Phanerochaete fulva s-lwz20190428-3b polysaccharide and application thereof in immunoregulation

Abstract

This invention belongs to the field of microbial polysaccharide and bioactive application technology, and discloses a polysaccharide from Phylloroporia pulla and its in vitro immunomodulatory activity. The polysaccharide is produced by Phylloroporia pulla strain S-LWZ20190428-3b, whose preservation number is CGMCC No. 42757. The weight-average molecular weight of the Phylloroporia pulla polysaccharide is 17.211 kDa, contains β-glycosidic bonds, and its monosaccharide composition includes mannose, galactose, glucose, fucose, glucuronic acid, xylose, and glucosamine hydrochloride in a molar ratio of 0.448:0.294:0.189:0.020:0.025:0.012:0.012. In vitro activity assays showed that this polysaccharide was non-toxic to RAW 264.7 cells within a concentration range of 25-800 μg / mL, significantly promoted cell proliferation, enhanced phagocytic capacity, increased NO secretion, and upregulated the expression of pro-inflammatory and anti-inflammatory factors. The *Pheretima asiatica* polysaccharide provided by this invention possesses significant immunomodulatory activity and can be used to prepare immunomodulators.

Description

Technical Field

[0001] This invention belongs to the field of microbial polysaccharide and bioactive application technology. Background Technology

[0002] Polysaccharides, as the core active ingredients in the aqueous extracts of medicinal fungi, have become a research hotspot in the modern biomedical field due to their diverse biological functions. They have been proven to have a variety of pharmacological activities such as anti-oxidation, anti-tumor, immunomodulation, and neuroprotection. Compared with chemically synthesized drugs, they have significant advantages such as fewer toxic side effects, better biocompatibility, and milder effects.

[0003] Phyllophoria fungi are an important class of medicinal fungi. They are rich in various active ingredients such as polysaccharides, sterols, acidic substances, and styrene compounds. Among them, polysaccharides have shown excellent activity in anti-oxidation, anti-tumor, and immunomodulation.

[0004] As an important member of the genus *Inonotus*, *Inonotus datura* possesses advantages over other species, including a wide host range and ease of fermentation cultivation, demonstrating great potential for the industrialization of its polysaccharides. However, current research on *Inonotus datura* polysaccharides is relatively scarce, and systematic validation of its immunomodulatory activity is still incomplete.

[0005] In modern society, the incidence of immune-related diseases is rising year by year, making the development of immunomodulators an important direction in the biomedical field. The immune system is the core of maintaining human homeostasis. Macrophages, as key effector cells in the immune system, play a crucial role in recognizing pathogens, clearing foreign substances, and regulating inflammatory responses. Their proliferation capacity, phagocytic function, NO secretion, and cytokine expression levels are core indicators for assessing immunomodulatory activity. Fungal polysaccharides, as natural immunomodulatory substances, can bind to pattern recognition receptors on the surface of macrophages, activate downstream signaling pathways, regulate the transcription and expression of immune-related genes, and thus regulate the body's immune function. Therefore, developing efficient and specific extraction and purification methods for *Inonotus obliquus* polysaccharides, systematically analyzing their structural characteristics, and comprehensively verifying their in vitro immunomodulatory activity will not only fill the research gap in *Inonotus obliquus* polysaccharides but also provide a new material basis and technical support for the development of novel natural immunomodulators. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a Phyllophoria pulla strain S-LWZ20190428-3b, with the accession number CGMCC No. 42757. This strain was deposited on April 17, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The accession number of this strain is CGMCC No. 42757.

[0007] The present invention also provides a polysaccharide of *Phoebe d'Argent*, the polysaccharide having a weight-average molecular weight of 17.211 kDa and being composed of mannose, galactose, glucose, fucose, glucuronic acid, xylose and glucosamine hydrochloride.

[0008] Furthermore, the molar ratio of mannose:galactose:glucose:fucose:glucuronic acid:xylose:glucosamine is 0.448:0.294:0.189:0.020:0.025:0.012:0.012.

[0009] The polysaccharide from *Phoebe dactylon* was non-toxic to RAW 264.7 cells in the concentration range of 25-800 μg / mL; at a concentration of 25-400 μg / mL, it significantly promoted cell proliferation, enhanced phagocytic capacity in a dose-dependent manner, increased NO secretion, and upregulated the expression of pro-inflammatory cytokines such as TNF-α, IL-6, IL-1α, and IL-1β, as well as the anti-inflammatory factor IL-10. Attached Figure Description

[0010] Figure 1 The DEAE-Sepharose Fast Flow column chromatography elution curve is for polysaccharides from *Phoebe dactylon*.

[0011] Figure 2 This is the ultraviolet spectrum of the polysaccharide component PPP-2.

[0012] Figure 3 This is the infrared spectrum of the polysaccharide component PPP-2.

[0013] Figure 4 This is a molecular weight spectrum of the polysaccharide component PPP-2.

[0014] Figure 5 This is a monosaccharide composition ion chromatogram of the polysaccharide component PPP-2.

[0015] Figure 6 The effect of polysaccharide component PPP-2 on the proliferation of RAW 264.7 cells.

[0016] Figure 7The effect of polysaccharide component PPP-2 on the phagocytic capacity of RAW 264.7 cells.

[0017] Figure 8 The effect of polysaccharide component PPP-2 on NO secretion in RAW 264.7 cells.

[0018] Figure 9 Effects of polysaccharide component PPP-2 on the secretion of TNF-α (a) and IL-6 (b) in RAW 264.7 cells.

[0019] Figure a shows the effect of PPP-2 on TNF-α secretion, and Figure b shows the effect of PPP-2 on IL-6 secretion.

[0020] Figure 10 Effects of polysaccharide component PPP-2 on the expression levels of cytokine mRNA in RAW 264.7 cells.

[0021] Figure a shows the dose-dependent upregulation of TNF-α mRNA expression level by PPP-2, Figure b shows the dose-dependent upregulation of IL-1α mRNA expression level by PPP-2, Figure c shows the dose-dependent upregulation of IL-1β mRNA expression level by PPP-2, Figure d shows the dose-dependent upregulation of IL-6 mRNA expression level by PPP-2, Figure e shows the dose-dependent upregulation of IL-10 mRNA expression level by PPP-2, and Figure f shows the dose-dependent upregulation of iNOS mRNA expression level by PPP-2. Detailed Implementation

[0022] Example 1

[0023] 1. Acquisition, isolation, and identification of bacterial strains

[0024] The strain was collected on April 28, 2019, in Yongquan Village, Zihe Town, Zichuan District, Zibo City, Shandong Province. It was obtained from fresh fruiting bodies growing on branches of living Prunus sp. trees using tissue isolation method and named strain S-LWZ20190428-3b. After purification, it was cultured at 26-28℃ for 10-15 days.

[0025] DNA from S-LWZ20190428-3 was extracted using a lysis method and a rapid fungal genome extraction kit. The nLSU sequence was obtained by PCR amplification, and its sequence information is shown in SEQ ID NO.1 in the sequence listing. By comparing with the BLAST database of NCBI and constructing a phylogenetic tree, combined with morphological analysis, the strain was identified as Phyllophoria pulla.

[0026] Phyllophoria pulla strain, named S-LWZ20190428-3b, was deposited on April 17, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The accession number for this strain is CGMCC No. 42757.

[0027] 2. Preparation of polysaccharides from *Phoebe d'Argent*

[0028] (1) Microbial culture:

[0029] PDA medium preparation: Weigh 200 g of peeled potatoes, cut them into small pieces, add 1 L of distilled water and boil for 30 min. Filter with double-layer gauze, add 20 g of glucose and 15 g of agar powder to the filtrate, stir to dissolve and bring the volume to 1 L. Autoclave at 121℃ for 20 min, cool and pour into plates for later use.

[0030] Preparation of seed culture medium: Weigh 35 g glucose, 10 g yeast extract, 1.0 g MgSO4·7H2O, 1.5 g KH2PO4, and 0.05 g VB1, add 1 L distilled water and stir to dissolve. Autoclave at 121℃ for 20 min and cool for later use.

[0031] Preparation of liquid fermentation medium: The formula and sterilization conditions are the same as those for seed culture medium.

[0032] The preserved *Porphyra yezoensis* strain was inoculated into the center of PDA medium and activated by incubation at 28°C for 7 days. A 1cm × 1cm mycelial block was inoculated into 100 mL of seed culture medium and cultured at 28°C and 140 rpm for 7 days to obtain seed culture. 300 mL of liquid fermentation medium was added to a 500 mL Erlenmeyer flask and autoclaved at 121°C for 20 min. After cooling, 30 mL of seed culture was inoculated and cultured at 28°C and 140 rpm for 7 days. The mycelium was collected by filtration, washed three times with distilled water, dried at 50°C to constant weight, and then pulverized.

[0033] (2) Polysaccharide extraction:

[0034] Weigh 10 g of mycelial powder, add 300 mL of distilled water at a material-to-liquid ratio of 1:30, extract in a boiling water bath for 3 h, filter with double-layer gauze, repeat the extraction twice with the filter residue, combine the filtrates from the three extractions, add 3 times the volume of anhydrous ethanol, let stand overnight at 4℃, centrifuge at 4500 rpm for 10 min, collect the precipitate, wash 3 times with anhydrous ethanol, and dry at 50℃ to constant weight to obtain crude polysaccharide.

[0035] (3) Deproteinization treatment:

[0036] The crude polysaccharide was dissolved in distilled water to prepare a 5% (w / w) polysaccharide solution. Chloroform and n-butanol were mixed in a 4:1 (v / v) ratio to prepare Sevage's reagent. The polysaccharide solution and Sevage's reagent were mixed in a specific ratio (polysaccharide solution: Sevage's reagent = 4:1), inverted and mixed for 20 min, and then allowed to stand for 30 min until separation occurred. The mixture was centrifuged at 4500 rpm for 5 min, and the upper polysaccharide layer was collected. This deproteinization process was repeated 7 times. The polysaccharide solution was concentrated to 20 mL under reduced pressure at 60 °C, transferred to a 3.5 kDa dialysis bag, placed in distilled water, and dialyzed at 4 °C for 2 days, changing the distilled water during this period. After dialysis, the solution was freeze-dried under vacuum to obtain the deproteinized polysaccharide.

[0037] (4) Column chromatography purification:

[0038] Weigh 20 mg of deproteinized polysaccharide, dissolve it in 10 mL of deionized water, filter it through a 0.22 μm aqueous filter membrane, and load it onto a DEAE-Sepharose Fast Flow anion exchange column (2.6 × 30 cm). Perform gradient elution at a flow rate of 1 mL / min, first eluting with distilled water until the baseline stabilizes, then eluting sequentially with 0.1 mol / L NaCl and 0.2 mol / L NaCl solutions. Collect 10 mL of eluent from each tube, and determine the absorbance at 490 nm using the phenol-sulfuric acid method. Plot the elution curve. Figure 1 The eluents corresponding to each peak were combined and transferred to 3.5 kDa dialysis bags, and dialyzed at 4°C for 48 h, with distilled water replaced during the process. After dialysis, the mixtures were freeze-dried under vacuum to obtain PPP-1 (yield 0.01%), PPP-2 (yield 0.036%) and PPP-3 (yield 0.005%), wherein PPP-2 is the polysaccharide of *Phoebe dactylon* described in this invention.

[0039] Example 2: Structural characterization of the polysaccharide PPP-2 from *Phoebe dactylon*.

[0040] (1) Ultraviolet spectroscopy analysis:

[0041] A 1.0 mg / mL PPP-2 solution was prepared, and the wavelength range of 200-800 nm was scanned using a UV spectrophotometer. The results showed no obvious absorption peaks at 260 nm and 280 nm, indicating that PPP-2 is free of nucleic acid and protein impurities and has high purity. Figure 2 ).

[0042] (2) Infrared spectroscopy analysis:

[0043] Weigh 2 mg of PPP-2 powder and 200 mg of KBr, mix, grind, and compress into tablets. Spectroscopy the results using a Fourier transform infrared spectrometer at 4000-400 cm⁻¹. -1Range scanning results showed that PPP-2 exhibited typical polysaccharide characteristic absorption peaks, with a peak at 890 cm⁻¹. -1 A β-glycosidic bond signal was detected at ( ). Figure 3 ).

[0044] (3) Molecular weight determination:

[0045] Using 0.5 mol / L NaCl as the mobile phase, high-performance gel permeation chromatography was employed at a column temperature of 40℃, a flow rate of 0.7 mL / min, and an injection volume of 25 μL. A standard curve was plotted using dextran standards, and the weight-average molecular weight of PPP-2 was calculated to be 17.211 kDa. Figure 4 ).

[0046] (4) Monosaccharide composition analysis:

[0047] Weigh 5 mg of PPP-2, add 2 mL of 3 mol / L TFA, hydrolyze at 120℃ for 3 h, dry under nitrogen, reconstitute with deionized water, and detect by ion chromatography (column: Dionex Carbopac). TM PA20, 3×150 mm; detector: electrochemical detector; flow rate 0.3 mL / min; column temperature 30℃), the results showed that PPP-2 was composed of mannose, galactose, glucose, fucose, glucuronic acid, xylose and glucosamine hydrochloride in a molar ratio of 0.448:0.294:0.189:0.020:0.025:0.012:0.012 (Table 1; Figure 5 ).

[0048] Table 1 Monosaccharide composition of PPP-2

[0049]

[0050] Example 3: Validation of the in vitro immunomodulatory activity of Polysaccharide PPP-2 from *Porphyra yezoensis*

[0051] (1) Cell culture:

[0052] Mouse macrophages RAW 264.7 were seeded in DMEM complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics and cultured in a 37°C, 5% CO2 incubator. Cells were passaged every 2-3 days, and cells in the logarithmic growth phase were used for experiments.

[0053] (2) Cytotoxicity test:

[0054] The cells were prepared into 1×102 5Cell suspensions of PPP-2 at concentrations of 25, 50, 100, 200, 400, 600, and 800 μg / mL were seeded into 96-well plates. After 12 h of culture, 100 μL of complete culture medium containing PPP-2 at concentrations of 25, 50, 100, 200, 400, 600, and 800 μg / mL were added to each well. The blank group received cell-free complete culture medium, and the negative control group received an equal volume of complete culture medium. Each group was divided into three replicates. After 24 h of culture, the culture medium was discarded, and 100 μL of complete culture medium containing 10% CCK-8 reagent was added to each well. After incubation for 2 h, the absorbance at 450 nm was measured using a microplate reader. The results showed that PPP-2 had no significant toxicity to RAW 264.7 cells in the concentration range of 25-800 μg / mL, and significantly promoted cell proliferation at concentrations of 25-600 μg / mL. At a concentration of 100 μg / mL, the cell viability reached 194.76%. Figure 6 ).

[0055] (3) Phagocytic capacity assay: Cells were prepared at a ratio of 1×10⁻⁶. 4 Cells were seeded per well in 96-well plates and cultured for 12 h. The experiment was divided into a negative control group (complete culture medium), a positive control group (2 μg / mL LPS), and drug-treated groups (25, 50, 100, 200, and 400 μg / mL PPP-2), with three replicates per group. After 24 h of culture, the old culture medium was discarded, and 100 μL of physiological saline containing 0.1% neutral red was added to each well. The cells were cultured for another 4 h, and the supernatant was discarded. The cells were washed three times with pre-warmed PBS, and 200 μL of lysis buffer was added to each well. After lysis in the dark for 2 h, the absorbance at 540 nm was measured. The results showed that PPP-2 significantly enhanced the phagocytic ability of RAW 264.7 cells. At 400 μg / mL, the phagocytic ability was 98.19% higher than the blank control group, showing a dose-dependent effect. Figure 7 ).

[0056] (4) NO secretion measurement: cells were prepared at a ratio of 3×10 5 PPP-2 was seeded at 1 / well in a 6-well plate and cultured for 12 h. Afterward, the plates were divided into groups as described above, with 3 replicates per group. After 24 h of culture, the supernatant was collected, and the absorbance at 540 nm was measured and the NO content was calculated according to the NO detection kit instructions. Results showed that PPP-2 at concentrations of 25-400 μg / mL significantly increased NO secretion in a concentration-dependent manner. At 400 μg / mL, the NO release reached 42.50 μmol / mL, which was higher than that of the positive control group. Figure 8 ).

[0057] (5) Cytokine secretion assay: The culture supernatant was collected, and the cytokine secretion levels were detected using a TNF-α and IL-6 ELISA kit. The absorbance at 450 nm and 630 nm was measured and the concentrations were calculated according to the kit instructions. The results showed that PPP-2 significantly promoted the secretion of TNF-α and IL-6 within the concentration range of 25-400 μg / mL. At 400 μg / mL, the secretion of TNF-α reached 42788.63 pg / mL, and the secretion of IL-6 was 526.44 pg / mL. Figure 9 ).

[0058] (6) Cytokine mRNA expression assay: Cell treatment was the same as described above. After culturing for 24 h, total RNA was extracted using the Trizol method, and cDNA was synthesized using the Evo M-MLV reverse transcription kit. GAPDH was used as an internal reference gene, and real-time quantitative PCR was performed using the SYBR Green ProTaq HS kit. Primer sequences are shown in Table 2. ⁻ΔΔCt The relative expression levels of the target genes were calculated using a method. The results showed that PPP-2 dose-dependently upregulated the mRNA expression levels of TNF-α, IL-1α, IL-1β, IL-6, IL-10, and iNOS. At 200 μg / mL, the expression levels of each gene were 4.81 times, 567.28 times, 40.86 times, 1073.96 times, 10.23 times, and 12.87 times that of the control group, respectively. Figure 10 ).

[0059] Table 2 Primer sequences for RT-qPCR of cytokine-related genes

[0060]

Claims

1. Phyllophoria pulla S-LWZ20190428-3b, with accession number CGMCCNo.42757.

2. Dark-colored leaf-infused polysaccharide, characterized in that, The weight-average molecular weight of the *Phoebe zhennan* polysaccharide is 17.211 kDa. The polysaccharide from *Phoebe d'Argent* is composed of mannose, galactose, glucose, fucose, glucuronic acid, xylose, and glucosamine hydrochloride.

3. The *Phoebe datura* polysaccharide according to claim 2, characterized in that, The molar ratio of mannose:galactose:glucose:fucose:glucuronic acid:xylose:glucosamine hydrochloride is 0.448:0.294:0.189:0.020:0.025:0.012:0.

012.

4. The use of the polysaccharide from *Phoebe datura* according to any one of claims 2-3 in in vitro immunomodulation.

Images