Schizophyllan polysaccharide and its preparation method and application

Abstract

The present invention discloses 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, with the molar ratio of glucose, mannose, galactose, and fucose being 86:8:5:1. The schizophyllan is a non-homopolymeric polysaccharide with glucose as its main component, which can promote the proliferation of HaCaT cells and HUVEC cells. At the same time, the schizophyllan can significantly accelerate the healing rate of wounds in mice with full-thickness skin wound models, 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%.

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, crucial for maintaining human health and function. However, complications such as infection or excessive inflammation can hinder or delay normal wound healing. Therefore, developing highly effective wound dressings to enhance wound healing has become a key area of ​​biomedical research in recent years.

[0003] Existing wound dressings are primarily made from natural or synthetic materials. Common protein materials include gelatin, collagen, and fibrin, while polysaccharide materials include seaweed polysaccharides, chitosan, and hyaluronic acid. These materials must not only possess good mechanical strength, appropriate porosity, and sufficient flexibility, but also provide a microenvironment conducive to cell migration and proliferation, supporting wound repair and tissue regeneration.

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

[0005] Therefore, the development of biomaterials that can biomimic 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 in the prior art and to provide a schizophyllan polysaccharide and a preparation method and application thereof.

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

[0008] The second object of the present invention is to provide the use of the above-mentioned Schizophyllan in the preparation of a product that promotes 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 through 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, and collect the supernatant after fermentation to obtain Schizophyllum exopolysaccharide fermentation broth;

[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: during fermentation for 0 to 36 hours, the dissolved oxygen is controlled at 40%; during fermentation for 36 to 84 hours, the dissolved oxygen is controlled at 30%; during fermentation 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: Use an inoculating loop to pick aerial hyphae of Schizophyllum and inoculate them onto a solid plate medium. Place the inoculated solid plate medium in a constant temperature incubator and incubate it upside down at 28°C for 5 days. Activate Schizophyllum culture by culturing the entire plate until the hyphae have grown. This culture is then preserved for future use. The solid plate medium contains 30 g / L glucose, 3 g / L yeast extract, 1 g / L KH2PO4, 0.5 g / L MgSO4·H2O, and 18 g / L agar powder.

[0024] S12. Liquid culture of Schizophyllum: Activated Schizophyllum strains were picked with an inoculating loop and inoculated into 100 mL of liquid culture medium. The culture was then placed in a 250 mL culture flask and incubated at 28°C and 160 rpm for 3 days to obtain a primary seed solution, which was retained for later use. The primary seed solution was inoculated into the liquid culture medium at a 5.0% (v / v) inoculum volume. When spikes were observed on the periphery of the bacteria, a secondary seed solution was obtained. The liquid culture medium contained 30 g / L glucose, 3 g / L yeast extract, 1 g / L KH2PO4, and 0.5 g / L MgSO4·H2O.

[0025] S13. Schizophyllum scale-up: The secondary seed solution was inoculated at a 5% (v / v) inoculum into a 5.0 L fermentor containing 3.5 L of culture medium containing 30 g / L glucose, 3 g / L yeast extract, 1 g / L KH2PO4, 0.5 g / L MgSO4·H2O, and 0.05-0.1% (v / v) F1075 (defoaming agent).

[0026] S14. During fermentation h 0-36, the dissolved oxygen (DO) was set to 40.0%. During fermentation h 36-84, the DO was set to 30.0%. At 48 h, glucose was added in batches at a rate of 5.0 g / L, once every 12 h, for a total of three additions. At 84-108 h, the DO was set to 45%. The stirring speed during fermentation was 100-600 rpm, the temperature 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). Fermentation product was obtained after 6 d.

[0027] S15. Preparation of fermentation supernatant: The fermentation product was passed through 8 layers of sterile gauze to remove the Schizophyllum cells, 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 broth.

[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 the Sevag method.

[0029] The Schizophyllum polysaccharide claimed in the present invention is purified and extracted from the fermentation supernatant of the Schizophyllum strain GDMCC NO.5.43. The polysaccharide is a heteropolymeric 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 the use of any of the above-mentioned Schizophyllan in the preparation of a product that promotes HUVEC cell migration.

[0032] At the same time, the schizophyllan polysaccharide claimed 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 protection for 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 protection for the use of any of the above-mentioned Schizophyllan in the preparation of products promoting wound healing.

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

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

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

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

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

[0040] Preferably, it contains 4 mg / mL of any one 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-homopolymeric 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 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 are shown in Figure a; 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 Graphs showing the results of the EdU-594 test evaluation in Example 2; a showing the fluorescence photography results for HaCaT cells; and b showing the fluorescence photography results for HUVEC cells.

[0048] Figure 6 This is a photographic result of different incubation times for the same scratched 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 after treatment with SPG5.43 in Example 3;

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

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

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

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

[0055] Figure 13 The figure shows the wound healing rate of each group of mice in Example 4;

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

[0057] Figure 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 with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0059] All animal experimental procedures involved in the examples of the present invention were approved by the Animal Care 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 aerial hyphae of Schizophyllum GDMCC No. 5.43 and inoculate them onto a solid plate medium. Place the inoculated solid plate medium in a constant temperature incubator and incubate it upside down at 28°C for 5 days. When the hyphae have grown all over the plate, activated Schizophyllum strains are obtained and preserved for future use. 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.

[0065] Liquid culture of Schizophyllum: Use an inoculation loop to pick activated Schizophyllum strains, inoculate them into 100 mL of liquid culture medium, and place them in a 250 mL culture flask. Cultivate 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 a 5.0% (v / v) inoculum and culture until spikes are observed on the periphery of the bacteria, thereby obtaining a second-level seed solution. 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] Schizophyllum scale-up culture: The secondary seed solution was inoculated at a 5% (v / v) inoculum into a 5.0 L fermentor containing 3.5 L of culture medium for fermentation; wherein the culture medium contained 30 g / L glucose, 3 g / L yeast extract, 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%; at the fermentation period of 48 hours, glucose was added in batches at an addition rate of 5.0 g / L, once every 12 hours, for a total of three additions; at the fermentation period of 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;

[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 broth of Schizophyllum obtained in step (1) was centrifuged at 20,000 r / min for 20 min using a high-speed tubular centrifuge, and the supernatant was collected. 500 mL of the supernatant was added to an activated macroporous resin containing 1 mg / mL of 5% macroporous resin AMP-277 (w / w), and the mixture was stirred in a constant temperature water bath at 70°C for 30 min. The mixture was centrifuged at 4,000 r / min for 20 min, the supernatant was collected, and vacuum filtration was performed 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 the free protein was removed by the sevag method. Then, 2 volumes of 95% ethanol (v / v) were added and precipitated at 4°C for 24 hours to obtain a flocculent polysaccharide free of protein. 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 using a tablet press. Fourier transform infrared spectroscopy (FTIR spectroscopy) 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 three times, and the solid was collected and dissolved in deionized water. The solid was filtered through a 0.22 μm microporous filter membrane to obtain the treated sample.

[0077] At Dionex TM CarboPac TM The treated sample was injected into a PA-20 (150×3.0 mm, 10 μm) anion exchange column with a flow rate of 0.5 mL / min, a column temperature of 30°C, and an injection volume of 5.0 μL 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 it, transfer it to a 2.0 L volumetric flask, ultrasonicate it for 10 minutes, and filter it with a 0.22 μm filter membrane to obtain a NaCl solution.

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

[0085] Accurately weigh 5 mg of sample (Schizophyllan SPG5.43 prepared in step 1), dissolve it in 1 mL of NaCl solution to prepare a 5 mg / mL sample solution, vortex 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) using a TSK-gelG3000 PWXL column (column temperature 40°C) for determination. 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 of the schizophyllan prepared in step 1 is 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, which was caused by the stretching vibration of OH. -1 The characteristic sugar absorption peak attributed to CH stretching vibration was detected near 1650cm -1 Nearby is the characteristic absorption peak of bound water in SPG5.43; 1000~1200cm -1 The signal peak near 886cm is the characteristic absorption peak of pyran ring; 1 The characteristic signal peak of β-type glycosidic bond is near it. The results show that the Schizophyllan SPG5.43 prepared in step 1 is a pyranose polysaccharide mainly composed of β-type glycosidic bond.

[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) is the main monosaccharide in Schizophyllan SPG5.43, with a relative content exceeding 80%. In addition to glucose, Schizophyllan SPG5.43 also contains mannose, galactose, and fucose, with relative contents ranging from 1 to 15%. Based on the monosaccharide composition test results shown in Table 2, Schizophyllan SPG5.43 is composed of glucose, mannose, galactose, and fucose in a molar ratio of 86:8:5:1, indicating that SPG5.43 is a glucose-rich heteropolysaccharide.

[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, namely 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 described 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 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 seeded into different 96-well cell plates at 5000 cells / well to obtain 96-well cell plates seeded with cells. Experimental group 1, blank control group, negative control group, and positive control group 1 were set up 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. Then, 10 μL of CCK-8 reagent and 100 μL of serum-free medium were added. After incubation at 37°C for 1 hour, the absorbance 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 consisted of an empty 96-well cell plate, and the absorbance OD at 450 nm was measured using a microplate 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 450 nm was measured using a microplate 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). 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 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 and cultured at 37°C for 24 hours. Then, 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 hours, 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] Control group (Control): 100 μL of high-glucose DMEM and DMEM / F12 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, the culture medium of experimental group 2 (SPG5.43 group), positive control group 2 (1 mg / mL hyaluronic acid group) and half of the control group was 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, the cells were permeabilized with 0.3% (v / v) Triton X-100 in PBS for 15 min. Then, they were co-cultured with click reaction solution in the dark at 25°C for 30 min, stained with Hoechst 33342 for 1 h, and then photographed and observed using a fluorescence microscope (Olympus IX50).

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

[0115] The blank group (Control) was as follows: 25,000 healthy and well-growing HUVEC cells were seeded into 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, a sterile pipette tip (200 μL) was used to vertically scratch the wells. The wells 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 The wound area at the time of 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 three 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 was that after washing with PBS solution three 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 Schizophyllan 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 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%, both of 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 50 μg / mL SPG5.43 solution, 300 μg / 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 50 μg / mL SPG5.43 solution and 300 μg / 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 Schizophyllan 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 as follows 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 reached 86.13% after 48 hours of culture (P < 0.05); while the scratch closure rate of the positive control group 3 treated with 1000 μg / mL HA was 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 Effects of Schizophyllan SPG5.43 on Antioxidative Stress in 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 96-well plates 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. 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 cultured for 24 hours. Then, CCK-8 reagent was added at 10 μL / well and incubated for 1 hour. The absorbance of each well at 450 nm was measured using a microplate reader to obtain the OD value. 实验组4 .

[0141] Blank group (ctl): The blank group is an empty 96-well plate, and the absorbance OD at 450 nm is measured using a microplate 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 a microplate 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 a microplate 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 a microplate 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 after SPG5.43 treatment was calculated according to the above method.

[0148] 2. Enzyme activity level test

[0149] (1) Glutathione peroxidase activity test

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

[0151] Healthy and well-growing HaCaT cells were cultured at a rate of 4 × 10 5 The cells were inoculated at 100 μg / mL in a 6-well plate and cultured for 24 h. After that, the cells were treated with H2O2 for 4 h, and then 50 μg / mL, 300 μg / mL, and 800 μg / mL of SPG5.43 solution were added to different wells at a rate of 2 mL / well. 1000 μg / mL of hyaluronic acid culture medium solution was added to the positive wells (PC) at a rate of 2 mL / well. No SPG5.43 solution was 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 protein concentration of each well was determined using a BCA protein concentration assay kit according to the kit instructions in combination with the cell supernatant of each well to obtain the protein concentration of each well.

[0154] Based on the number of samples, prepare an appropriate amount of BCA working solution by adding 50 volumes of BCA Reagent A to 1 volume of BCA Reagent B (50:1) and mix thoroughly. Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of the standard to the standard wells of a 96-well plate. Add standard diluent to a total of 20 μL, corresponding to standard concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL, respectively. Add the appropriate volume of sample to the sample wells of the 96-well plate. If the sample volume is less than 20 μL, add standard diluent to a total of 20 μL. Be sure to record the sample volume. Add 200 μL of BCA working solution to each sample well and incubate at 37°C for 20-30 minutes. Measure the absorbance at wavelength A562 using a microplate reader. 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 kit instructions using a glutathione kit (S0056, Beyotime).

[0156] (2) Superoxide dismutase activity test

[0157] Healthy and well-growing HaCaT cells were cultured at a rate of 4 × 10 5 Cells were inoculated at 100 μg / mL in 6-well plates and cultured for 24 h. After treating the cells in each well with H2O2 (600 μM) for 4 h, 50 μg / mL, 300 μg / mL and 800 μg / mL of SPG5.43 solution were added to different wells, and high molecular weight hyaluronic acid HA (1000 μg / mL) was added to another well as a positive control well. After culturing at 37°C for 24 h, 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 after treatment 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 the figure Figure 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 Figure 10 As shown in the figure, the superoxide dismutase activity test results are as follows Figure 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, regardless of whether it was HaCaT cells or 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, alleviate 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 18-22 g were selected to construct an animal model. The hair on the back of the mice was shaved with an electric shaver, and fine hairs were removed with a depilatory cream. A sterile 8-mm diameter punch was used to create a circular full-thickness skin wound with a diameter of 8 mm on the back of the mice to obtain the 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 wounds of model mice were 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 dressing, and the 3M transparent dressing was 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, 3rd, 7th, 14th and 18th day after treatment. The wound area of ​​each mouse was measured using 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 the 0th day after treatment, M i is the wound area on the i-th day after treatment, where i=3, 7, 14 or 18.

[0179] 2. Experimental Results

[0180] The wounds of mice in each group were photographed after treatment. Figure 12 The wound healing rates of mice in each group are shown in the figure below. Figure 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 administration, and the wound healing rate reached 96.65% on the 18th day after administration, 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 PBS solution to obtain 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) were prepared using complete medium to obtain 100 μg / mL, 400 μg / mL, and 800 μg / mL SPG5.46 solutions.

[0186] Laminarin (laminarin, G304913) were 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 culture 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 seeded into different 96-well cell plates at 5000 cells / well to obtain 96-well cell plates seeded with cells. Blank group, negative group, SPG-5.43 group, SPG-5.46 group, Laminarin group, and Pullulan group were set up 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 of each well at 450 nm was measured using a microplate 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. Then, 10 μL of CCK-8 reagent and 100 μL of serum-free medium were added. After incubation at 37°C for 1 hour, the absorbance 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, SPG-5.46, Laminarin, and Pullulan groups 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. Figure 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. Figure 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, when HaCaT 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 lower than 100%; and after HaCaT cells were treated with laminarin and Prussian polysaccharide, although they had a certain promoting effect on cell proliferation, 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 of cells 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 lower than 100%; and after HUVEC cells were treated with laminarin and Prussian polysaccharide, although they had a certain promoting effect on cell proliferation, the cell proliferation rate was generally low.

[0203] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art will readily appreciate that other variations or modifications may be made based on the above descriptions and concepts. It is not necessary and impossible to provide an exhaustive list of all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. Application of Schizophyllan in the preparation of products promoting wound healing; The schizophyllan polysaccharide has two components with molecular weights of 1.54×107Da and 1.85×104Da, respectively, and is composed of glucose, mannose, galactose and fucose, with a molar ratio of glucose, mannose, galactose and fucose of 86:8:5:

1. The preparation method of Schizophyllan comprises the following steps: S1. Fully ferment using Schizophyllum GDMCC NO.5.43, and collect the supernatant after fermentation to obtain Schizophyllum exopolysaccharide fermentation broth; S2. The Schizophyllum exopolysaccharide fermentation broth obtained in step S1 was centrifuged at 20,000 rpm for 20 minutes using a high-speed tubular centrifuge, and the supernatant was collected. 500 mL of the supernatant was added to an activated macroporous resin containing 1 mg / mL of 5% macroporous resin AMP-277. The mixture was stirred in a 70°C water bath for 30 minutes, and then centrifuged at 4,000 rpm for 20 minutes. The supernatant was collected and vacuum filtered to obtain a fermentation supernatant. The fermentation supernatant was concentrated using a 70°C vacuum pump at a volume ratio of 5:2 between the original solution and the concentrated solution. Free protein was then removed using the sevag method. Twice the volume of 95% ethanol was added, and the mixture was precipitated at 4°C for 24 hours to obtain a flocculent polysaccharide free of protein. The polysaccharide was washed with 5 mL of 95% ethanol, redissolved in 15 mL of distilled water, and dialyzed with running water and distilled water for 24 hours, respectively. The polysaccharide was then freeze-dried to obtain schizophyllan.

2. The use according to claim 1, characterized in that The fermentation conditions in step S1 are as follows: during fermentation for 0 to 84 hours, the dissolved oxygen is controlled at 20% to 40%; during fermentation 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.

3. A product for promoting the proliferation of HaCaT cells and / or HUVEC cells, characterized in that: Contains ≥50 μg / mL of the Schizophyllan shown in claim 1.

4. The product according to claim 3, characterized in that The invention contains 50 to 800 μg / mL of the Schizophyllan according to claim 1.

5. A product for promoting wound healing, characterized in that: The invention contains 1 to 4 mg / mL of the Schizophyllan according to claim 1.

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