Schizophyllum commune fermentation broth polysaccharide-selenium nanoparticle compound with immunoregulation and oxidative damage resisting activity as well as preparation and application of schizophyllum commune fermentation broth polysaccharide-selenium nanoparticle compound

Abstract

The invention discloses a schizophyllum commune fermentation broth polysaccharide-selenium nanoparticle compound with immunoregulation and oxidative damage resisting activity as well as preparation and application thereof. The polysaccharide-selenium nanoparticle compound is prepared from schizophyllum commune fermentation broth polysaccharide, sodium selenite and L-ascorbic acid through an oxidation-reduction method. The reaction conditions are mild, preparation of the polysaccharide-selenium nanoparticle compound can be achieved through a one-step method, and the obtained polysaccharide-selenium nanoparticle compound has remarkable growth promotion and immunoregulation effects on macrophages, has a good repairing effect on oxidative damage nematodes, and has a good application prospect. Important development and utilization prospects are realized in the fields of health-care foods and medicines.

Description

Technical Field

[0001] The invention relates to a Schizophyllum fermentation broth polysaccharide-selenium nanoparticle complex having both immunomodulatory and antioxidant damage activities, a preparation method and application thereof, and belongs to the technical field of food and medicine. Background Art

[0002] The immune system is the most effective "weapon" for the human body to defend against pathogen invasion. Its immune surveillance function can identify foreign invading antigens and remove mutated cells, aging cells, dead cells and other harmful components in the body, thereby protecting the human body from viruses, bacteria and diseases. While removing pathogens, the immune system will suffer certain damage, but it can repair itself under the regulation of the body. Therefore, it is crucial to promote the improvement of the body's self-immunity. There are many products on the market that can improve human immunity, but the types and quantities of natural immunomodulators are limited. The research and development of efficient and non-toxic natural immunomodulators is of great significance.

[0003] Selenium, as an important component of glutathione peroxidase, is one of the essential trace elements for the human body. It has multiple biological activities such as improving the body's immunity, anti-tumor, and anti-oxidation. Selenium deficiency may cause cancer, cardiovascular, cataract and other diseases. Common inorganic selenium includes selenite, selenide and elemental selenium, which have certain toxicity, and selenium nanoparticles can reduce the toxicity of selenium to a certain extent. However, due to the high surface energy of selenium nanoparticles, pure selenium nanoparticles will quickly aggregate to form black or gray elemental selenium, and its biological activity, biocompatibility and bioavailability will be significantly reduced. Therefore, a certain concentration of stabilizer is required to maintain the structure of selenium nanoparticles. Natural polysaccharide molecules contain a large number of hydroxyl, carboxyl and amino groups. These groups can well enhance the stability of selenium nanoparticles. At the same time, the combination of polysaccharides and selenium nanoparticles can also have a nano-size effect. For example, polysaccharide-selenium nanoparticles can enhance the endocytosis of cells and increase their uptake capacity to better exert the immunomodulatory effect.

[0004] Schizophyllum commune, also known as white ginseng fungus, is a large fungus with high nutritional and medicinal value. However, its yield is limited by traditional agricultural cultivation and is restricted by seasons and environment. Studies have found that the polysaccharide of Schizophyllum commune fermentation broth prepared by liquid fermentation technology has good pharmacological effects, such as effectively inhibiting the growth of gastric cancer, rectal cancer and other digestive tract cancer cells, lowering blood pressure, blood lipids, blood sugar, resisting thrombosis, improving cellular immune function, promoting the formation of immunoglobulins, stimulating the activity of mononuclear phagocytes, enhancing macrophage phagocytosis and other physiological activities. At present, the polysaccharide of Schizophyllum commune fermentation broth, namely schizophyllum commune, has been commercialized and developed. In some countries such as Japan, it is used as a clinical drug for immune regulation of the body and has a good development prospect. However, the polysaccharide of Schizophyllum commune fermentation broth has a large molecular weight, high viscosity and poor water solubility, which seriously restricts its wide application. In the present invention, polysaccharide-selenium composite nanoparticles of Schizophyllum fermentation broth are prepared by a one-step method, and it is confirmed that the composite nanoparticles have both immunomodulatory and antioxidant damage effects, which have not been reported so far, and thus have important development and utilization value. Summary of the invention

[0005] On the basis of the above-mentioned prior art, the present invention provides a Schizophyllum fermentation broth polysaccharide-selenium nanoparticle complex having both immunomodulatory and antioxidant damage activities and a preparation method thereof, aiming to enhance the immunological activity of Schizophyllum fermentation broth polysaccharide and provide another selenium nanoparticle stabilizer.

[0006] In order to achieve the purpose, the present invention adopts the following technical scheme:

[0007] A method for preparing a polysaccharide-selenium nanoparticle complex of Schizophyllum fermentation broth having both immunomodulatory and antioxidant damage activities comprises the following steps:

[0008] Step 1, fully stirring 0.4-5 mg / mL of Schizophyllum fermentation broth polysaccharide solution and 5-25 mg / mL of sodium selenite solution at 25-60° C. for 0.5-1.5 h to obtain a mixed solution;

[0009] Step 2, adding 5-20 mg / mL of L-ascorbic acid solution to the mixed solution obtained in step 1, and continuing to stir and react at 25-60° C. for 3-5 hours to obtain a Schizophyllum fermentation broth polysaccharide-selenium nanoparticle complex having both immunomodulatory and antioxidant damage activities;

[0010] Furthermore, the volume ratio of the Schizophyllum fermentation broth polysaccharide solution, the sodium selenite solution and the L-ascorbic acid solution is 1-2 mL: 2-4 mL: 4-7 mL.

[0011] Furthermore, the polysaccharide from the fermentation broth of Schizophyllum in step 1 is obtained according to the following steps:

[0012] (1) Take a diameter of 1 to 2 cm2 The solid plate of Schizophyllum mycelium was inoculated into 150 mL YPD liquid medium, cultured at 25-30° C. with shaking for 5-7 days, centrifuged at 1000-5000 rpm for 3-5 minutes, and the supernatant was collected to obtain the Schizophyllum fermentation liquid;

[0013] (2) After the Schizophyllum fermentation liquid obtained in step (1) is concentrated to 1 / 12 to 1 / 5 of its volume, anhydrous ethanol is added to the concentrated liquid until the final volume concentration of ethanol is 60 to 80%, the liquid is allowed to stand at 4° C. for 6 to 8 hours, and centrifuged at 3000 to 7000 rpm for 5 to 10 minutes to collect the precipitate;

[0014] (3) The precipitate obtained in step (2) was dissolved in deionized water, dialyzed using a 1-5 kDa dialysis bag, and then freeze-dried or vacuum-dried to obtain a precipitate with a molecular weight mainly distributed in the range of 2.5×10 5 to 5.6×10 5 The polysaccharide of Schizophyllum fermentation broth is mainly composed of five monosaccharides, namely fucose, rhamnose, galactose, mannose and glucose, with a molar ratio of 1:1.21:3.36:26.2:26.45.

[0015] The polysaccharide-selenium nanoparticle complex of the fermentation broth of Schizophyllum obtained by the preparation method of the present invention has a particle size between 20 and 80 nm and can be used to prepare an immunomodulator and / or an oxidative damage repair agent.

[0016] The beneficial effects of the present invention are embodied in:

[0017] 1. The polysaccharide obtained from the fermentation broth of Schizophyllum truncatum has good immune activity and a stable polysaccharide structure, and can be combined with selenium nanoparticles to form a Schizophyllum truncatum fermentation broth polysaccharide-selenium nanoparticle complex with good dispersion, and the complex has significantly enhanced immune activity.

[0018] 2. The polysaccharide-selenium nanoparticle complex prepared by the method of the present invention has a nanoparticle size of 20 to 80 nm as measured by a Malvern nano-laser particle size analyzer, and has a concentrated particle size distribution and good stability.

[0019] 3. The polysaccharide-selenium nanoparticle complex prepared by the method of the present invention further improves the immune activity and anti-oxidative damage ability on the basis of the polysaccharide of Schizophyllum fermentation broth, and can be used to improve the antioxidant capacity of the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the liquid phase diagram of SPG-1 obtained in Example 1. It can be seen that the molecular weight of the polysaccharide is distributed in the range of 2.5×10 5 to 5.6×10 5 Between big.

[0021] Figure 2 This is the monosaccharide composition analysis of SPG-1 obtained in Example 1, where (A) is the liquid phase diagram of the mixed monosaccharide standard, and (B) is the liquid phase diagram of the monosaccharide composition of SPG-1.

[0022] Figure 3 This is a photograph of the SPG-1 / SeNPs solution obtained in Example 3 and a graph showing its particle size distribution.

[0023] Figure 4 This is a graph showing the particle size distribution of the polysaccharide-selenium nanoparticle complex obtained from the fermentation broth of Schizophyllum corytatum in Example 4.

[0024] Figure 5 The TEM and SEM images of the Schizophyllum fermentation broth polysaccharide-selenium nanoparticles composite obtained in Example 4. (A) and (B) are TEM images of the composite, and (C) is a SEM image of the composite.

[0025] Figure 6 This is the toxicity test of SPG-1 and SPG-1 / SeNPs on RAW264.7 cells in Example 5.

[0026] Figure 7 This is the detection of the NO release amount secreted by RAW264.7 cells by SPG-1 and SPG-1 / SeNPs in Example 5.

[0027] Figure 8 This is the detection of the phagocytic effect of SPG-1 / SeNPs on RAW264.7 cells in Example 5.

[0028] Fig. 9 This is the morphological observation of SPG-1 / SeNPs after phagocytosis of RAW264.7 cells in Example 5.

[0029] Fig.10 This is the toxicity test of SPG-1 and SPG-1 / SeNPs to Caenorhabditis elegans in Example 6.

[0030] Fig.11 In Example 6, SPG-1 / SeNPs were used to treat H 2 O 2 Effects of damaged Caenorhabditis elegans on SOD enzyme activity.

[0031] Fig.12 The SPG-1 / SeNPs to H 2 O 2 Effects of injury on GSH-Px enzyme activity in Caenorhabditis elegans.

[0032] Fig.13 The SPG-1 / SeNPs to H 2 O2 Effects of injury on CAT enzyme activity in Caenorhabditis elegans.

[0033] Fig.14 The SPG-1 / SeNPs to H 2 O 2 Effects of damaged Caenorhabditis elegans on MDA content.

[0034] Fig.15 The SPG-1 / SeNPs to H 2 O 2 Effects of inducing ROS activity (ROS) in Caenorhabditis elegans.

[0035] Fig.16 The SPG-1 / SeNPs to H 2 O 2 Effects of inducing lipofuscin in Caenorhabditis elegans . DETAILED DESCRIPTION

[0036] The technical solution of the present invention is further described below in conjunction with specific embodiments. The following embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0037] Example 1: Preparation of polysaccharides from Schizophyllum fermentation broth

[0038] 1. Place a 1cm diameter 2 The solid plate of Schizophyllum mycelium was inoculated into 150 mL YPD liquid culture medium, cultured at 28°C with shaking for 7 days, centrifuged at 1000 rpm for 5 min, and the supernatant was taken to obtain the Schizophyllum fermentation broth.

[0039] 2. Concentrate the fermentation liquid of Schizophyllum to 1 / 8 of its volume, add anhydrous ethanol to the obtained concentrated liquid until the final volume concentration of ethanol is 75%, let it stand at 4°C for 8h, centrifuge at 4000rpm for 10min, discard the supernatant and collect the precipitate.

[0040] 3. Add 200 mL of deionized water to the precipitate obtained in step 2 to dissolve it, dialyze it using a 3.5 kDa dialysis bag for 3 days, and freeze-dry it to obtain the Schizophyllum fermentation broth polysaccharide (SPG-1).

[0041] 4. Prepare the SPG-1 obtained in step 3 into a 5 mg / mL solution, filter it with a 0.22 μm microporous membrane, and analyze its molecular weight by high performance liquid chromatography. Figure 1 SPG-1 is a heterogeneous polysaccharide, mainly composed of a large peak, with a peak time of about 12.521-13.967 min. The molecular weight of SPG-1 is mainly distributed in the range of 2.5×10 5~5.6×10 5 Da. Compare the liquid phase diagram of SPG-1 degraded by trifluoroacetic acid with the liquid phase diagram of monosaccharide standard, such as Figure 2 As shown, it is speculated that SPG-1 is mainly composed of fucose, rhamnose, galactose, mannose and glucose with a molar ratio of 1:1.21:3.36:26.2:26.45.

[0042] Example 2: Preparation of polysaccharides from Schizophyllum fermentation broth

[0043] 1. Place a 1cm diameter 2 The solid plate of Schizophyllum mycelium was inoculated into 150 mL YPD liquid culture medium and cultured with shaking at 25°C for 5 days. Centrifuged at 3000 rpm for 3 minutes, and the supernatant was taken to obtain the Schizophyllum fermentation broth.

[0044] 2. Concentrate the fermentation broth of Schizophyllum to 1 / 10 of its volume, add anhydrous ethanol to the concentrated solution until the final volume concentration of ethanol is 80%, let it stand at 4°C for 6 hours, centrifuge at 6000 rpm for 5 minutes, discard the supernatant, and collect the precipitate.

[0045] 3. Add 100 mL of deionized water to the precipitate obtained in step 2 to dissolve it, dialyze it using a 1 kDa dialysis bag for 4 days, and vacuum dry it to obtain the polysaccharide of the fermentation broth of Schizophyllum.

[0046] 4. The polysaccharide in the fermentation broth of Schizophyllum obtained in step 3 was prepared into a 5 mg / mL solution, filtered with a 0.22 μm microporous filter membrane, and analyzed by high performance liquid chromatography. The peak time of the polysaccharide in the fermentation broth of Schizophyllum was mainly concentrated in 12.521-13.967 min.

[0047] Example 3: Preparation of Schizophyllum fermentation broth polysaccharide-selenium nanoparticle complex

[0048] 1. 1.5 mL of 0.5 mg / mL Schizophyllum fermentation broth polysaccharide solution (prepared using SPG-1 prepared in Example 1) and 2.6 mL of 5 mg / mL sodium selenite solution were stirred and mixed at 40° C. for 1 h to obtain a mixed solution.

[0049] 2. Add 5.2 mL of 10 mg / mL L-ascorbic acid solution to the mixture obtained in step 1, and continue stirring the reaction at 40°C for 4 h to obtain a Schizophyllum fermentation broth polysaccharide-selenium nanoparticle complex (SPG-1 / SeNPs) with both immunomodulatory and antioxidant damage activities. The particle size of the obtained nanoparticles was measured using a Malvern nano-laser particle size analyzer. The results are as follows: Figure 3 As shown, it can be seen that the particle size of the polysaccharide-selenium nanoparticle complex of the Schizophyllum fermentation broth is 30-65 nm, and the particle size distribution is concentrated.

[0050] Example 4: Preparation of Schizophyllum fermentation broth polysaccharide-selenium nanoparticle complex

[0051] 1. 1.8 mL of 1 mg / mL Schizophyllum fermentation broth polysaccharide solution (prepared using SPG-1 prepared in Example 1) and 2.8 mL of 10 mg / mL sodium selenite solution were fully stirred at 40° C. for 1 h to obtain a mixed solution.

[0052] 2. Add 5.6 mL of 20 mg / mL L-ascorbic acid solution to the mixture obtained in step 1, and continue stirring the reaction at 40°C for 3 h to obtain a Schizophyllum fermentation broth polysaccharide-selenium nanoparticle complex with both immunomodulatory and antioxidant damage activities. The particle size of the obtained nanoparticles was measured using a Malvern nano-laser particle size analyzer. The results are as follows: Figure 4 As shown in the figure, it can be seen that the particle size of the polysaccharide-selenium nanoparticle complex of the fermentation broth of Schizophyllum is 20-70 nm, and the particle size distribution is concentrated. The morphology of the obtained nanoparticles was observed by scanning electron microscopy and transmission electron microscopy. The results are shown in Figure 5 As shown, it can be seen that the morphology of the Schizophyllum fermentation broth polysaccharide-selenium nanoparticle complex is approximately spherical, with a diameter of about 67nm. It is speculated that the obtained nanoparticles are Schizophyllum fermentation broth polysaccharides attached to the outside of the selenium core to form a thin film, thereby stabilizing the selenium nanoparticles.

[0053] Example 5: In vitro immunological activity determination of the polysaccharide-selenium nanoparticle complex obtained from the fermentation broth of Schizophyllum japonicum in Example 3

[0054] 1. Cytotoxicity Assay

[0055] RAW264.7 cells were cultured at 1×10 5 Cells / well were inoculated in a 96-well plate (100 μL per well). After culturing for 24 hours, 100 μL of increasing concentrations of Schizophyllum fermentation broth polysaccharide or Schizophyllum fermentation broth polysaccharide-selenium nanoparticle complex was added (measured according to the concentration of polysaccharide in the complex, 2-10 μg / mL). 40 μg / mL SeNPs was used as the control group, and an equal amount of complete culture medium was used as the blank control group for 24 hours. The cell survival rate was determined by the MTT method. After 24 hours, the drug was aspirated, washed 2-3 times with PBS, 100 μL of MTT solution (0.5 mg / mL) was added to each well, and the culture was continued for 4 hours. The supernatant was discarded, 150 μL of dimethyl sulfoxide was added to each well, and the shaker was shaken at low speed for 10 minutes to fully dissolve the crystals. The absorbance at 570 nm was measured by an enzyme marker. The results of the toxic effects of Schizophyllum fermentation broth polysaccharide and Schizophyllum fermentation broth polysaccharide-selenium nanoparticle complex on macrophages are shown in the figure. Figure 6As shown, when the concentration of Schizophyllum fermentation broth polysaccharide in the nanoparticles was 2-10 μg / mL, SPG-1 and SPG-1 / SeNPs had no toxic effect on macrophages, while SeNPs without SPG-1 had obvious toxicity to macrophages, inhibiting the growth of macrophages with cell viability at only 61.3%. After adding SPG-1, the stability of SeNPs was increased while its toxic effect on cells was reduced, and it promoted the proliferation of macrophages at a certain concentration.

[0056] 2. Determination of NO generation

[0057] The amount of NO produced was detected by Griess method. RAW264.7 cells were cultured at 1×10 4 Cells / well were inoculated in a 96-well plate (100 μL per well), cultured for 24 h, and treated with different concentrations of Schizophyllum polysaccharide and Schizophyllum fermentation broth polysaccharide-selenium nanoparticle complexes (measured according to the concentration of polysaccharide in the complex, 2-10 μg / mL) for 24 h. A positive control group (1 μg / mL) and a blank control group were set up. The cell culture supernatant was collected and mixed with an equal volume of Griess reagent, fully reacted at room temperature for 30 min, and the absorbance was measured at 540 nm with an enzyme reader. The results of the detection of the amount of NO secreted by RAW264.7 cells by Schizophyllum fermentation broth polysaccharide and Schizophyllum fermentation broth polysaccharide-selenium nanoparticle complexes are shown in Figure 2. Figure 7 As shown, the production of NO in the blank control was lower than that in the LPS positive control group, Schizophyllum polysaccharide and Schizophyllum fermentation broth polysaccharide-selenium nanoparticle complex treatment groups. At the same time, after treatment with different concentrations of Schizophyllum fermentation broth polysaccharide-selenium nanoparticle complex, the release of NO from macrophages was concentration-dependent, indicating that the Schizophyllum fermentation broth polysaccharide-selenium nanoparticle complex had a promoting effect on the production of NO in RAW264.7 cells, and Schizophyllum polysaccharide was prepared into selenium nanoparticles, which improved the immune activity of Schizophyllum polysaccharide.

[0058] 3. Phagocytic activity assay and morphological observation

[0059] The phagocytic ability of macrophages was detected by neutral red uptake assay. RAW264.7 cells (1×10 4 Cells / well) were placed in a 96-well plate and cultured for 24 hours. The Schizophyllum fermentation broth polysaccharide-selenium nanoparticle complex (measured at the concentration of polysaccharide in the complex, 2-10 μg / mL) was used for treatment for 24 hours, and a positive control group LPS (1 μg / mL) and a blank control group were set up. The drug was discarded, and 100 μg / L 0.05% neutral red solution was added and cultured for another 20 minutes. The neutral red solution was discarded, the cells were washed three times with PBS, and observed under a fluorescent inverted microscope. The results of the phagocytic effect of the Schizophyllum fermentation broth polysaccharide-selenium nanoparticle complex on RAW264.7 cells are shown in Figure 2. Figure 8As shown, the phagocytic index of the group treated with polysaccharide-selenium nanoparticle complex of Schizophyllum fermentation broth was significantly higher than that of the blank control, and showed good concentration dependence. At the same time, the maximum concentration of polysaccharide-selenium nanoparticle complex treated with Schizophyllum fermentation broth (10 μg / mL) was higher than that of the LPS positive control group, indicating that the polysaccharide-selenium nanoparticle complex of Schizophyllum fermentation broth can significantly promote the phagocytic ability of RAW264.7 cells.

[0060] In order to further observe the changes in cell morphology during phagocytosis, the morphology of RAW264.7 cells was detected. Fig. 9 It can be seen that the cells in the blank control group are round and have a normal morphology. The cell morphology after treatment with LPS and Schizophyllum fermentation broth polysaccharide-selenium nanoparticle complexes changed significantly. The cells extended pseudopodia, the climbing ability was enhanced, the tentacles increased, and the adhesion ability was enhanced. This structure is conducive to increasing the contact area between the cells and the external environment.

[0061] Example 6: In vivo immunological activity determination of the polysaccharide-selenium nanoparticle complex obtained from the fermentation broth of Schizophyllum japonicum in Example 3

[0062] 1. Toxicity test of polysaccharide-selenium nanoparticle complex from Schizophyllum fermentation broth on Caenorhabditis elegans

[0063] The toxicity was determined by the insect immersion method. After the Caenorhabditis elegans was synchronized, different concentrations of SPG-1 and SPG-1 / SeNPs containing OP50 diluted in M9 (measured according to the concentration of polysaccharides in the complex, 5-40 μg / mL) were cultured for 24 hours. The SeNPs group with a Se concentration of 40 μg / mL was used as a control, and the OP50 bacterial solution diluted in M9 was used as a blank group. The survival of Caenorhabditis elegans was observed under a microscope and statistical analysis was performed. The results are shown in Figure 2. Fig.10 As shown, compared with the C. elegans control group, the treatment of 5-40μg / mL SPG-1 and SPG-1 / SeNPs had no significant effect on the number of C. elegans, indicating that SPG-1 and SPG-1 / SeNPs were not toxic to C. elegans within this concentration range. However, SeNPs were toxic to C. elegans, with a survival rate of only 30.4%, indicating that the combination of SPG-1 and selenium nanoparticles reduced the toxicity of selenium nanoparticles and significantly increased the survival rate of C. elegans. Subsequent experiments were conducted using 40μg / mL SPG-1 and SPG-1 / SeNPs.

[0064] 2. Effect of polysaccharide-selenium nanoparticle complex of Schizophyllum fermentation broth on H 2 O 2 Effects of damaged Caenorhabditis elegans on SOD, MDA, GSH-Px and CAT enzyme activities

[0065] After synchronization, C. elegans were cultured with 40 μg / mL SPG-1 and SPG-1 / SeNPs containing OP50 for 24 h, and the blank group was OP50 bacterial solution. 2 O 2 After 4 hours of injury treatment, the superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) enzyme activities and malondialdehyde (MDA) content in C. elegans were detected using a kit. Figures 11 to 14 As shown in the figure, compared with the blank group, the SPG-1 and SPG-1 / SeNPs groups significantly upregulated the activity levels of SOD, CAT and GSH-Px after administration, indicating that SPG-1 / SeNPs can improve the activity of SOD, CAT and GSH-Px in Caenorhabditis elegans. Fig.14 It can be seen that the MDA content in the SPG-1 and SPG-1 / SeNPs groups was lower than that in the control group, indicating that SPG-1 / SeNPs can reduce the degree of lipid peroxidation cell damage in Caenorhabditis elegans, thereby improving the nematode's ability to resist oxidative damage.

[0066] 3. Effect of polysaccharide-selenium nanoparticle complex of Schizophyllum fermentation broth on H 2 O 2 Effects of ROS in damaged Caenorhabditis elegans

[0067] After synchronization, C. elegans were cultured with different concentrations of SPG-1 and SPG-1 / SeNPs containing OP50 for 24 h, and the blank group was cultured with OP50 solution. 2 O 2 After 4 hours of injury treatment, H2DCFDA probe staining was performed for 2 hours, and confocal microscopy was used to observe and the fluorescence intensity was quantified using ImageJ software. Fig.15 It can be seen that compared with the blank group, the green fluorescence intensity in the SPG-1 / SeNPs group of Caenorhabditis elegans was significantly weakened, and the ROS level was significantly reduced. Fluorescence quantitative analysis showed that the ROS level in the SPG-1 / SeNPs-treated group of Caenorhabditis elegans was reduced by 30.7%. This shows that SPG-1 / SeNPs can reduce the ROS level in Caenorhabditis elegans and enhance the ability of Caenorhabditis elegans to resist oxidative damage.

[0068] 4. Effect of polysaccharide-selenium nanoparticle complex of Schizophyllum fermentation broth on H 2 O 2 Effects of Lipid Nucleotides on the Liver of Caenorhabditis elegans

[0069] After synchronization, C. elegans were cultured with different concentrations of SPG-1 and SPG-1 / SeNPs containing OP50 for 24 h, and the OP50 culture solution was used as the blank group. 2 O 2 After 4 h of injury treatment, the cells were observed under a confocal microscope (360 nm excitation wavelength / 450 nm emission wavelength), and the image fluorescence was analyzed using ImageJ software. Fig.16 It can be seen that compared with the control group, the blue fluorescence intensity of the SPG-1 / SeNPs group was significantly reduced, and the cumulative amount was reduced. The results of fluorescence quantitative analysis showed that the lipofuscin level in the SPG-1 / SeNPs-treated group was reduced by 12.1%, which reduced the H-induced 2 O 2 The resulting increase in lipofuscin increases the sensitivity to H 2 O 2 The ability to defend against chemical damage.

[0070] The above are merely exemplary embodiments of the present invention and are not intended to limit the present invention. 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 present invention.

Claims

1. A method for preparing a polysaccharide-selenium nanoparticle complex from Schizophyllum fermentation broth having both immunomodulatory and antioxidant activities, characterized in that: The steps include: Step 1, fully stirring and mixing 0.4-5 mg / mL of Schizophyllum fermentation broth polysaccharide solution and 5-25 mg / mL of sodium selenite solution at 25-60° C. for 0.5-1.5 h to obtain a mixed solution; Step 2: Add 5-20 mg / mL of L-ascorbic acid solution to the mixed solution obtained in step 1, and continue stirring the reaction at 25-60° C. for 3-5 hours to obtain a Schizophyllum fermentation broth polysaccharide-selenium nanoparticle complex having both immunomodulatory and antioxidant damage activities.

2. The method for preparing the polysaccharide-selenium nanoparticle complex of Schizophyllum fermentation broth according to claim 1, characterized in that: The volume ratio of the polysaccharide solution of the Schizophyllum fermentation broth, the sodium selenite solution and the L-ascorbic acid solution is 1-2 mL: 2-4 mL: 4-7 mL.

3. The method for preparing the polysaccharide-selenium nanoparticle complex of Schizophyllum fermentation broth according to claim 1, characterized in that: The polysaccharide in the fermentation broth of Schizophyllum in step 1 is obtained by the following steps: (1) Take a diameter of 1 to 2 cm 2 The solid plate of Schizophyllum mycelium is inoculated into 150 mL YPD liquid medium, cultured at 25-30° C. with shaking for 5-7 days, centrifuged at 1000-5000 rpm for 3-5 minutes, and the supernatant is collected to obtain the Schizophyllum fermentation liquid; (2) After the Schizophyllum fermentation broth obtained in step (1) is concentrated to 1 / 12 to 1 / 5 of its volume, anhydrous ethanol is added until the final volume concentration of ethanol is 60 to 80%, the mixture is allowed to stand at 4° C. for 6 to 8 hours, and centrifuged at 3000 to 7000 rpm for 5 to 10 minutes to collect the precipitate; (3) After dissolving the precipitate obtained in step (2) in deionized water, dialyzing it using a 1-5 kDa dialysis bag, and then drying it, the polysaccharide of the fermentation broth of Schizophyllum is obtained.

4. The method for preparing the polysaccharide-selenium nanoparticle complex of Schizophyllum fermentation broth according to claim 1 or 3, characterized in that: The molecular weight of the polysaccharide in the fermentation broth of Schizophyllum is mainly distributed in the range of 2.5×10 5 to 5.6×10 5 Between big.

5. The method for preparing the polysaccharide-selenium nanoparticle complex of Schizophyllum fermentation broth according to claim 1 or 3, characterized in that: The polysaccharide of the Schizophyllum fermentation broth is composed of five monosaccharides, namely, fucose, rhamnose, galactose, mannose and glucose, in a molar ratio of 1:1.21:3.36:26.2:26.

45.

6. A polysaccharide-selenium nanoparticle complex from Schizophyllum fermentation broth obtained by the preparation method according to any one of claims 1 to 5.

7. The Schizophyllum fermentation broth polysaccharide-selenium nanoparticle complex according to claim 6, characterized in that: The particle size of the Schizophyllum fermentation broth polysaccharide-selenium nanoparticle complex is between 20 and 80 nm.

8. An application of the polysaccharide-selenium nanoparticle complex of Schizophyllum fermentation broth according to claim 6 or 7, characterized in that: Used for preparing immunomodulators and / or oxidative damage repair agents.

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