 g / L agar powder;

[0024] S12. Liquid culture of Schizophyllum: Use an inoculation loop to pick up the activated Schizophyllum strains, inoculate them into 100 mL of liquid culture medium, and place them in a 250 mL culture bottle, and culture them at 28°C and 160 rpm for 3 days to obtain a primary seed solution, which is retained for later use; inoculate the primary seed solution into the liquid culture medium at an inoculation rate of 5.0% (v / v), and observe the appearance of thorns on the periphery of the bacteria to obtain a secondary seed solution; wherein the liquid culture medium contains 30 g / L glucose, 3 g / L yeast powder, 1 g / L KH2PO4 and 0.5 g / L MgSO4·H2O;

[0025] S13. Amplification of Schizophyllum: The secondary seed solution was inoculated at a 5% (v / v) inoculum into a 5.0 L fermenter containing 3.5 L of culture medium for fermentation; wherein the culture medium contained 30 g / L glucose, 3 g / L yeast powder, 1 g / L KH2PO4, 0.5 g / L MgSO4·H2O and 0.05-0.1% (v / v) F1075 (defoaming agent);

[0026] S14. During the fermentation period of 0 to 36 hours, the dissolved oxygen (DO) was set to 40.0%; during the fermentation period of 36 to 84 hours, the DO was set to 30.0%; during the fermentation period of 48 hours, glucose was added in batches, wherein the amount of glucose added was 5.0 g / L, and it was added once every 12 hours, for a total of three times; when the fermentation period was 84 to 108 hours, the DO was set to 45%; the stirring speed during the fermentation process was 100 to 600 rpm, the temperature during the fermentation process was maintained at 28°C, and the pH of the fermentation system was maintained at 6.0 using 0.1 M NaOH and 0.1 M acetic acid, the concentration of glucose added in batches was 50.0% (m / v), and the fermentation product was obtained after 6 days of fermentation;

[0027] S15. Preparation of fermentation supernatant: The fermentation product was passed through 8 layers of sterile gauze to remove the Schizophyllum bacteria, and then centrifuged at 4°C and 10,000 g for 30 min. The supernatant was collected and passed through a 100 nm ceramic membrane to obtain the Schizophyllum exopolysaccharide fermentation liquid.

[0028] Preferably, the purification using macroporous resin in step S2 is specifically as follows: adding the supernatant to an activated macroporous resin containing 1 mg / mL of 5% AMP-277 (w / w), after constant temperature water bath at 70°C, collecting the supernatant by solid-liquid separation, performing vacuum filtration, concentrating and removing free protein by sevag method.

[0029] The schizophyllan polysaccharide claimed in the present invention is purified and extracted from the fermentation supernatant of the schizophyllan strain GDMCC NO.5.43. The polysaccharide is a non-homogeneous polysaccharide with glucose as the main component, which can promote the proliferation of HaCaT cells and HUVEC cells, and can promote the migration of HUVEC cells.

[0030] Therefore, the present invention also claims to protect the use of any of the above-mentioned Schizophyllan in the preparation of products that promote the proliferation of HaCaT cells and / or HUVEC cells.

[0031] The present invention also claims to protect the use of any of the above-mentioned Schizophyllan in the preparation of products that promote HUVEC cell migration.

[0032] At the same time, the schizophyllan polysaccharide claimed for protection in the present invention can significantly promote the wound healing speed of model mice (mice with full-thickness skin wounds), and has excellent effects in epithelial re-formation, granulation tissue formation and tissue remodeling.

[0033] Therefore, the present invention also claims to protect the use of any of the above-mentioned Schizophyllan in the preparation of products that promote collagen fiber deposition.

[0034] The present invention also claims to protect the use of any of the above-mentioned Schizophyllan in the preparation of products promoting wound healing.

[0035] The present invention also claims a product for promoting the proliferation of HaCaT cells and / or HUVEC cells, which contains ≥50 μg / mL of any of the above-mentioned Schizophyllan.

[0036] Preferably, it contains 50-800 μg / mL of any one of the above-mentioned Schizophyllan.

[0037] More preferably, it contains 300-500 μg / mL of any one of the above-mentioned Schizophyllan.

[0038] More preferably, it contains 300 μg / mL of any of the above-mentioned Schizophyllan.

[0039] The present invention also claims a product for promoting wound healing, which contains 1-4 mg / mL of any one of the above-mentioned Schizophyllan.

[0040] Preferably, it contains 4 mg / mL of any of the above-mentioned Schizophyllan.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] The present invention provides a schizophyllan polysaccharide having two molecular weights of 1.54×10 7 Da and 1.85×10 4 The components of Da are composed of glucose, mannose, galactose and fucose, and the molar ratio of glucose, mannose, galactose and fucose is 86:8:5:1. The schizophyllan is a non-homogeneous polysaccharide with glucose as the main component, which can promote the proliferation of HaCaT cells and HUVEC cells, and can promote the proliferation of HUVEC cells; at the same time, the schizophyllan can significantly promote the wound healing speed of model mice (mice with full-thickness skin wounds), and has excellent effects in epithelial re-formation, granulation tissue formation and tissue remodeling. When 4 mg / mL of schizophyllan is used to treat mouse wounds, the healing rate reaches 96.65%. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the FTIR spectrum result of the Schizophyllan prepared in step 1 of Example 1;

[0044] Figure 2 is the chromatogram of the monosaccharide standard;

[0045] Figure 3 This is the chromatogram of Schizophyllan SPG5.43 prepared in step 1 of Example 1;

[0046] Figure 4 The cell proliferation rate results in Example 2; a is the effect of Schizophyllan SPG5.43 on the proliferation rate of HaCaT cells; b is the effect of Schizophyllan SPG5.43 on the proliferation rate of HUVEC cells;

[0047] Figure 5 a is a graph showing the results of the EdU-594 test evaluation in Example 2; a is a graph showing the results of the fluorescence photography of HaCaT cells; b is a graph showing the results of the fluorescence photography of HUVEC cells;

[0048] Figure 6 This is a picture showing the photographic results of different culture times for the same scratch area in Example 2;

[0049] Figure 7 The mobility result diagram in Example 2;

[0050] Figure 8 This is a graph showing the survival rate of HaCaT cells treated with SPG5.43 in Example 3;

[0051] Fig. 9This is a graph showing the survival rate of HUVEC cells treated with SPG5.43 in Example 3;

[0052] Fig.10 This is a graph showing the results of the glutathione peroxide activity test in Example 3;

[0053] Fig.11 This is a graph showing the results of the superoxide dismutase activity test in Example 3;

[0054] Fig.12 The wound surfaces of the mice in each group after treatment in Example 4 were photographed;

[0055] Fig.13 The figure is the result diagram of wound healing rate of each group of mice in Example 4;

[0056] Fig.14 This is the result graph of the effects of Schizophyllan SPG5.43, Schizophyllan SPG5.46, laminarin and Prussian polysaccharide on the proliferation rate of HaCaT cells;

[0057] Fig.15 This is a graph showing the effects of Schizophyllan SPG5.43, Schizophyllan SPG5.46, laminarin and Prussian polysaccharide on the proliferation rate of HUVEC cells. DETAILED DESCRIPTION

[0058] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0059] All animal experimental procedures involved in the embodiments of the present invention were approved by the Animal Protection Committee and were performed in accordance with the regulations on experimental animal management of Guangdong Pharmaceutical University (Guangzhou, China).

[0060] Example 1 Preparation and structural identification of Schizophyllan

[0061] 1. Experimental Methods

[0062] 1. Preparation method

[0063] (1) Preparation of Schizophyllum exopolysaccharide fermentation broth:

[0064] Solid culture of Schizophyllum: Use an inoculation loop to pick up aerial hyphae of Schizophyllum GDMCC NO.5.43 and inoculate them into a solid plate medium. Place the inoculated solid plate medium in a constant temperature incubator and invert and culture it at 28°C for 5 days. When the hyphae cover the entire plate, activated Schizophyllum strains are obtained and preserved for future use. The solid plate medium contains 30g / L glucose, 3g / L yeast powder, 1g / L KH2PO4, 0.5g / L MgSO4·H2O and 18g / L agar powder.

[0065] Liquid culture of Schizophyllum: Use an inoculation loop to pick up the activated Schizophyllum strains, inoculate them into 100 mL of liquid culture medium, and place them in a 250 mL culture bottle, and culture them at 28°C and 160 rpm for 3 days to obtain a first-level seed solution, which is retained for later use; inoculate the first-level seed solution into 100 mL of liquid culture medium at an inoculation amount of 5.0% (v / v) for culture until thorns are observed on the periphery of the bacteria, thereby obtaining a second-level seed solution; wherein the liquid culture medium contains 30 g / L glucose, 3 g / L yeast powder, 1 g / L KH2PO4 and 0.5 g / L MgSO4·H2O;

[0066] Amplification of Schizophyllum: The secondary seed solution was inoculated at a 5% (v / v) inoculum into a 5.0 L fermentation tank containing 3.5 L culture medium for fermentation; wherein the culture medium contained 30 g / L glucose, 3 g / L yeast powder, 1 g / L KH2PO4, 0.5 g / L MgSO4·H2O and 0.05-0.1% (v / v) F1075 (defoaming agent);

[0067] During the fermentation period of 0 to 36 hours, the dissolved oxygen (DO) was set to 40.0%; during the fermentation period of 36 to 84 hours, the DO was set to 30.0%; during the fermentation period of 48 hours, glucose was added in batches, wherein the amount of glucose added was 5.0 g / L, and the glucose was added once every 12 hours, for a total of three times; when the fermentation period was 84 to 108 hours, the DO was set to 45%; the stirring speed during the fermentation process was 100 to 600 rpm, the temperature during the fermentation process was maintained at 28°C, and 0.1 M NaOH and 0.1 M acetic acid were used to maintain the pH of the fermentation system at 6.0, the concentration of glucose added in batches was 50.0% (m / v), and the fermentation product was obtained after 6 days of fermentation;

[0068] Preparation of fermentation supernatant: The fermentation product was passed through 8 layers of sterile gauze to remove the Schizophyllum bacteria, and then centrifuged at 4°C and 10,000 g for 30 min. The supernatant was collected and passed through a 100 nm ceramic membrane to obtain the Schizophyllum exopolysaccharide fermentation liquid.

[0069] (2) Preparation of Schizophyllan SPG5.43:

[0070] The extracellular polysaccharide fermentation liquid of Schizophyllum obtained in step (1) is centrifuged at 20000r / min for 20min using a high-speed tubular centrifuge, and the supernatant is collected. 500mL of the supernatant is added to an activated macroporous resin containing 1mg / mL of 5% macroporous resin AMP-277 (w / w), and the mixture is stirred in a constant temperature water bath at 70°C for 30min, and then centrifuged at 4000r / min for 20min using a centrifuge, and the supernatant is collected and vacuum filtered to obtain a fermentation supernatant.

[0071] The fermentation supernatant was concentrated using a 70°C vacuum pump at the ratio of original solution: concentrate = 5:2 (v / v), and then free protein was removed using the sevag method. Subsequently, 2 volumes of 95% ethanol (v / v) were added, and after precipitation at 4°C for 24 hours, the protein-free flocculent polysaccharide was obtained. The polysaccharide was washed with 5 mL of 95% ethanol (v / v), redissolved in 15 mL of distilled water, dialyzed with running water and distilled water for 24 hours respectively, and freeze-dried to obtain schizophyllan SPG5.43.

[0072] 2. Schizophyllan polysaccharide structure identification

[0073] (1) Infrared scanning

[0074] The Schizophyllan SPG5.43 prepared in step 1 was freeze-dried, and 0.5 mg of the freeze-dried Schizophyllan SPG5.43 was weighed and mixed with 50 mg of KBr powder. After mixing, the mixture was ground and tableted with a tablet press, and then Fourier transform-infrared spectroscopy (FTIR spectrum) was performed in the wavelength range of 4000-400 cm -1 .

[0075] (2) Monosaccharide composition test

[0076] The schizophyllan SPG5.43 prepared in step 1 was placed in a sealed tube, and schizophyllan was hydrolyzed with 2.0 M trifluoroacetic acid (TFA) at 121°C for 2 h. The solid-liquid separation was performed and the solid was dried with nitrogen (N2). The solid was then washed with methanol for 3 times. The solid was then collected and dissolved in deionized water, and filtered through a 0.22 μm microporous filter membrane to obtain a treated sample.

[0077] At Dionex TM CarboPac TM On a PA-20 (150×3.0 mm, 10 μm) anion exchange column, the flow rate was set to 0.5 mL / min, the column temperature was set to 30°C, and the injection volume was set to 5.0 μL. The treated sample was injected for gradient elution, and the chromatogram of the treated sample was recorded.

[0078] During gradient elution, mobile phase A was H2O, mobile phase B was 0.1 M NaOH, and mobile phase C was an aqueous solution containing 0.1 M NaOH and 0.2 M NaAc; the gradient elution conditions are shown in Table 1.

[0079] Table 1 Gradient elution conditions

[0080] time Mobile phase A / %(v) Mobile phase B / %(v) Mobile phase C / %(v) 0min 95 5 0 26min 85 5 10 42.1min 60 0 40 52.1min 95 5 0

[0081] Analytically pure fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, mannuronic acid and guluronic acid (all products of Sigma, analytically pure) were used as monosaccharide standards. The chromatograms of the monosaccharide standards were recorded according to the above method, and the monosaccharide composition in the treated samples was analyzed.

[0082] (3) Molecular weight test

[0083] Weigh 5.844 g of NaCl, add Wahaha water (ultrapure water) to dissolve, transfer to a 2.0 L volumetric flask, ultrasonicate for 10 min, and filter with a 0.22 μm filter membrane to obtain a NaCl solution.

[0084] Accurately weigh 5 mg of the standard (pullulan standard, P121048), dissolve it in 1 mL of the mobile phase solution to prepare a 5 mg / mL standard solution, and transfer the standard solution to a 1.8 mL injection vial.

[0085] Accurately weigh 5 mg of sample (SPG5.43 prepared in step 1), dissolve it in 1 mL of NaCl solution to prepare a 5 mg / mL sample solution. After vortexing to dissolve, centrifuge at 12000 rpm for 10 min, aspirate the supernatant, and filter the supernatant with a 0.22 μm water-based microporous filter membrane. Then transfer the supernatant to a 1.8 mL injection vial.

[0086] After filtering the supernatant with a filter membrane, 20 μL of the filtrate was subjected to high performance gel permeation chromatography (HPGPC) and measured using a TSK-gelG3000 PWXL column (column temperature 40°C). The detector was a differential refractometer with a flow rate of 0.5 mL / min. Pullulan (product of Aladdin, analytical grade, P121048) was used to draw a standard curve.

[0087] 2. Experimental Results

[0088] 1. Infrared scanning results

[0089] The FTIR spectrum results of the schizophyllan prepared in step 1 are shown in the figure below: Figure 1As shown in the results, the schizophyllan polysaccharide SPG5.43 prepared in step 1 has a typical polysaccharide characteristic absorption peak at 3318 cm -1 A strong and broad signal absorption peak appeared near 2928cm -1 The characteristic sugar absorption peak attributed to CH stretching vibration was detected nearby; 1650cm -1 Nearby is the characteristic absorption peak of bound water in SPG5.43; 1000~1200cm -1 The signal peak near is the characteristic absorption peak of the pyran ring; 886cm 1 Nearby is the characteristic signal peak of β-glycosidic bond. The results show that the schizophyllan SPG5.43 prepared in step 1 is a pyranose polysaccharide mainly composed of β-glycosidic bonds.

[0090] 2. Monosaccharide composition test results

[0091] The chromatogram of the monosaccharide standard is as follows Figure 2 As shown, the chromatogram of Schizophyllan SPG5.43 prepared in step 1 is as shown Figure 3 The monosaccharide composition test results of Schizophyllan SPG5.43 are shown in Table 2.

[0092] Table 2 Monosaccharide composition test results of Schizophyllan SPG5.43

[0093] Monosaccharides Relative content*(%) Glucose (Glc) 85.63±0.6 Mannose (Man) 8.06±0.1 Galactose (Gal) 5.11±0.2 Fucose (Fuc) 1.2±0.0

[0094] The results showed that glucose (Glc) was the main monosaccharide in Schizophyllan SPG5.43, and the relative content in Schizophyllan SPG5.43 exceeded 80%. In addition to glucose, Schizophyllan SPG5.43 also contained mannose, galactose and fucose, and the relative content was between 1% and 15%. Based on the monosaccharide composition test results shown in Table 2, Schizophyllan SPG5.43 was a polysaccharide composed of glucose, mannose, galactose and fucose in a molar ratio of 86:8:5:1, indicating that Schizophyllan SPG5.43 was a heteropolysaccharide rich in glucose.

[0095] 3. Molecular weight test results

[0096] The molecular weight test results of Schizophyllan SPG5.43 showed that it contained two polysaccharides with different molecular weight ranges, 1.54×10 7 Da and 1.85×10 4 Da polysaccharide, indicating that SPG5.43 is a heteropolymeric polysaccharide.

[0097] Example 2 Effects of Schizophyllan SPG5.43 on HaCaT cells and HUVEC cells

[0098] 1. Experimental Methods

[0099] 1. Effect of SPG5.43 on the proliferation of HaCaT cells and HUVEC cells

[0100] (1) Cell proliferation rate test

[0101] According to the preparation method shown in Example 1, Schizophyllan SPG5.43 was prepared, and SPG5.43 was irradiated with ultraviolet light for 30 minutes and then dissolved in a PBS solution to obtain a polysaccharide mother solution. After the polysaccharide mother solution was sterilized by high-temperature steam, it was added to a complete culture medium to obtain SPG5.43 solutions with different SPG5.43 concentrations, including 50 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, 600 μg / mL and 800 μg / mL SPG5.43 solutions.

[0102] Healthy and well-growing HaCaT cells and HUVEC cells were inoculated into different 96-well cell plates at 5000 cells / well to obtain 96-well cell plates inoculated with cells, and experimental group 1, blank control group, negative control group and positive control group 1 were set up respectively, as follows:

[0103] Experimental group 1: SPG5.43 solutions with different SPG5.43 concentrations were added to 96-well cell plates seeded with cells at 100 μL / well for 24 hours, followed by 10 μL of CCK-8 reagent and 100 μL of serum-free medium. After incubation at 37°C for 1 hour, the absorbance OD of each well at 450 nm was measured using a microplate reader (SpectraMax iD3). 实验组 .

[0104] Blank control group: The blank control group was an empty 96-well cell plate, and the absorbance OD at 450 nm was measured using an ELISA reader. 空白 .

[0105] Negative control group (Ctl): The negative control group was a 96-well cell plate inoculated with cells, and the absorbance OD at 450nm was measured using an ELISA reader. 阴性 .

[0106] Positive control group 1 (PC): The difference between the positive control group 1 and the experimental group is that the SPG5.43 solution is replaced with an equal amount of hyaluronic acid (McLean, H909938), and the rest of the treatment is the same. The absorbance OD of the positive control group 1 is obtained. 阳性1 .

[0107] Then, the cell proliferation rates in the experimental group, negative control group, and positive control group 1 were calculated respectively according to formula 1;

[0108] Formula I: Cell proliferation rate (%) = (OD n -OD 空白 ) / (OD 阴性 -OD 空白 )×100%; where OD n For OD 实验 OD 阴性 or OD 阳性1 .

[0109] (2) EdU-594 assay to assess cell proliferation

[0110] Healthy and well-growing HaCaT cells and HUVEC cells were seeded at 12,500 cells / well in a 24-well plate. After culturing at 37°C for 24 h, 50 μg / mL and 300 μg / mL of SPG5.43 solution were added to different wells at 500 μL / well, respectively, and cultured at 37°C for 24 h, as experimental group 2 (SPG50 and SPG300).

[0111] Positive control group 2 (1 mg / mL hyaluronic acid group, PC) was as follows: 100 μL of 0.1 mg / mL high molecular weight hyaluronic acid HA was added to the wells seeded with HaCaT cells and HUVEC cells, respectively, and cultured at 37° C. for 24 h.

[0112] The control group (Control) was as follows: 100 μL of high-glucose DMEM and DMEM / F12 culture medium were added to the wells seeded with HaCaT cells and HUVEC cells, respectively, and cultured at 37°C for 24 h.

[0113] After the culture was completed, the experimental group 2 (SPG5.43 group), the positive control group 2 (1 mg / mL hyaluronic acid group) and half of the culture medium in the control group were removed, and then incubated with 100 μL of EdU reagent for 2 h, the liquid was removed, and the cells were fixed with 4% (w / v) paraformaldehyde for 15 min, then permeabilized with 0.3% (v / v) Triton X-100 in PBS for 15 min, and then co-cultured with the click reaction solution at 25°C in the dark for 30 min, and then stained with Hoechst 33342 for 1 h, and then photographed and observed using a fluorescence microscope (Olympus IX50) for each group of cells.

[0114] 2. Effect of Schizophyllan SPG5.43 on HUVEC cell migration

[0115] The blank group (Control) was as follows: 25,000 healthy HUVEC cells in good growth state were seeded in a 24-well plate, and cultured at 37° C. using complete medium until the cells attached to the wall to form a continuous membrane (complete fusion).

[0116] After removing the complete culture medium, the cells were pretreated with mitomycin C for 2 h. After removing the liquid in the wells, vertical scratches were made using a sterile pipette tip (200 μL). The cells were then washed three times with PBS solution. Images of the same scratched area were taken at 0 h, 24 h, 36 h, and 48 h of culture, and the wound area (scratch area) was measured using Image J software. The migration rate of HUVEC was calculated according to Formula II.

[0117] Formula II: Mobility (%) = {(W0-W n ) / W0}×100%;

[0118] Where W0 is the wound area at 0h of culture, W n is the wound area after culture for n hours, n=24, 36 or 48.

[0119] The difference between experimental group 3 and the blank group is that after washing with PBS solution for 3 times, 50 μg / mL, 300 μg / mL, 500 μg / mL and 800 μg / mL of SPG5.43 solution were added respectively.

[0120] The difference between the positive control group 3 (PC) and the blank group is that after washing with PBS solution for 3 times, 1 mg / mL hyaluronic acid solution was added.

[0121] 2. Experimental Results

[0122] 1. Effects on the proliferation of HaCaT cells and HUVEC cells

[0123] (1) Cell proliferation rate test results

[0124] The cell proliferation rate results are shown in the figure Figure 4 As shown, Figure 4 a is the effect of Schizophyllan SPG5.43 on the proliferation rate of HaCaT cells. Figure 4 b shows the effect of SPG5.43 on the proliferation rate of HUVEC cells.

[0125] The results showed that for HaCaT cells, compared with the negative control group (Ctl), after 24 hours of treatment with schizophyllan SPG5.43, 50 μg / mL of SPG5.43 solution significantly increased the viability of HaCaT cells, and the cell proliferation rate reached 152.63%; when the concentration of SPG5.43 reached 200 μg / mL, the cell proliferation rate reached 163.63%, and when the concentration of SPG5.43 reached 300 μg / mL, the cell proliferation rate reached 161.22%.

[0126] For HUVEC cells, compared with the negative control group (Ctl), after 24 hours of treatment with schizophyllan SPG5.43, 50 μg / mL of SPG5.43 solution significantly increased the viability of HUVEC cells, and the cell proliferation rate reached 141.44%; when the concentration of SPG5.43 reached 300 μg / mL, the cell proliferation rate reached 156.57%.

[0127] Moreover, when the concentration of SPG5.43 reached 800 μg / mL, the proliferation rate of HaCaT cells was still 135.12%, and the proliferation rate of HUVEC cells was still 141.50%, which were much higher than the proliferation rate of cells in the positive control group 1 (PC) (122.60% and 131.20%), and there was a significant difference (P < 0.001).

[0128] (2) EdU-594 trial evaluation results

[0129] The results of the EdU-594 test are shown in the figure Figure 5 As shown, Figure 5 a is the fluorescence photography result of HaCaT cells. Figure 5 b is the fluorescence photography result of HUVEC cells.

[0130] The results showed that whether it was the fluorescence photography results for HaCaT cells or HUVEC cells, the proportion of EdU-positive cells (red cells) in the cells treated with 50μg / mL SPG5.43 solution, 300μg / mL SPG5.43 solution and positive control group 2 was significantly higher than that in the control group, and the proportion of EdU-positive cells in the cells treated with 50μg / mL SPG5.43 solution and 300μg / mL SPG5.43 solution was also significantly higher than that in the positive control group.

[0131] It shows that treatment with schizophyllan SPG5.43 can significantly promote the proliferation of HaCaT cells and HUVEC cells.

[0132] 2. Effect of SPG5.43 on the migration of HUVEC cells

[0133] The photographic results of different culture times for the same scratch area are shown in the figure below. Figure 6 As shown in the figure, the mobility results are shown in Figure 7 shown.

[0134] The results showed that after 24 hours of culture, the scratch closure rate (HUVEC cell migration rate) of the blank group was 33.56%, while the scratch closure rate (HUVEC cell migration rate) treated with 500 μg / mL SPG5.43 solution reached 62.16%, and was able to reach 86.13% after 48 hours of culture (P < 0.05); while the positive control group 3 treated with 1000 μg / mL HA had a scratch closure rate of 51.54 after 48 hours of culture, which was not significantly different from the blank group (49.91%).

[0135] The results showed that Schizophyllan SPG5.43 could significantly promote the migration of HUVEC cells and thus promote wound healing.

[0136] Example 3 Effect of Schizophyllan SPG5.43 on Antioxidative Stress of HaCaT Cells and HUVEC Cells

[0137] 1. Experimental Methods

[0138] 1. Effect on the survival rate of cells treated with H2O2

[0139] Healthy and well-growing HaCaT cells were seeded into a 96-well plate at 5000 cells / well, and experimental group 4, blank group, negative control group 1 (NC), model group (MC) and positive control group 4 (PC) were set up.

[0140] H2O2 was added to the cell wells of experimental group 4 to a final concentration of 800 μM, and then 50 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL and 600 μg / mL of SPG5.43 solution were added to the cells in different wells at 100 μL / well and co-cultured for 24 h, and then CCK-8 reagent was added at 10 μL / well and incubated for 1 h. The absorbance of each well at 450 nm was measured using an enzyme reader to obtain the OD 实验组4 .

[0141] Blank group (ctl): The blank group is an empty 96-well plate, and the absorbance OD at 450nm is tested using an ELISA reader 空白组 .

[0142] Negative control group 1 (NC): The negative control group 1 was a 96-well plate inoculated with HaCaT cells, and the absorbance OD at 450nm was measured using an ELISA reader. 阴性 .

[0143] The difference between the model group (MC) and the H2O2 treatment group is that no SPG5.43 solution was added to the cell wells, and the absorbance OD at 450nm was measured using an ELISA reader. 模型 .

[0144] The difference between the positive control group 4 (PC) and the H2O2 treatment group is that the SPG5.43 solution was replaced with an equal amount of 1 mg / mL high molecular weight hyaluronic acid HA, and the absorbance OD at 450 nm was measured using an ELISA reader. 阳性对照组4 .

[0145] The survival rate of cells in each group was calculated according to formula III;

[0146] Formula III: Cell survival rate (%) = (OD m -OD 空白组 ) / (OD 阴性 -OD 空白组 )×100%.

[0147] HaCaT cells were replaced with HUVEC cells, and the survival rate of HUVEC cells treated with SPG5.43 was calculated according to the above method.

[0148] 2. Enzyme activity level test

[0149] (1) Glutathione peroxidase activity test

[0150] The glutathione peroxide detection kit (DTNM method, Beyotime) was used to detect the glutathione peroxide activity of HaCaT cells and HUVEC cells treated with SPG5.43 as follows:

[0151] Healthy and well-growing HaCaT cells were cultured at a rate of 4 × 10 5 / mL were inoculated in a 6-well plate and cultured for 24h. Then, after the cells were treated with H2O2 for 4h, 50μg / mL, 300μg / mL and 800μg / mL of SPG5.43 solution were added to different wells at 2mL / well respectively; 1000μg / mL of hyaluronic acid culture medium solution was added to the positive wells (PC) at 2mL / well; SPG5.43 solution was not added to the model wells (MC); healthy and well-growing HaCaT cells were added to the blank wells (Ctl) without treatment.

[0152] After culturing at 37°C for 24 h, 200 μL of cell lysate was added to each well. After standing on ice for 30 min, the cells were centrifuged at 4°C and 12,000×g for 10 min. The cell supernatant of each well was collected to measure protein concentration and enzyme activity.

[0153] The BCA protein concentration assay kit was used to measure the protein concentration of each well in combination with the cell supernatant according to the kit instructions.

[0154] According to the number of samples, prepare an appropriate amount of BCA working solution by adding 1 volume of BCA reagent B to 50 volumes of BCA reagent A (50:1) and mix thoroughly. Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of the standard to the standard wells of the 96-well plate, and add the standard diluent to make up to 20 μL, which is equivalent to the standard concentration of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL, respectively. Add an appropriate volume of sample to the sample wells of the 96-well plate. If the sample is less than 20 μL, add the standard diluent to make up to 20 μL. Please remember to record the sample volume. Add 200 μL of BCA working solution to each sample well and place at 37°C for 20-30 minutes. Use an enzyme reader to measure the absorbance at A562 wavelength. Calculate the protein concentration of the sample based on the standard curve and the sample volume used.

[0155] The glutathione peroxide activity of the sample was detected by combining the BCA reagent and the protein concentration of the sample according to the instructions of the kit (S0056, Biotime).

[0156] (2) Superoxide dismutase activity test

[0157] Healthy and well-growing HaCaT cells were cultured at a rate of 4 × 10 5 / mL were inoculated in a 6-well plate and cultured for 24h. After the cells in each well were treated with H2O2 (600μM) for 4h, 50μg / mL, 300μg / mL and 800μg / mL of SPG5.43 solution were added to different wells, and high molecular weight hyaluronic acid HA (1000μg / mL) was added to another well as a positive control well. After culturing at 37℃ for 24h, the SOD enzyme activity in each well was detected using a total SOD activity detection kit (WST-8 method, S0101S, Beyotime) according to its instructions.

[0158] 2. Experimental Results

[0159] 1. Survival rate affects results

[0160] The survival rate of HaCaT cells treated with SPG5.43 is shown in the figure below. Figure 8 As shown in the figure, the survival rate of HUVEC cells after treatment with SPG5.43 is shown in Fig. 9 shown.

[0161] The results showed that after HaCaT cells and HUVEC cells were treated with H2O2, the survival rate of cells treated with SPG5.43 was significantly higher than that of cells not treated (model group). After HaCaT cells were treated with 300 μg / mL SPG5.43, the survival rate increased significantly by 168.99% compared with the MC group, and was also significantly better than the survival rate of cells treated with high molecular weight hyaluronic acid HA; while the survival rate of SHUVEC cells increased with the increase of SPG5.43 concentration, and there was no significant difference in the survival rate of cells (PC) treated with high molecular weight hyaluronic acid HA.

[0162] The results showed that Schizophyllan SPG5.43 could significantly improve the survival rate of cells treated with H2O2.

[0163] 2. Enzyme activity test results

[0164] Glutathione peroxide activity test results are shown in the figure Fig.10 As shown in the figure, the superoxide dismutase activity test results are shown in Fig.11 shown.

[0165] The results showed that the activity levels of GSH-Px and SOD in the cells of the model well (MC) were significantly downregulated for both HaCaT cells and HUVEC cells.

[0166] However, the GSH-Px enzyme activity levels and SOD activity levels in cells treated with different concentrations of SPG5.43 showed a significant increase; and the GSH-Px and SOD enzyme activity levels in HaCaT cells and HUVEC cells treated with 300 μg / mL SPG5.43 reached the maximum value. The SOD activity levels in HaCaT cells and HUVEC cells were 2.18 and 7.24 times that of the MC group, respectively, and the GSH-Px activity levels in HaCaT cells and HUVEC cells were 1.61 and 2.54 times that of the MC group, respectively.

[0167] The results showed that SPG5.43 could increase the SOD activity and GSH-Px activity of cells, reduce cell apoptosis, and increase the enzyme activity of cells to counteract the oxidative stress caused by H2O2.

[0168] Example 4 Effect of Schizophyllan SPG5.43 on Wound Healing

[0169] 1. Experimental Methods

[0170] Eight-week-old male BALB / c mice weighing 18 to 22 g were selected to construct an animal model. The hair on the back of the mice was shaved with an electric shaver, and fine hair was removed with a depilatory cream. A circular full-thickness skin wound with a diameter of 8 mm was constructed on the back of the mice using a sterile punch with a diameter of 8 mm to obtain model mice.

[0171] 1 mg treatment group: The wounds of the model mice were treated with a 1 mg / mL SPG5.43 solution, and the SPG5.43 solution was replaced every 3 days.

[0172] 2 mg treatment group: The wounds of the model mice were treated with a 2 mg / mL SPG5.43 solution, and the SPG5.43 solution was replaced every 3 days.

[0173] 4 mg treatment group: The wounds of the model mice were treated with a 4 mg / mL SPG5.43 solution, and the SPG5.43 solution was replaced every 3 days.

[0174] Blank group (Ctl): The wound surface of the model mice was treated with PBS solution, and the PBS solution was replaced every 3 days.

[0175] Positive group (PC): The wounds of the model mice were treated with 3M transparent dressings, and the 3M transparent dressings were changed every 3 days.

[0176] Three mice were set up in parallel in each group, and the wound surface of each group of mice was photographed and observed on the 0th day, 3rd day, 7th day, 14th day and 18th day after treatment. The wound area of ​​each mouse was measured by ImageJ (v1.8.0) software, and the wound healing rate of each mouse was calculated according to Formula IV; the wound healing rate of each group of mice was the average wound healing rate of the mice in the same group.

[0177] Formula IV: Wound healing rate (%) = {(M0-M i ) / M0}×100%;

[0178] Where M0 is the wound area on day 0 after treatment, M i is the wound area on the i-th day after treatment, i=3, 7, 14 or 18.

[0179] 2. Experimental Results

[0180] The wounds of mice in each group were photographed after treatment. Fig.12 The wound healing rate of each group of mice is shown in the figure Fig.13 shown.

[0181] The results showed that compared with the blank group, the wounds of the mice in the 1mg treatment group, 2mg treatment group, 4mg treatment group and positive group were significantly repaired, indicating that SPG5.43 and 3M transparent dressing can promote wound healing; and the wounds of the mice in the 4mg treatment group remained moist on the 3rd day after medication, and the wound healing rate reached 96.65% on the 18th day after medication, and the effect of promoting wound healing was better than that of the positive group and other treatment groups.

[0182] The results show that Schizophyllan SPG5.43 can promote wound healing and can be used to prepare products that promote wound healing. Comparative Example 1 Effects of different polysaccharides on HaCaT cells and HUVEC cells

[0183] 1. Experimental Methods

[0184] According to the preparation method shown in Example 1, Schizophyllan SPG5.43 was prepared. After SPG5.43 was irradiated with ultraviolet light for 30 minutes, it was dissolved in a PBS solution to obtain an SPG5.43 mother liquor. After the SPG5.43 mother liquor was sterilized by high-pressure steam, 100 μg / mL, 400 μg / mL and 800 μg / mL SPG5.43 solutions were prepared using complete culture medium.

[0185] Homopolymer Schizophyllan SPG5.46 (Schizophyllan homopolysaccharide, BA-2005B) was prepared using complete medium to obtain 100 μg / mL, 400 μg / mL and 800 μg / mL SPG5.46 solutions.

[0186] Laminarin (laminarin, G304913) was prepared using complete culture medium to obtain 100 μg / mL, 400 μg / mL and 800 μg / mL laminarin solutions.

[0187] Pullulan (Pullulan, P121048) was prepared using complete medium to obtain 100 μg / mL, 400 μg / mL and 800 μg / mL Pullulan solutions.

[0188] Healthy and well-growing HaCaT cells and HUVEC cells were inoculated into different 96-well cell plates at 5000 cells / well to obtain 96-well cell plates inoculated with cells, and blank group, negative group, SPG-5.43 group, SPG-5.46 group, Laminarin group and Pullulan group were set up respectively, as follows:

[0189] Blank group: Empty 96-well cell plate, and use a microplate reader to measure the absorbance OD of each well at 450nm空白 .

[0190] Negative group: 96-well cell plates were inoculated with cells, and the absorbance OD of each well at 450 nm was measured using an ELISA reader. 阴性 .

[0191] SPG-5.43 group: 100 μg / mL, 400 μg / mL and 800 μg / mL SPG5.43 solutions were added to different 96-well cell plates seeded with cells at 100 μL / well for 24 hours, followed by 10 μL of CCK-8 reagent and 100 μL of serum-free medium. After incubation at 37°C for 1 hour, the absorbance OD of each well at 450 nm was measured using a microplate reader (SpectraMax iD3) SPG-5.43 .

[0192] SPG-5.46 group: The SPG5.43 solution in the SPG-5.43 group was replaced with 100 μg / mL, 400 μg / mL, and 800 μg / mL SPG5.46 solutions, and the same treatment was performed. The absorbance OD of each well at 450 nm was measured using a microplate reader. SPG-5.46 .

[0193] Laminarin group: The SPG5.43 solution in the SPG-5.43 group was replaced with 100 μg / mL, 400 μg / mL, and 800 μg / mL laminarin solutions, and the same treatment was performed. The absorbance OD of each well at 450 nm was measured using a microplate reader. Laminarin .

[0194] Pullulan group: The SPG5.43 solution in the SPG-5.43 group was replaced with 100 μg / mL, 400 μg / mL, and 800 μg / mL Pullulan solutions, and the same treatment was performed. The absorbance OD of each well at 450 nm was measured using a microplate reader. Pullulan .

[0195] Then, the cell proliferation rates in the SPG-5.43 group, SPG-5.46 group, Laminarin group, and Pullulan group were calculated according to formula V;

[0196] Formula V: Cell proliferation rate (%) = (OD a -OD 空白 ) / (OD 阴性 -OD 空白 )×100%; where OD a For OD SPG-5.43 OD SPG-5.46 OD Laminarin or OD Pullulan .

[0197] 2. Experimental Results

[0198] The results of the effects of Schizophyllan SPG5.43, Schizophyllan SPG5.46, laminarin and Prussian polysaccharide on the proliferation rate of HaCaT cells are shown in the figure below. Fig.14 As shown in the figure, the effects of Schizophyllan SPG5.43, Schizophyllan SPG5.46, laminarin and Prussian polysaccharide on the proliferation rate of HUVEC cells are shown in the figure Fig.15 shown.

[0199] The results showed that: (1) The proliferation rate of HaCaT cells treated with 100 μg / mL, 400 μg / mL and 800 μg / mL of Schizophyllan SPG5.43 was significantly higher than that of cells treated with the other three groups of polysaccharides. Among them, the proliferation rate of HaCaT cells treated with 100 μg / mL of SPG5.43 solution was the highest, reaching 155.83%, P < 0.01.

[0200] However, for HaCaT cells treated with homopolymeric schizophyllan SPG5.46, the cell proliferation rate decreased significantly with the increase of SPG5.46 concentration, and the cell proliferation rate was less than 100%; and after HaCaT cells were treated with laminarin and Prussian polysaccharide, although they had a certain proliferation-promoting effect on cells, the cell proliferation rate was generally low.

[0201] (2) The proliferation rates of HUVEC cells treated with 100 μg / mL, 400 μg / mL and 800 μg / mL of Schizophyllan SPG5.43 were significantly higher than those treated with the other three groups of polysaccharides. Among them, the proliferation rate of HUVEC cells treated with 400 μg / mL of SPG5.43 solution was the highest, reaching 157.20%, P < 0.01.

[0202] However, when HUVEC cells were treated with homopolymeric schizophyllan SPG5.46, the cell proliferation rate decreased significantly with the increase of SPG5.46 concentration, and the cell proliferation rate was less than 100%; and after HUVEC cells were treated with laminarin and Prussian polysaccharide, although they had a certain proliferation-promoting effect on cells, the cell proliferation rate was generally low.

[0203] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A Schizophyllan polysaccharide, characterized in that It has two molecular weights of 1.54×10 7 Da and 1.85×10 4 The components of Da are composed of glucose, mannose, galactose and fucose, and the molar ratio of glucose, mannose, galactose and fucose is 86:8:5:

1.

2. A Schizophyllan polysaccharide, characterized in that The preparation method of Schizophyllan comprises the following steps: S1. Fully ferment using Schizophyllum GDMCC NO.5.43, collect the supernatant after the fermentation to obtain Schizophyllum exopolysaccharide fermentation liquid; S2. The Schizophyllum exopolysaccharide fermentation liquid obtained in step S1 is separated into solid and liquid, purified and protein removed to obtain Schizophyllum polysaccharide.

3. The Schizophyllan according to claim 2, characterized in that The fermentation conditions in step S1 are: when fermenting for 0 to 84 hours, the dissolved oxygen is controlled at 20% to 40%; when fermenting for 84 to 108 hours, the dissolved oxygen is controlled at 25% to 50%; and the temperature is controlled at 25 to 30° C. during the fermentation process.

4. Use of the Schizophyllan according to any one of claims 1 to 3 in the preparation of a product for promoting the proliferation of HaCaT cells and / or HUVEC cells.

5. Use of the Schizophyllan according to any one of claims 1 to 3 in the preparation of a product for promoting HUVEC cell migration.

6. Use of the Schizophyllan according to any one of claims 1 to 3 in the preparation of products that promote collagen fiber deposition.

7. Use of the Schizophyllan according to any one of claims 1 to 3 in the preparation of a product that promotes wound healing.

8. A product for promoting the proliferation of HaCaT cells and / or HUVEC cells, characterized in that: Contains ≥50 μg / mL of the Schizophyllan according to any one of claims 1 to 3.

9. The product according to claim 8, characterized in that Contains 50 to 800 μg / mL of the Schizophyllan according to any one of claims 1 to 3.

10. A product for promoting wound healing, characterized in that: Contains 1 to 4 mg / mL of the Schizophyllan according to any one of claims 1 to 3.

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