Preparation method and application of gelatin sponge soot polysaccharide

Abstract

The invention discloses a preparation method and application of gelatin soot polysaccharide. The cavernous soot fungus polysaccharide is prepared by adopting cavernous soot fungus degreased powder as a raw material, adopting ultrasonic-assisted alkali extraction, and carrying out solid-liquid separation, concentration, alcohol precipitation, drying and purification to obtain the cavernous soot fungus polysaccharide. The spongy soot fungus polysaccharide prepared by the preparation method is uniform polysaccharide, has obvious effects of resisting oxidation and protecting myocardial cells, can obviously reverse myocardial cell damage caused by oxidative stress and remove excessive reactive oxygen species (ROS), and can be applied to preparation of myocardial cell protective agents.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and more specifically, relates to a preparation method and application of spongiostilbe polysaccharide. Background Art

[0002] Cardiovascular diseases are an important cause of high global mortality and medical costs. Research reports that excessive reactive oxygen species (ROS), including hydrogen peroxide and superoxide anion, are generated during the pathological processes of many cardiovascular diseases, and oxidative stress is a common mechanism for cardiovascular damage caused by many pathological factors. As an important cause of cardiomyocyte death, oxidative stress is an important link in the occurrence of ischemic heart disease and can trigger atherosclerosis, myocardial ischemia / reperfusion injury, etc. Therefore, reducing oxidative stress injury of cardiomyocytes is one of the important strategies for myocardial protection.

[0003] Spongiostilbe is also known as bamboo bird's nest. As a rare edible and medicinal fungus that can form fruiting bodies in the family Capnodiaceae, spongiostilbe has edible, economic and medicinal values and is famous for its unique effects in anti-inflammatory, antibacterial and anti-cancer aspects. It is mainly distributed in the United States, Canada, Thailand, India, Pakistan, and in Yibin, Mianyang, Leshan, Deyang, Mianyang and other places in Guizhou, Jiangsu, Zhejiang and Sichuan provinces of China. Spongiostilbe polysaccharide (SSPs), as its most important metabolite, has significant antioxidant and anti-inflammatory abilities. However, the extraction optimization of SSPs and its efficacy in improving oxidative stress injury of cardiomyocytes have not been reported. Moreover, traditional extraction methods such as hot water extraction have the disadvantages of low yield and low efficiency, which greatly limit the industrial development and application of polysaccharides, and the antioxidant effect of polysaccharides prepared by traditional extraction methods is limited. Summary of the Invention

[0004] The purpose of the present invention is to overcome the above-mentioned deficiencies or defects in the prior art and provide a preparation method of spongiostilbe polysaccharide.

[0005] Another purpose of the present invention is to provide the spongiostilbe polysaccharide.

[0006] Another purpose of the present invention is to provide the application of the spongiostilbe polysaccharide.

[0007] To achieve the above purposes, the present invention adopts the following technical solutions:

[0008] A preparation method of spongiostilbe polysaccharide, comprising the following steps:

[0009] Using defatted powder of spongiostilbe as raw material, adopting ultrasonic-assisted alkali extraction, and obtaining spongiostilbe polysaccharide through solid-liquid separation, concentration, alcohol precipitation, drying and purification.

[0010] The present invention provides a method for preparing spongiostatin polysaccharide. The preparation method uses spongiostatin as a raw material, adopts ultrasonic-assisted alkali extraction, and obtains spongiostatin polysaccharide through solid-liquid separation, concentration, alcohol precipitation, drying, and purification. The obtained spongiostatin polysaccharide is a homogeneous polysaccharide mainly composed of glucose, and has significant antioxidant and cardiomyocyte protection effects, which are significantly superior to other spongiostatin polysaccharides.

[0011] The present invention also provides the preparation method of the spongiostatin polysaccharide, which includes the following steps:

[0012] Using defatted powder of spongiostatin as a raw material, adopting ultrasonic-assisted alkali extraction, and obtaining spongiostatin polysaccharide through solid-liquid separation, concentration, alcohol precipitation, drying, and purification.

[0013] Further, the ultrasonic-assisted alkali extraction is specifically as follows: soaking and swelling the defatted powder of spongiostatin in water, adding sodium hydroxide, and heating for ultrasonic extraction.

[0014] Further, the swelling time is not less than 72 hours.

[0015] Further, the mass-volume ratio of the defatted powder of spongiostatin to water is 1:(50 - 70).

[0016] In some preferred specific embodiments, the mass-volume ratio of the defatted powder of spongiostatin to water is 1:60.

[0017] Further, the heating temperature is 75 - 85 °C.

[0018] Further, the power of the ultrasonic extraction is 300 - 700 W.

[0019] Furthermore, the power of the ultrasonic extraction is 500 - 700 W.

[0020] Furthermore, the power of the ultrasonic extraction is 500 W.

[0021] Further, the time of the ultrasonic extraction is 40 - 80 min.

[0022] The defatted powder of spongiostatin in the present invention can be prepared according to the existing technology, for example, but not limited to, by cleaning, drying the spongiostatin, and then using ethanol for defatting, drying, and sieving.

[0023] In some preferred specific embodiments, the defatted powder of spongiostatin is obtained by cleaning, drying, and pulverizing the spongiostatin, then adding 5 - 6 times (mass-volume ratio) of 95% ethanol for reflux defatting 3 times, each time for 1 hour, filtering, drying, and sieving through a 60-mesh sieve.

[0024] Specifically, the temperature of the reflux degreasing is 65-75 °C.

[0025] The solid-liquid separation in the present invention can adopt common methods, such as but not limited to filtration and / or centrifugation.

[0026] The concentration in the present invention can adopt common methods, such as but not limited to rotary evaporation.

[0027] In some preferred specific embodiments, the concentration is to rotary evaporate and concentrate the supernatant after solid-liquid separation to 1 / 10 of the original volume.

[0028] The alcohol precipitation in the present invention can refer to the prior art, such as but not limited to adding ethanol and standing overnight, and performing solid-liquid separation to obtain a precipitate.

[0029] In some preferred specific embodiments, the alcohol precipitation is to add the concentrated product to 3 times the volume of 95% ethanol, refrigerate and stand overnight, and centrifuge at 5000 r / min to obtain a precipitate.

[0030] Further, the purification is obtained by chromatography column and dialysis purification.

[0031] Further, the eluent of the chromatography column is 0.5 mol / L NaCl solution.

[0032] Further, the dialysis cut-off molecular weight of the dialysis is 3500 Da.

[0033] In some preferred specific embodiments, the purification is to prepare the product after drying the alcohol precipitation into an aqueous solution of 10 mg / mL, load it onto a chromatography column (DEAE Sepharose FF chromatography column, 40*500 mm), elute with 0.5 mol / L NaCl solution, control the flow rate at about 1 mL / min, collect the eluent, concentrate it to 20% of the original volume, and dialyze for 24 h (the dialysis bag cut-off molecular weight is 3500 Da, and deionized water is changed every 8 h); the dialysate is freeze-dried to obtain the purified polysaccharide.

[0034] The present invention also protects the spongiostelium polysaccharide prepared by the above preparation method.

[0035] Further, the spongiostelium polysaccharide is composed of glucose, mannose and galactose in a molar ratio of 100:(0.1-2):(0.1-1), and is a homogeneous polysaccharide, and the molecular weight of the spongiostelium polysaccharide is 30-50 kDa.

[0036] In some preferred specific embodiments, the spongiostelium polysaccharide is composed of glucose, mannose and galactose in a molar ratio of 100:0.47:0.23, specifically as Figure 4 shown.

[0037] In some preferred embodiments, the molecular weight of the spongiostatin polysaccharide is 40-41 kDa.

[0038] Furthermore, the IC 50 value of the spongiostatin polysaccharide for scavenging DPPH free radicals is 0.013±0.011 mg / mL.

[0039] The present invention also protects the application of the spongiostatin polysaccharide prepared by the above preparation method as an antioxidant.

[0040] The present invention also protects the application of the spongiostatin polysaccharide prepared by the above preparation method in the preparation of a cardiomyocyte protectant.

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

[0042] The present invention provides a preparation method of spongiostatin polysaccharide. The preparation method uses spongiostatin defatted powder as a raw material, adopts ultrasonic-assisted alkali ultrasound, and obtains spongiostatin polysaccharide through solid-liquid separation, concentration, alcohol precipitation, drying, and purification. The polysaccharide prepared by the preparation method is mainly composed of glucose, is a homogeneous polysaccharide, has significant antioxidant and cardiomyocyte protection effects, can significantly reverse cardiomyocyte damage caused by oxidative stress, scavenge excessive reactive oxygen species (ROS), and can be applied to the preparation of a cardiomyocyte protectant. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Shows the scavenging effect of spongiostatin polysaccharide prepared by different extraction methods on DPPH free radicals;

[0044] Figure 2 Shows the effect of ultrasonic power on the yield of spongiostatin polysaccharide extracted by ultrasonic-assisted alkali and the scavenging effect on DPPH free radicals; (Polysaccharide yield = mass of spongiostatin polysaccharide / mass of spongiostatin polysaccharide extract

[0045] *100%);

[0046] Figure 3 Shows the molecular weight distribution diagram of spongiostatin polysaccharide prepared in Example 1 and Comparative Examples 1-4;

[0047] Figure 4 Shows the monosaccharide composition diagram of spongiostatin polysaccharide prepared in Example 1 and Comparative Examples 1-4;

[0048] Figure 5 Shows the ultraviolet spectrum diagram of spongiostatin polysaccharide prepared in Example 1;

[0049] Figure 6Infrared spectra of the spongy-gelatinous Capnodium polysaccharide prepared in Example 1 and Comparative Examples 1-4;

[0050] Figure 7 Effects of H2O2 at different concentrations and different stimulation times on the viability of H9C2 cardiomyocytes (*p < 0.05 vs control group (0 μmol / L H2O2 stimulation group or 0 h stimulation group), **p < 0.01 vs control group (0 μmol / L H2O2 stimulation group or 0 h stimulation group));

[0051] Figure 8 Effects of the spongy-gelatinous Capnodium polysaccharide prepared in Example 1 on the viability of H9C2 cardiomyocytes (*p < 0.05 vs

[0052] control group, **p < 0.01 vs control group; #p < 0.05 vs H2O2 group, ##p < 0.01 vs H2O2 group);

[0053] Figure 9 Fluorescence images of the detection of reactive oxygen species (ROS) in H9C2 cardiomyocytes under different treatment conditions.

[0054] Figure 10 Effects of the spongy-gelatinous Capnodium polysaccharide prepared in the example on the fluorescence intensity of reactive oxygen species (ROS) in H9C2 cardiomyocytes (*p < 0.05 vs control group, **p < 0.01 vs control group; #p < 0.05 vs H2O2 group, ##p < 0.01

[0055] vs H2O2 group). Detailed implementation manners

[0056] The present invention will be further elaborated in detail below in conjunction with specific examples. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0057] Capnodium spongiosum is purchased from Yibin, Sichuan.

[0058] Example 1

[0059] A preparation method of a spongy-gelatinous Capnodium polysaccharide, comprising the following steps:

[0060] S1. Wash, dry, and pulverize Capnodium spongiosum to obtain Capnodium spongiosum powder. Take the powder and add 5-6 times the amount of 95% ethanol, reflux and degrease at 70 °C for 3 times, 1 hour each time, then filter, dry, and pass through a 60-mesh sieve to obtain Capnodium spongiosum defatted powder;

[0061] S2. Take 20 g of defatted powder of Hypoxylon spongiosum, add water according to a solid-liquid ratio of 1:60, soak and swell for 72 hours, add sodium hydroxide to adjust the concentration to 1%, perform ultrasonic extraction in water at 80 °C for 1 hour, and then centrifuge to obtain the supernatant; the power of the ultrasonic extraction is 500 W;

[0062] S3. After rotary evaporation and concentration of the supernatant to 1 / 10 of the original volume, add it to 3 volumes of 95% ethanol, refrigerate and let stand overnight, and centrifuge at 5000 r / min to obtain the precipitate part;

[0063] S4. Add 2 volumes of anhydrous ethanol, ether, and acetone (the three solvents are added separately) to the precipitate part, stir and wash each 2 times and centrifuge, and then dry to obtain the polysaccharide extract of Hypoxylon spongiosum;

[0064] S5. Prepare an aqueous solution of the polysaccharide extract of Hypoxylon spongiosum at 10 mg / mL, and load it onto a DEAE Sepharose FF chromatography column (40×500 mm); after column equilibration, elute with a 0.5 mol / L NaCl solution, control the flow rate at about 1 mL / min, collect the eluate, concentrate it to 20% of the original volume, and dialyze for 24 h (the molecular weight cut-off of the dialysis bag is 3500 Da, and the deionized water is changed every 8 h); the dialysate is freeze-dried to obtain, abbreviated as SSPs-UAE-OH.

[0065] Example 2

[0066] A preparation method of polysaccharide of Hypoxylon spongiosum, comprising the following steps:

[0067] The preparation method and parameters are the same as those in Example 1, except that the ultrasonic powers in step S2 are 300 W, 400 W, 600 W, and 700 W respectively.

[0068] Comparative Example 1

[0069] A preparation method of polysaccharide of Hypoxylon spongiosum, comprising the following steps:

[0070] The preparation method and parameters are the same as those in Example 1, except that an equal amount of citric acid is used instead of sodium hydroxide in step S2, and the polysaccharide of Hypoxylon spongiosum prepared is abbreviated as SSPs-UAE-H.

[0071] Comparative Example 2

[0072] A preparation method of polysaccharide of Hypoxylon spongiosum, comprising the following steps:

[0073] The preparation method and parameters are the same as those in Example 1, except that no sodium hydroxide is added in step S2, and the polysaccharide of Hypoxylon spongiosum prepared is abbreviated as SSPs-UAE.

[0074] Comparative Example 3

[0075] A preparation method of spongiocladium polypore polysaccharide, comprising the following steps:

[0076] The preparation method and parameters are the same as those in Example 1, except that in step S2, 20 g of defatted spongiocladium polypore powder is added to water according to a solid-liquid ratio of 1:60, soaked and swollen for 72 hours, stirred at room temperature for 4 hours, and then centrifuged to obtain the supernatant; the obtained spongiocladium polypore polysaccharide is abbreviated as SSPs-HWE.

[0077] Comparative Example 4

[0078] A preparation method of spongiocladium polypore polysaccharide, comprising the following steps:

[0079] The preparation method and parameters are the same as those in Example 1, except that in step S2, 20 g of defatted spongiocladium polypore powder is added to water according to a solid-liquid ratio of 1:60, soaked and swollen for 72 hours, stirred in a water bath at 90 °C for 1 hour, and then centrifuged to obtain the supernatant; the obtained spongiocladium polypore polysaccharide is abbreviated as SSPs-RWE.

[0080] Experimental Example 1 Chemical Composition Analysis

[0081] Using glucose as the standard, the total sugar content of the spongiocladium polypore polysaccharide extract was determined by the sulfuric acid-phenol method, and the standard equation was Y = 1.013X, R 2 = 0.9976; using bovine serum albumin as the standard, the protein content in the spongiocladium polypore polysaccharide extract was determined by the Coomassie brilliant blue method, and the standard equation was Y = 7.8029X - 0.00395, R 2 = 0.9983; using gallic acid as the standard, the polyphenol content in the spongiocladium polypore polysaccharide extract was determined by the Folin-Ciocalteu method, and the standard equation was Y = 12.089X, R 2 = 0.9927; using galacturonic acid as the standard, the uronic acid content in the spongiocladium polypore polysaccharide extract was determined by the sulfuric acid-carbazole method, and the standard equation was Y = 25.7823X - 0.02448, R 2 = 0.9515. The results are shown in Table 1.

[0082] Table 1 Component Analysis of Spongiocladium Polypore Polysaccharide Extract

[0083]

[0084] Note: Different lowercase superscript letters represent statistical differences.

[0085] As can be seen from Table 1, ultrasonic-assisted extraction significantly improved the purity of spongiocladium polypore polysaccharide. Compared with traditional ultrasonic-assisted water extraction, the purity of polysaccharide extracted by ultrasonic-assisted alkali extraction was significantly improved, reaching 76.46 ± 2.75%.

[0086] Experimental Example 2 Antioxidant Effect of Spongiococcum polysaccharide

[0087] (1) 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging ability: Add 2 mL of Spongiococcum polysaccharide aqueous solution (0.5 - 5 mg / mL) to 2 mL of DPPH-ethanol solution (0.1 mmol / L), mix well, and keep in the dark at room temperature for 30 min. Measure the absorbance (A1) at 517 nm. Each sample is tested in triplicate. Calculate the scavenging rate (%) using the formula: Scavenging rate (%) = [1 - (A1 - A2) / A0] × 100% (where A1 is the absorbance value of the Spongiococcum polysaccharide sample, A2 is the absorbance value of the control group with absolute ethanol instead of the DPPH solution, and A0 is the absorbance value of the blank group with distilled water instead of the sample); The sample concentration corresponding to a scavenging rate of 50% is defined as the half-inhibitory concentration (IC 50 ), and the smaller the IC 50 , the higher the activity. The results are as Figure 1 shown, and the test results at different ultrasonic powers are as Figure 2 shown;

[0088] Table 2 IC 50 values of Spongiococcum polysaccharide for scavenging DPPH radicals

[0089]

[0090] As can be seen from Figure 1 , within the concentration range of 0.5 - 5 mg / mL, Spongiococcum polysaccharide extracted by different methods has scavenging effects on DPPH radicals; except for the polysaccharide SSPs-UAE-OH extracted by ultrasonic-assisted alkali method, the scavenging effect of others increases with the increase of sample concentration; for the polysaccharide SSPs-UAE-OH extracted by ultrasonic-assisted alkali method, the scavenging effect reaches the highest at 1 mg / ml. Considering the scavenging IC 50 value, the Spongiococcum polysaccharide provided by the present invention has the best effect.

[0091] From Figure 2 it can be seen that within the ultrasonic power range of 300 - 700 W, with the increase of ultrasonic power, the polysaccharide yield first increases and then slightly decreases, and the antioxidant activity of the polysaccharide gradually increases; when the ultrasonic power reaches 500 W, the polysaccharide yield is the highest, and then it decreases slightly. The preferred ultrasonic power is 500 - 700 W, and the most preferred is 500 W.

[0092] Experimental Example 3 Structure Determination of SSPs-UAE-OH

[0093] (1) Molecular weight determination: The sponge Colletotrichum polysaccharide prepared in Example 1 and Comparative Examples 1-4 was dissolved in deionized water to prepare a 5 mg / mL solution, and the sample was injected through a 0.22 μm microporous filter membrane with an injection volume of 20 μL. The analytical instrument was an Agilent 1260 high-performance liquid chromatograph equipped with a TSKgel GMPWxl (300×7.8 mm i.d.) gel chromatography column and an OpenLAB CDS workstation. The mobile phase was 0.1 mol / L Na2SO4, the flow rate was 0.5 mL / min, the column temperature was 35 °C, and the detector was a differential refractive index detector. Dextrans with different molecular weights (180-135350 Da) were used as standard polysaccharides, and a standard curve was plotted based on the retention time and the logarithm of the molecular weight (LogMp). The linear regression equation of the standard curve was: y = -0.4321x + 12.19, R 2 = 0.992. The results are shown in Figure 3 and Table 3.

[0094] It can be seen from Figure 3 and Table 3 that the sponge Colletotrichum polysaccharide provided by the present invention belongs to a homogeneous polysaccharide with a molecular weight of 40.6151 kDa.

[0095] Table 3 Molecular weight of sponge Colletotrichum polysaccharide

[0096]

[0097] (2) Monosaccharide composition determination: The sponge Colletotrichum polysaccharide prepared in Example 1 and Comparative Examples 1-4 was configured into a 20 mg / mL solution, which was mixed with trifluoroacetic acid aqueous solution (4 mol / L) in a volume ratio of 1:1, sealed, and degraded at about 110 °C for 4 h; after the degradation was completed, 0.2 mL was taken, dried, redissolved, and derivatized with PMP-methanol solution at 70 °C under alkaline conditions; after the reaction was completed, the pH was adjusted to neutral, and chloroform was extracted three times repeatedly. The aqueous solution was filtered through a 0.22 μm microporous filter membrane for HPLC analysis;

[0098] Analytical instrument and chromatographic conditions: An Agilent 1260 high-performance liquid chromatograph equipped with a Zorbax KP-C 18 chromatographic column (150 mm×4.6 mm, 5 μm) (including a quaternary pump, an autosampler, a column oven, an ultraviolet detector, Agilent, USA); the mobile phase was phosphate buffer solution (0.1 mol / L, pH 7.0) / acetonitrile (83 / 17, V / V); the flow rate was 0.8 mL / min; the column temperature was 30 °C; the injection volume: 20 μL; ultraviolet detector (254 nm), and the test results are shown in Figure 4 and Table 4.

[0099] It can be seen from Figure 4It can be seen that the polysaccharide of Capnodium spongiaeforme mainly consists of glucose, mannose and galactose in a molar ratio of 100:0.47:0.23.

[0100] Table 4 Molar ratio of monosaccharide composition in the polysaccharide of Capnodium spongiaeforme

[0101]

[0102] (3) Ultraviolet spectrum analysis: The polysaccharide of Capnodium spongiaeforme prepared in Example 1 was formulated into a 0.5 mg / mL solution, and the ultraviolet spectrum was scanned at 1900 - 800 nm. The instrument was an ultraviolet-visible spectrophotometer (T600, Purkinje General Instrument Co., Ltd.), and the results are as Figure 5 shown.

[0103] From Figure 5 it can be seen that the polysaccharide of Capnodium spongiaeforme prepared in the present invention has a strong absorption peak at 200 nm, which is the characteristic ultraviolet absorption peak of polysaccharides; there is a very weak absorption peak at 280 nm, indicating that the polysaccharide extract of Capnodium spongiaeforme in the present invention contains a small amount of protein, which is consistent with the component analysis results in Table 1.

[0104] (4) Infrared spectrum analysis: 2 mg of the polysaccharide of Capnodium spongiaeforme prepared in Example 1 and Comparative Examples 1 - 4 was mixed with 100 mg of dry spectroscopic pure potassium bromide and pressed into a tablet, and the infrared spectrum was scanned in the range of 4000 - 400 cm -1 The analysis instrument was a Fourier transform infrared spectrometer (FTIR-650, Gangdong), and the test results are as Figure 6 shown.

[0105] From Figure 6 it can be seen that the strong absorption peak near 3305 cm -1 is caused by the stretching vibration of OH; the absorption peak at 2904 cm -1 is caused by the stretching or bending vibration of C-H; the absorption peak at 1607 cm -1 represents COOH; the absorption peak at 1374 cm -1 represents symmetric COO-; the absorption peak at 1076 cm -1 is the characteristic absorption peak of the main chain pyranose ring; the absorption peak at 1018 cm -1 is the absorption peak of the side chain furanose ring. Infrared spectrum analysis further confirms that the polysaccharide of Capnodium spongiaeforme in the present invention conforms to the polysaccharide structure characteristics.

[0106] Experimental Example 4 Protective effect of the polysaccharide of Capnodium spongiaeforme on oxidative stress injury of cardiomyocytes

[0107] Construction of cardiomyocyte oxidative damage model: Rat cardiomyocytes H9C2 were inoculated in a culture dish. After the cell confluence reached 80% - 90%, cells in the logarithmic growth phase were taken at 3×103 Cells were inoculated in a cell culture plate at a density of cells / well and divided into 5 groups: blank control group, H2O2 treatment group, and H2O2 + Sphaeropsis sapinea polysaccharide treatment groups with different concentrations. They were adherently cultured in a conventional incubator (37 °C, 5% CO2) with DMEM complete medium containing 10% serum for 12 h. After pretreatment with different concentrations of SSPs-UAE-OH for a certain time, a certain concentration of H2O2 was added to induce cell damage. Cells were collected after 12 h for subsequent experiments.

[0108] The effect of Sphaeropsis sapinea polysaccharide on the proliferation ability of H9C2 cells was detected by CCK8 method: The cell culture medium and CCK8 reagent were mixed at a ratio of 9:1 to prepare a detection reagent. 100 μL was added to each well and incubated in the incubator for 2 h, and the absorbance value at 450 nm was detected.

[0109] Cell viability = average optical density value of the drug administration well / average optical density value of the control group × 100%

[0110] H9C2 cardiomyocytes were treated with different concentrations (0, 100, 200, 400, 800 μmol / L) of H2O2 for 12 h, and the activity of cardiomyocytes was measured by CCK-8. The results are shown in Figure 7 Figure A. The results showed that compared with the blank group, after intervention with 100 μmol / L of H2O2, the cell activity of cardiomyocytes decreased slightly, with no statistical difference. When the concentration increased to 200 μmol / L, the decrease in cardiomyocyte activity was statistically different from that of the control group. When the H2O2 concentration was 400 μmol / L, the activity of cardiomyocytes was significantly inhibited, and the cell viability decreased to about 50%. When the H2O2 concentration was 800 μmol / L, most cardiomyocytes had died. Therefore, the H2O2 concentration in this study was set at 400 μmol / L.

[0111] When the H2O2 concentration was 400 μmol / L, H9C2 cardiomyocytes were treated for different durations (0, 6, 12, 24 h). The results are shown in Figure 7 Figure B. Compared with the blank group, the activity of cardiomyocytes decreased with the prolongation of the action time of H2O2. When the treatment time exceeded 12 h, the activity of cardiomyocytes decreased significantly. In summary, in subsequent experiments, the H2O2 concentration was 400 μmol / L and the stimulation time was 12 h.

[0112] After pretreatment with Sphaeropsis sapinea polysaccharide at different concentrations (50, 100, 200, 400, 1000 μg / ml) for 2 h, H9C2 cells were stimulated with 400 μmol / L H2O2 for 12 h, and the activity of cardiomyocytes in each group was measured by CCK-8. The results are shown in Figure 8As shown, the experimental results indicate that compared with the blank group, the viability of H9C2 cells in the H2O2 group decreased, showing a statistically significant difference. Compared with the H2O2 group, the viability of H9C2 cells in the sponge coal tar polysaccharide intervention groups with concentrations of 100, 200, 400, and 1000 μg / ml increased significantly, showing a statistically significant difference.

[0113] Detection of intracellular reactive oxygen species (ROS): Remove the cell culture medium, add serum-free medium with a final concentration of 10 μmol / L DCFH-DA, and incubate in a 37°C cell culture incubator for 20 min. Wash the cells three times with serum-free medium, and observe the fluorescence intensity of cells in each group using an inverted fluorescence microscope.

[0114] After pretreating H9C2 cells with sponge coal tar polysaccharide at different concentrations (50, 100, 200, 400 μg / ml) for 2 h, stimulate the H9C2 cells with 400 μmol / L H2O2 for 12 h, perform fluorescence staining and observe with an inverted fluorescence microscope. The fluorescence images are as Figure 9 shown. Quantify using Image Pro Plus image analysis software ( Figure 10 ). The experimental data show that compared with the blank group, the intracellular reactive oxygen species (ROS) in H9C2 cells in the H2O2 group increased significantly, showing a statistically significant difference. Compared with the H2O2 group, the intracellular reactive oxygen species (ROS) in H9C2 cells in the sponge coal tar polysaccharide intervention groups with concentrations of 100, 200, 400 μg / ml decreased significantly, showing a statistically significant difference. Among them, the sponge coal tar polysaccharide at 100 μg / ml had the best activity.

[0115] In summary, the sponge coal tar polysaccharide of the present invention is simple to prepare and has the best antioxidant activity; the ultrasonic-assisted alkali extraction sponge coal tar polysaccharide is a homogeneous polysaccharide, has a good inhibitory effect on oxidative damage of cardiomyocytes, is a leading compound for the development of an ideal cardioprotective agent, and has good application prospects.

[0116] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for preparing sponge glue coal soot fungus polysaccharide, characterized in that: The steps include: The defatted powder of coal soot fungus was used as raw material, and the polysaccharide of coal soot fungus was obtained by ultrasonic-assisted alkali extraction, solid-liquid separation, concentration, alcohol precipitation, drying and purification.

2. The preparation method according to claim 1, characterized in that: The ultrasonic-assisted alkali extraction is specifically as follows: soaking the defatted powder of coal sponge soot bacteria in water to swell it, adding sodium hydroxide, and heating and ultrasonic extraction.

3. The preparation method according to claim 2, characterized in that: The mass volume ratio of the sodium hydroxide and water is (0.5-1.5):

100.

4. The preparation method according to claim 2, characterized in that: The heating temperature is 75-85°C.

5. The preparation method according to claim 2, characterized in that: The power of the ultrasonic extraction is 300-700W.

6. The preparation method according to claim 2, characterized in that: The ultrasonic extraction time is 40 to 80 minutes.

7. The preparation method according to claim 2, characterized in that: The purification is performed by chromatography column and dialysis.

8. A sponge glue coal soot fungus polysaccharide, characterized in that: The method is prepared according to any one of claims 1 to 7.

9. Use of the sponge glue coal soot fungus polysaccharide prepared by the preparation method according to any one of claims 1 to 7 as an antioxidant.

10. Use of the polysaccharide of Coal Marrow obtained by the preparation method according to any one of claims 1 to 7 in the preparation of a myocardial cell protective agent.

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