A method for preparing polyphenols from sugarcane endophytic fungi with high immunogenicity by solid-state fermentation

Abstract

This invention discloses a method for preparing highly immunomodulatory polyphenols from sugarcane endophytic fungi via solid-state fermentation. The sugarcane endophytic fungal polyphenols are obtained by solid-state fermentation of the sugarcane endophytic fungus *Fusarium proliferatum* 15, followed by purification and extraction. Identification analysis confirmed that it is a cyclic (hydroxyproline-leucine) dipeptide. Experimental verification showed that it enhances the body's immune activity. This invention features a simple preparation process, mild culture conditions, and uses lignocellulose as a substrate for solid-state fermentation to obtain endophytic fungal polyphenols, offering significant cost advantages and avoiding the pollution problems caused by lignocellulose resources. The preparation process is more green and environmentally friendly, providing a cost-effective and efficient method for preparing highly active immunomodulatory polyphenols.

Description

Technical Field

[0001] This invention belongs to the field of fungal extracts and their applications, specifically relating to a method for preparing polyphenols from sugarcane endophytic fungi with high immunogenicity through solid-state fermentation. Background Technology

[0002] Since the 21st century, research on endophytic fungi has focused on the study and development of plant endophytic fungi. my country possesses abundant and diverse plant resources, and utilizing its endophytic fungal resource bank to discover low-toxicity, highly effective, and inexpensive anticancer drugs, antibacterial drugs, pesticides, antioxidants, plant hormones, and immunosuppressants from their metabolites is of great significance. Traditional Chinese medicine has long regarded sugarcane (Saccharum sinensis Roxb.) as a valuable remedy for clearing heat and detoxifying. Modern scientific research shows that the main active ingredient in sugarcane is polyphenols. Sugarcane polyphenols possess various physiological and pharmacological activities, including promoting digestion, enhancing immunity, antioxidation, strengthening blood vessel walls, preventing arteriosclerosis, lowering blood pressure, and inhibiting cancer cell growth, making them a hot research topic in the field of polyphenol research. Currently, the preparation of sugarcane polyphenols mainly uses sugarcane as the base material. However, the growth process of sugarcane is greatly affected by environmental factors and has a long cycle, resulting in excessively high production costs and limiting the large-scale application of sugarcane polyphenols. Furthermore, the complex structures of polyphenolic compounds make them difficult to synthesize through conventional chemical methods. Therefore, there is an urgent need to find an alternative way to quickly obtain sugarcane polyphenols.

[0003] Recent studies have shown that plant endophytic fungi are an important resource of novel bioactive components, capable of producing bioactive components identical or similar to those of their symbiotic plants. Compared to preparing compounds through plant extraction, obtaining these compounds through plant endophytic fungi offers significant advantages, such as rapid cell growth, ease of cultivation, convenient downstream processing, cost savings, and green sustainability. Currently, several endophytic fungal polyphenols have been reported, such as those from ginger endophytic fungi (Aspergillus austroafricanus) and palm root endophytic fungi (Penicillium citrinum TDPEF34).

[0004] In related technologies, the production of polyphenols by sugarcane endophytic fungi is rarely mentioned, and there is a lack of research and understanding of this polyphenol. Therefore, developing an efficient and inexpensive method for preparing polyphenols from sugarcane endophytic fungi is of great significance. Summary of the Invention

[0005] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes a method for preparing polyphenols from sugarcane endophytic fungi and their applications, which can improve the yield and activity of the polyphenols, and the polyphenols obtained by this preparation method have high immunomodulatory activity.

[0006] The first aspect of this invention provides a method for preparing polyphenols from sugarcane endophytic fungi, comprising the following steps:

[0007] (1) Activate sugarcane endophytic fungi to obtain sugarcane endophytic fungal fermentation seed liquid;

[0008] (2) Inoculate the sugarcane endophytic fungal fermentation seed liquid into the sugarcane endophytic fungal solid fermentation medium and ferment for 25-30 days, then collect the mycelium;

[0009] (3) The mycelium was extracted with methanol solution, the mycelium was collected by filtration, the alcohol was precipitated, the precipitate was centrifuged and dialyzed, and then dried to obtain crude polyphenols.

[0010] (4) Use an eluent to re-dissolve the crude polyphenols, remove proteins and pigments, and use a semi-preparative liquid phase to elute the pure polyphenols from the sugarcane endophytic fungi.

[0011] The sugarcane endophytic fungal polyphenol is a cyclic (hydroxyproline-leucine) dipeptide.

[0012] In some specific embodiments of the present invention, the sugarcane endophytic fungus is Fusarium proliferatum15.

[0013] In this invention, the sugarcane endophytic fungus Fusarium proliferatum 15 is disclosed in the prior Chinese patent CN114410479A (publication date: April 29, 2022), with the corresponding accession number GDMCC No: 62048.

[0014] In some embodiments of the present invention, the bacterial activity in the seed culture of the fermentation broth in step (1) is 10. 7 ~10 8 CFU / mL.

[0015] In some embodiments of the present invention, the bacterial activity in the seed culture of the fermentation broth in step (1) is 10. 8 CFU / mL.

[0016] In some embodiments of the present invention, the composition of the solid fermentation culture medium in step (2) includes: 75-90 wt% bagasse, 5-10 wt% corn bran, and 5-10 wt% rice bran.

[0017] In some embodiments of the present invention, the moisture content of the solid fermentation medium in step (2) is 5-15 wt%.

[0018] In some specific embodiments of the present invention, the solid fermentation culture medium in step (2) is composed of: 80 wt% bagasse, 5 wt% corn bran, 5 wt% rice bran, and 10 wt% moisture content.

[0019] In some embodiments of the present invention, the solid fermentation culture medium is prepared by mixing 75-90 wt% bagasse, 5-10 wt% corn bran, and 5-10 wt% rice bran, then adding water to adjust the moisture content to 10 wt%, and adjusting the pH to 6.5 using 1M HCl.

[0020] In this invention, the solid fermentation medium is dispensed into 1L Erlenmeyer flasks, 200mL per flask, and then sterilized at 121℃ for 20min after sealing.

[0021] In some specific embodiments of the present invention, the volume ratio of the inoculated and activated sugarcane endophytic fungus fermentation seed liquid to the sugarcane endophytic fungus fermentation culture medium in step (2) is 1:5 to 15.

[0022] In some specific embodiments of the present invention, the volume ratio of the inoculated and activated sugarcane endophytic fungus fermentation seed liquid to the sugarcane endophytic fungus fermentation culture medium in step (2) is 1:10.

[0023] In some embodiments of the present invention, the fermentation temperature in step (2) is 23-27°C.

[0024] In some specific embodiments of the present invention, the fermentation temperature in step (2) is 25°C.

[0025] In some embodiments of the present invention, the volume fraction of the methanol solution in step (3) is 40-60%.

[0026] In some embodiments of the present invention, the volume fraction of the methanol solution in step (3) is 50%.

[0027] In some embodiments of the present invention, the eluent in step (4) includes methanol.

[0028] In some embodiments of the present invention, the eluent is a 40-60% methanol solution.

[0029] In some embodiments of the present invention, the eluent is a 50% methanol solution.

[0030] In some embodiments of the present invention, the flowability of the semi-preparative liquid phase elution in step (4) is aqueous acetic acid and methanol, and the column used is a C18 column.

[0031] In some embodiments of the present invention, the specific method for preparing sugarcane endophytic fungal polyphenols is as follows: Sugarcane endophytic fungus *Fusarium proliferatum* 15 is inoculated into PDB medium and activated for 8–12 days to obtain sugarcane endophytic fungal fermentation seed liquid; 5–15% (v / v) of the sugarcane endophytic fungal fermentation seed liquid is inoculated into a sugarcane endophytic fungal solid fermentation medium and fermented at 23–27°C for 25–35 days; after fermentation, the mycelium is collected, vacuum dried and ground, extracted with methanol, the cell body is collected by filtration, precipitated with alcohol, centrifuged, dialyzed, and freeze-dried to obtain crude polyphenols; the obtained crude polyphenol sample is dissolved in an eluent, and proteins and pigments are removed using H103 macroporous adsorption resin, followed by methanol elution, collection of the eluent, and concentration under reduced pressure to obtain protein- and pigment-free crude polyphenols; the protein- and pigment-free crude polyphenols are dissolved in a mobile phase and eluted with a semi-preparative liquid phase to obtain pure polyphenols.

[0032] In some embodiments of the present invention, the semi-preparative liquid elution uses an Elite P3500 semi-preparative liquid chromatography system, with a mobile phase (v / v) of 70 (98% aqueous acetic acid): 30 (methanol), a 10 μm, 250 × 4.6 mm C18 column (octadecyl bonded silica gel column), a flow rate of 1 mL / min, and a processing time of 50 min.

[0033] A second aspect of the present invention provides a sugarcane endophytic fungal polyphenol prepared by the preparation method described in the first aspect of the present invention. The sugarcane endophytic fungal polyphenol is a cyclic (hydroxyproline-leucine) dipeptide.

[0034] In this invention, after preparation by the method described in the first aspect of this invention, the final product is identified as a cyclic (hydroxyproline-leucine) dipeptide after material structure identification and analysis.

[0035] In this invention, the terms "cyclic dipeptide" and "sugarcane endophytic fungal polyphenol" both refer to cyclic (hydroxyproline-leucine) dipeptides.

[0036] In this invention, the effects of the prepared sugarcane endophytic fungal polyphenols were verified by testing their influence on mouse mononuclear macrophages, demonstrating that the polyphenol extract has no toxic effect on cells and can enhance cellular immune activity.

[0037] In this invention, the sugarcane endophytic fungal polyphenols prepared have no toxic effect on mouse monocyte macrophage RAW264.7 cells, can promote the proliferation of mouse monocyte macrophage RAW264.7 cells, and can activate the cytokines TNF-α and IL-6 secreted by RAW264.7 cells to varying degrees.

[0038] A third aspect of the present invention provides its use in the preparation of products containing polyphenols from sugarcane endophytic fungi as described in the second aspect of the present invention to enhance the body's immunity.

[0039] In some embodiments of the present invention, the products that enhance the body's immunity include food and medicine.

[0040] In some embodiments of the present invention, the dosage form of the medicine includes suppositories, creams, lotions, tablets, effervescent tablets, capsules, soft capsules, and sprays.

[0041] In some embodiments of the present invention, the pharmaceutical product also includes other pharmaceutically acceptable excipients.

[0042] In some embodiments of the present invention, the pharmaceutically acceptable excipients include, but are not limited to, adhesives, disintegrants, lubricants, emulsifiers, plasticizers, and stabilizers.

[0043] The beneficial effects of this invention are as follows:

[0044] 1. Compared with traditional liquid fermentation methods, the solid-state fermentation method provided by this invention has advantages such as higher productivity, lower water and energy requirements, easier aeration, lower sterility requirements, easier downstream processing, use of cheaper lignocellulosic materials as solid substrates, and environmental friendliness. Various lignocellulosic materials, mainly derived from agricultural residues such as sugarcane bagasse and corn bran, can serve as excellent substrates in solid-state fermentation. Fungi and their hyphae can penetrate into the solid substrate, contributing to the expansion pressure at the mycelial tips. Furthermore, using agricultural waste as a substrate helps address pollution issues.

[0045] 2. The polyphenols obtained by this invention through steps such as strain activation, solid-state fermentation, purification and extraction have been tested and certified as cyclic dipeptides. Verification through examples shows that they are non-toxic to cells and have high immune activity, and can be used to enhance the body's immunity. Attached Figure Description

[0046] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0047] Figure 1 This is a semi-preparative liquid phase spectrum;

[0048] Figure 2 It is a cyclic (hydroxyproline-leucine) dipeptide 1 H spectrum;

[0049] Figure 3 It is a cyclic (hydroxyproline-leucine) dipeptide 13 C-spectrum;

[0050] Figure 4Schematic diagram of COSY, HMBC and HSQC related spectra of cyclic (hydroxyproline-leucine) dipeptide;

[0051] Figure 5 The viability of mouse monocyte-macrophage RAW264.7 cells under different concentrations of cyclic dipeptide was determined by the MTT assay.

[0052] Figure 6 The effects of different concentrations of cyclic dipeptide and the positive control group LPS on the phagocytic capacity of mouse monocyte macrophages RAW264.7 cells were investigated.

[0053] Figure 7 The effects of different concentrations of cyclic dipeptide and the positive control group LPS on the secretion of interleukin-6 by mouse monocyte macrophage RAW264.7 cells;

[0054] Figure 8 The effects of different concentrations of cyclic dipeptide and the positive control group LPS on tumor necrosis factor-α in mouse monocyte / macrophage RAW264.7 cells were investigated. Detailed Implementation

[0055] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with embodiments, so as to fully understand the purpose, features and effects of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, conditions described in a laboratory manual, or conditions recommended by the manufacturer. Unless otherwise specified, the raw materials, reagents, etc. used in the present invention are all commercially available. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0056] The sugarcane endophytic fungus involved in this embodiment of the invention is Fusarium proliferatum 15, which is disclosed in the prior Chinese patent CN114410479A (publication date: April 29, 2022), and the corresponding accession number is GDMCC No: 62048.

[0057] In the following examples, the culture medium used for activation was potato dextrose agar (PDB) medium. The formula of the solid fermentation medium for sugarcane endophytic fungi used was as follows: 75-90 wt% sugarcane bagasse (80 wt% in the examples of this invention), 5-10 wt% corn bran (5 wt% in the examples of this invention), and 5-10 wt% rice bran (5 wt% in the examples of this invention). Water was added to adjust the moisture content to 10 wt%, and 1M HCl was used to adjust the pH to 6.5. The solution was dispensed into 1L Erlenmeyer flasks, 200 mL per flask, sealed, and sterilized at 121°C for 20 min. After sterilization, the solution was allowed to stand at room temperature.

[0058] Example 1

[0059] This embodiment prepares a polyphenol from sugarcane endophytic fungi with high immunogenicity. The specific process is as follows:

[0060] The sugarcane endophytic fungus Fusarium proliferatum 15 was inoculated into PDB medium at pH 6.5 and activated at 25°C for 10 days to obtain the sugarcane endophytic fungal fermentation seed liquid.

[0061] 10% (v / v) of sugarcane endophytic fungal fermentation seed culture was inoculated into a solid fermentation medium and fermented at 25℃ for 30 days. After fermentation, mycelia were collected, vacuum dried, and ground. Then, the mycelia were extracted with 50% methanol at room temperature for 6 hours. The mycelial cells were collected by filtration, and this step was repeated three times. The extracts were combined, concentrated under reduced pressure, and precipitated with 50% methanol at room temperature. The mixture was centrifuged at 8000 rpm for 10 minutes, dialyzed four times with ultrapure water, concentrated under reduced pressure, and freeze-dried to obtain crude polyphenols. The yield of crude polyphenols was 5 g / mL of fermentation seed culture. The obtained crude polyphenols were dissolved in 50% methanol to obtain a 1 mg / mL crude polyphenol solution. Proteins and pigments were removed using H103 macroporous adsorption resin, followed by elution with 50% methanol. The eluent was collected and concentrated under reduced pressure to obtain protein- and pigment-free crude polyphenols. The crude polyphenols were dissolved in a mobile phase and eluted with a semi-preparative liquid phase to obtain single components and pure polyphenols (e.g., [missing information]). Figure 1 (As shown). The semi-preparative liquid chromatography (HPLC) elution was performed using an Elite P3500 semi-preparative HPLC system. The mobile phase (v / v) was 70 (98% aqueous acetic acid): 30 (methanol). The column was a 10 μm, 250 × 4.6 mm C18 column (octadecyl bonded silica gel HPLC column). The flow rate was 1 mL / min, and the processing time was 50 min. The polyphenols were then freeze-dried at low temperature, and the yield of pure polyphenols was found to be approximately 50 mg / L of fermentation seed broth.

[0062] Example 2

[0063] This embodiment prepares a polyphenol from sugarcane endophytic fungi with high immunogenicity. The specific process is as follows:

[0064] The sugarcane endophytic fungus Fusarium proliferatum strain 15 was activated and cultured in PDB medium at pH 6.5 at 25°C for 10 days to obtain the seed culture of the strain fermentation broth.

[0065] A 9% (v / v) fermentation seed culture of sugarcane endophytic fungi was inoculated into a solid fermentation medium and fermented at 25°C for 30 days. After fermentation, the mycelia were collected, vacuum dried, and ground. Then, the mycelia were extracted with 50% methanol at room temperature for 6 hours. The mycelial cells were collected by filtration, and this step was repeated three times. The extracts were combined, concentrated under reduced pressure, and precipitated with 50% methanol at room temperature. The mixture was centrifuged at 8000 rpm for 10 minutes, dialyzed four times with ultrapure water, concentrated under reduced pressure, and freeze-dried to obtain a crude polyphenol sample. The yield of crude polyphenols was 4.68 g / mL of the fermentation seed culture. The obtained crude polyphenol sample was dissolved in 50% methanol as an eluent to obtain a 1 mg / mL crude polyphenol solution. Proteins and pigments were removed using an H103 macroporous adsorption resin, followed by elution with 50% methanol. The eluent was collected and concentrated under reduced pressure to obtain protein- and pigment-free crude polyphenols. The crude polyphenols were dissolved in a mobile phase and eluted with a semi-preparative liquid phase to obtain single components and pure polyphenols. The semi-preparative liquid chromatography (HPLC) elution was performed using an Elite P3500 semi-preparative HPLC system. The mobile phase (v / v) was 70 (98% aqueous acetic acid): 30 (methanol). The column was a 10 μm, 250 × 4.6 mm C18 column (octadecyl bonded silica gel HPLC column). The flow rate was 1 mL / min, and the processing time was 50 min. The polyphenols were then freeze-dried at low temperature, and the yield of pure polyphenols was found to be approximately 46.8 mg / L of fermentation seed broth.

[0066] Example 3

[0067] This embodiment prepares a polyphenol from sugarcane endophytic fungi with high immunogenicity. The specific process is as follows:

[0068] The sugarcane endophytic fungus Fusarium proliferatum strain 15 was activated and cultured in PDB medium at pH 6.5 at 25°C for 10 days to obtain the seed culture of the strain fermentation broth.

[0069] 8% (v / v) of sugarcane endophytic fungal fermentation seed culture was inoculated into a solid fermentation medium and fermented at 25°C for 30 days. After fermentation, mycelia were collected, vacuum dried, and ground. The mycelia were then extracted with 50% methanol for 6 hours at room temperature. The mycelial cells were collected by filtration, and this step was repeated three times. The extracts were combined, concentrated under reduced pressure, and precipitated with 50% methanol at room temperature. The mixture was centrifuged at 8000 rpm for 10 minutes, dialyzed four times with ultrapure water, concentrated under reduced pressure, and freeze-dried to obtain crude polyphenols. The yield of crude polyphenols was 4.56 g / mL of fermentation seed culture. The obtained crude polyphenols were dissolved in 50% methanol to obtain a 1 mg / mL crude polyphenol solution. Proteins and pigments were removed using H103 macroporous adsorption resin, followed by elution with 50% methanol. The eluent was collected and concentrated under reduced pressure to obtain protein- and pigment-free crude polyphenols. The crude polyphenols were dissolved in a mobile phase and eluted with a semi-preparative liquid phase to obtain single components and pure polyphenols. The semi-preparative liquid chromatography (HPLC) elution was performed using an Elite P3500 semi-preparative HPLC system. The mobile phase (v / v) was 70 (98% aqueous acetic acid): 30 (methanol). The column was a 10 μm, 250 × 4.6 mm C18 column (octadecyl bonded silica gel HPLC column). The flow rate was 1 mL / min, and the processing time was 50 min. The polyphenols were then freeze-dried at low temperature, and the yield of pure polyphenols was found to be approximately 45.6 mg / L of fermentation seed broth.

[0070] Example 4

[0071] To verify the stability of the methods in the above embodiments, the preparation method in Example 1 was repeated twice, and the preparation method in Example 2 was repeated once.

[0072] The results showed that, for the method in Example 1, the first replicate yielded a crude polyphenol sample yield of 4.76 g / mL and a pure product yield of 47.6 mg / L. The second replicate yielded a crude polyphenol sample yield of 4.81 g / mL and a pure product yield of 48.1 mg / L. For the method in Example 2, the first replicate yielded a crude polyphenol sample yield of 4.69 g / mL and a pure product yield of 46.9 mg / L. The three replicates showed no significant difference in crude polyphenol sample and pure product yields compared to the corresponding replicates in Example 1 or 2, indicating that the preparation methods in the above examples are stable and suitable for large-scale production.

[0073] Analysis and identification of pure polyphenols

[0074] The polyphenol obtained in Example 1 above was sent to a testing institution for material structure identification and analysis, and it was determined that the obtained polyphenol was a cyclic (hydroxyproline-leucine) dipeptide (e.g., ...). Figure 2 , Figure 3 , Figure 4 As shown in the figure, the testing institution is the Scientific Compass Research Service Platform. In the following examples, the cyclic dipeptides all refer to cyclic (hydroxyproline-leucine) dipeptides.

[0075] Cytotoxicity verification

[0076] This embodiment experimentally verifies the mechanism of action of the cyclic dipeptide on mouse macrophage RAW264.7 cells. The specific process is as follows:

[0077] Culture of mouse macrophage RAW264.7 cells: After thawing frozen RAW264.7 cells, they were placed in a 37°C water bath with constant shaking. The cells were then transferred to 10 mL of DMEM medium (containing 10% FBS + 1% antibiotics), mixed well, and centrifuged at 1000×g for 5 min to collect the cells. The cells were resuspended in 10 mL of fresh DMEM medium, transferred to cell culture dishes, and incubated at 37°C in a 5% CO2 incubator. Cell growth was observed regularly, and the DMEM medium was replaced as needed.

[0078] The effect of cyclic dipeptide concentrations of 15.625, 31.25, 62.5, 125, 500, and 1000 μg / mL on the survival rate of RAW264.7 cells was determined using the MTT assay. RAW264.7 cells in the logarithmic growth phase obtained in the above examples were used, and the cell density was adjusted to 5 × 10⁻⁶ cells / mL. 5 After mixing, the solution was seeded at a concentration of 100 μL / mL into 96-well plates and cultured at 37°C in 5% CO2 for 24 h. Cells were then treated according to pre-defined groups: the control group was treated with serum-free DMEM medium after discarding the supernatant; the experimental groups were treated with cyclic dipeptide solutions at concentrations of 15.625, 31.25, 62.5, 125, 500, and 1000 μg / mL, respectively, and cultured for another 24 h. Then, 20 μL of 5 mg / mL LMTT solution prepared with PBS was added to each well, and the cells were cultured for 4 h. After aspirating the supernatant from each well, 100 μL of DMSO was added, and the cells were incubated at 37°C with shaking for 10 min. The OD value was measured at 570 nm. The control group was not seeded, but all other procedures were the same as above, with 6 replicates per group. Cell viability was calculated using the following formula:

[0079] Cell viability (%) = (AC) / (BC) × 100%

[0080] Wherein, A - OD value of experimental group, B - OD value of control group, and C - OD value of blank group.

[0081] The cytotoxic effects of different concentrations (15.625, 31.25, 62.5, 125, 250, 500, and 1000 μg / mL) of cyclic dipeptide on RAW264.7 cells were evaluated using the MTT assay. The results are as follows: Figure 5As shown, the survival rates of RAW264.7 cells treated with cyclic dipeptide at concentrations of 15.625, 31.25, 62.5, 125, 250, 500, and 1000 μg / mL were 93.23±3.24%, 94.13±3.53%, 93.87±2.86%, 94.37±3.72%, 94.73±3.53%, 93.07±2.86%, and 94.67±3.72%, respectively. This indicates that the cyclic dipeptide did not significantly inhibit the survival of RAW264.7 cells. Furthermore, these results also demonstrate that the cyclic dipeptide is not cytotoxic to RAW264.7 cells.

[0082] Verification of the effect of cell phagocytosis rate

[0083] The mechanism of action of the cyclic dipeptide on mouse macrophage RAW264.7 was further demonstrated by referring to the methods in the above embodiments, and the effect of the cyclic dipeptide on the phagocytic capacity of RAW264.7 cells was found (e.g. Figure 6 (As shown in the figure). The positive control group, LPS (2 μg / mL), significantly increased the phagocytic rate of RAW264.7 cells. Simultaneously, treatment of RAW264.7 cells with cyclic dipeptide showed a similar effect to the LPS group; that is, the phagocytic capacity of RAW264.7 cells increased with increasing cyclic dipeptide concentration in a dose-dependent manner. Cyclic dipeptide significantly increased the phagocytic rate of RAW264.7 cells from 8.32 ± 0.63% (31.25 μg / mL) to 31.92 ± 1.28% (1000 μg / mL).

[0084] Verification of high immune activity

[0085] This embodiment experimentally verifies the mechanism of action of the cyclic dipeptide on mouse macrophage RAW264.7 cells. The specific process is as follows:

[0086] The culture of mouse macrophages RAW264.7 was the same as in the above examples.

[0087] Inoculate 1 mL 1×10 6 RAW264.7 cells per mL were cultured in 24-well cell culture plates at 37°C for 24 h in a 5% CO2 incubator, and the supernatant was removed. 1 mL of LDM basal medium and different concentrations (15.625, 31.25, 62.5, 125, 250, 500, and 1000 μg / mL) of cyclic dipeptide solution were added to different wells, and the cells were cultured again at 37°C for 24 h in a 5% CO2 incubator. The supernatant was then collected in 1.5 mL centrifuge tubes for subsequent experimental analysis.

[0088] The expression levels of IL-6 and TNF-α in RAW264.7 cells treated with cyclic dipeptide were determined using a commercially available ELISA kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.), following the kit instructions. Specifically, the absorbance of the samples was measured at 450 nm, and the levels of cytokines IL-6 and TNF-α (pg / mL) were calculated based on the standard curve.

[0089] It was observed that the production of major cytokines (IL-6 and TNF-α) in RAW264.7 cells significantly increased after macrophage activation. Since cyclic dipeptides significantly promote phagocytosis in RAW264.7 cells, the effect of cyclic dipeptides on the levels of IL-6 and TNF-α in RAW264.7 cells was measured. Figure 7-8 As shown, compared with the blank control group, cyclic dipeptide significantly promoted macrophage cytokine secretion. Even after RAW264.7 cells were treated with a low concentration of cyclic dipeptide (31.25 μg / mL), IL-6 and TNF-α levels rapidly increased to 700.43 pg / mL and 1433.25 pg / mL, respectively, far exceeding the cytokine secretion levels in the blank control group. Moreover, the promoting effect of cyclic dipeptide on IL-6 and TNF-α secretion was dose-dependent. In this example, the TNF-α concentration in the cyclic dipeptide-treated group was as high as 2269.25 ± 0.50 pg / mL, which was higher than the TNF-α secretion level of 1826.50 ± 0.38 pg / mL after LPS treatment in the positive control group.

[0090] The efficacy of cyclic dipeptides on mouse RAW264.7 mononuclear macrophages was verified, indicating that polyphenols, metabolites of solid-state fermentation by sugarcane endophytic fungi, have a high effect on enhancing the body's immunity.

[0091] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A method for preparing a sugarcane endophytic fungus polyphenol, characterized in that, The method comprises the following steps: (1) activating the sugarcane endophytic fungus to obtain a sugarcane endophytic fungus fermentation seed liquid; (2) inoculating the sugarcane endophytic fungus fermentation seed liquid into a sugarcane endophytic fungus solid fermentation medium to ferment for 25-30 days, and collecting mycelium; (3) using a methanol solution to extract the mycelium, filtering to collect the mycelium, alcohol precipitation, centrifugation to collect the precipitate, dialysis, and drying to obtain crude polyphenols; (4) using an eluent to redissolve the crude polyphenols, removing proteins and pigments, and using a semi-preparative liquid phase to elute pure sugarcane endophytic fungus polyphenols; The sugarcane endophytic fungus polyphenols are cyclo(hydroxyproline-leucine) dipeptides; The sugarcane endophytic fungus is Fusarium proliferatum 15; The composition of the sugarcane endophytic fungus solid fermentation medium is 75-90 wt% of bagasse, 5-10 wt% of corn bran, and 5-10 wt% of rice bran, wherein the water content is 10 wt%, and an amount of HCl is added to adjust the pH of the medium to 6.5; The volume fraction of the methanol solution in step (3) is 40-60%; The eluent in step (4) is 40-60% methanol; In step (4), the mobile phase for semi-preparative liquid phase elution is acetic acid aqueous solution and methanol, and a C18 chromatographic column is used; The fermentation culture conditions in step (2) are 23-27℃.

2. The production method according to claim 1, characterized by, The volume ratio of the inoculated activated sugarcane endophytic fungus fermentation seed liquid to the sugarcane endophytic fungus fermentation medium in step (2) is 1:5-15.

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