Unsaturated fatty acid in flammulina velutipes mononuclear mycelium and preparation method thereof

Abstract

The invention provides unsaturated fatty acid in flammulina velutipes mononuclear mycelium. The unsaturated fatty acid compound is one or more of structural formulas 1 and 2. Unsaturated fatty acid in flammulina velutipes mononuclear mycelium provided by the invention has an anti-inflammatory effect, and has a wide application prospect, # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the application field of edible fungi, and in particular to unsaturated fatty acids in monokaryotic mycelium of Flammulina velutipes and a preparation method thereof. Background Art

[0002] Flammulina filiformis, also known as winter mushroom, edible mushroom, green bar mushroom, and hairy-stem gold money mushroom, belongs to the phylum Basidomycota, class Agaricomycetes, order Agaricales, family Physalacriaceae, and genus Flammulina. It is rich in nutrients and active ingredients, exhibiting numerous pharmacological effects, including anti-tumor, antibacterial, hypoglycemic, lipid-lowering, and anti-inflammatory properties. It is a popular fungus used for both medicinal and edible purposes.

[0003] Inflammation is a physiological defense response of living tissue to harmful stimuli such as pathogens, damaged cells, or irritants. When the body is stimulated by inflammatory factors, it activates macrophages and other cells in the body to synthesize and release various inflammatory mediators, such as nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6). These mediators increase vascular permeability and promote chemotaxis of inflammatory cells, thereby inducing inflammation.

[0004] During the life cycle of Flammulina velutipes, nuclear phase changes are complex. Research has shown that gene expression varies significantly among monokaryotic strains of Flammulina velutipes. Differences in upstream gene expression influence the synthesis and expression of downstream secondary metabolites. Therefore, identifying anti-inflammatory drugs or lead compounds from the fermentation products of monokaryotic strains of Flammulina velutipes is a promising direction for the development of Flammulina velutipes resources. Summary of the Invention

[0005] The present invention first provides an unsaturated fatty acid in the monokaryotic mycelium of Flammulina velutipes, which is a compound of structural formula 1 or 2 or a pharmaceutically acceptable salt thereof:

[0006]

[0007] The present invention also provides a method for preparing unsaturated fatty acids from the monokaryotic mycelium of Flammulina velutipes, comprising the following steps:

[0008] (1) Fermentation of monokaryotic strains of Flammulina velutipes

[0009] The strain stored in a 4°C refrigerator was activated on a PDA plate medium and inoculated into a first-level triangular shake flask (medium formula: 20% potato, 2% glucose, natural pH). The culture was shaken at 150 rpm and 25°C for 7 days in the dark. The inoculum was then transferred to a second-level shake flask (using the same medium formula as the first-level strain) at a 10% (volume percentage) inoculum. The culture was shaken at 150 rpm and 25°C in the dark for 4 days. The culture was then transferred to a static culture for 30 days, and the mycelium was harvested.

[0010] (2) Extraction and extraction of mycelium

[0011] The freeze-dried mycelia are soaked and extracted with 80-95% by volume ethanol aqueous solution at room temperature for 1-4 times, each time for 24 hours, and the extracts are combined and concentrated to obtain an ethanol extract of Flammulina velutipes mycelia;

[0012] The ethanol extract of the Flammulina velutipes mycelia was further extracted with an equal volume of petroleum ether for 1-4 times, the petroleum ether phase was discarded, and then extracted with an equal volume of ethyl acetate for 1-4 times, the ethyl acetate extracts were combined, and concentrated under reduced pressure to dryness to obtain an ethyl acetate phase;

[0013] (3) Preparation of compounds

[0014] The ethyl acetate phase was packed with YMC ODS C18 (particle size 50 μm, pore size 12 nm) and separated by gradient elution with water (phase A) and acetonitrile (phase B) (0-40 min, B: 10%-70%; 40-60 min, B: 70%-100%) at a flow rate of 200 mL / min; one fraction was collected every 200 mL, for a total of 59 fractions, which were numbered in the order of receipt and analyzed by thin-layer chromatography. Similar fractions were combined to obtain Fr.1-Fr.15.

[0015] According to the results of liquid chromatography detection, Fr.15 is rich in the characteristic UV absorption peak of 210nm of unsaturated fatty acid compounds, so Fr.15 was further separated and purified;

[0016] A Zorbax Eclipse Plus C18 column (5 μm, 4.6 mm × 250 mm) was used for semi-preparative chromatography. Gradient elution was performed with water (phase A) and acetonitrile (phase B) (0-45 min, B: 65%-90%; 45-65 min, B: 90%-90%). Detection was performed at a wavelength of 210 nm. The chromatographic peak with a RT of 16-20 min was collected to obtain Fr.15-1.

[0017] A Zorbax Eclipse Plus C18 column (5μm, 4.6mm×250mm) semi-preparative column was selected to further separate Fr.15-1, with gradient elution of water (phase A) and acetonitrile (phase B) (0-45min, B: 65%-70%), detection at a wavelength of 210nm, and the chromatographic peak at 28.062min was collected to obtain compound 1 (9.7mg); the chromatographic peak at 37.082min was collected to obtain compound 2 (21.6mg).

[0018] The structures of the obtained compounds 1 and 2 were analyzed by mass spectrometry, optical rotation, ultraviolet, infrared, and nuclear magnetic resonance detection, and their precise chemical structures were determined to be the above-mentioned structural formula 1 and structural formula 2, respectively. The chemical names of the compounds of the above-mentioned structural formulas 1-2 are as follows:

[0019] Compound 1: (4S,10S,2E)-4,10-dihydroxy-2-octadecenoic acid

[0020] Compound 2: (4S,2E)-4-hydroxy-15-oxoicos-2-enoic acid

[0021] The present invention also provides the use of the unsaturated fatty acids in the monokaryotic mycelium of Flammulina velutipes as a drug for preventing or treating inflammation; wherein the unsaturated fatty acid compound is one or two of the compounds of the following structural formula 1-2.

[0022] The present invention also provides a composition for preventing and / or treating inflammation or playing an auxiliary role during or after the treatment of inflammation, which comprises one or two of the unsaturated fatty acids in the monokaryotic mycelium of Flammulina velutipes.

[0023] The present invention uses a monokaryotic strain of Flammulina velutipes as the research subject, conducting liquid static fermentation and isolating and purifying new unsaturated fatty acid compounds from the extract. Structural identification and anti-inflammatory activity studies of these compounds revealed that these unsaturated fatty acid compounds possess excellent anti-inflammatory activity and have potential value for development as medicines or health foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Fr.15 semi-preparative liquid chromatogram

[0025] Figure 2 Fr.15-1 semi-preparative liquid chromatogram

[0026] Figure 3 UV chromatogram of compound 1 (210 nm)

[0027] Figure 4UV chromatogram of compound 2 (210 nm)

[0028] Figure 5 Effects of compounds on LPS-induced TNF-α release in RAW264.7 cells

[0029] Figure 6 Effects of compounds on LPS-induced IL-6 release in RAW264.7 cells DETAILED DESCRIPTION

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0031] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0032] The monokaryotic strain of Flammulina filiformis (strain number: 5S) was provided by the Variety Innovation and Breeding Laboratory of the Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences. It was prepared from the dikaryotic strain of Flammulina filiformis 0990 by protoplast unikaryonization technology and preserved in the Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences.

[0033] Potato agar (PDA), dexamethasone, and Griess reagent were purchased from Beijing Solaibao Technology Co., Ltd.

[0034] Glucose, analytical grade methanol, petroleum ether, ethyl acetate, ethanol, acetonitrile, and chromatographic grade methanol and acetonitrile were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0035] Lipopolysaccharide (LPS) was purchased from Sigma, USA;

[0036] Mouse monocyte-macrophage RAW264.7 cells were purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, with the product catalog number SCSP-5036.

[0037] Mouse TNF-α and IL-6 kits were purchased from Beijing Sizhengbai Biotechnology Co., Ltd.

[0038] Example 1 Preparation of Compounds 1 and 2

[0039] (1) Fermentation of monokaryotic strains of Flammulina velutipes

[0040] The strain, stored in a 4°C refrigerator, was activated on PDA plates and inoculated into a first-stage triangular shake flask (20% potato, 2% glucose, natural pH). Culture was shaken at 25°C (150 rpm, 25°C) in the dark for 7 days. A 10% inoculum was then transferred to a second-stage shake flask (using the same medium formulation) and cultured at 25°C in the dark for 4 days. After static culture at 25°C in the dark for 30 days, the mycelium was harvested.

[0041] (2) Extraction and extraction of mycelium

[0042] The freeze-dried mycelia were soaked and extracted with 95% by volume ethanol aqueous solution at room temperature for 3 times, each time for 24 hours, and the extracts were combined and concentrated to obtain the enoki mushroom mycelia ethanol extract.

[0043] The ethanol extract of Flammulina velutipes mycelia was further extracted three times with an equal volume of petroleum ether, the petroleum ether phase was discarded, and then extracted three times with an equal volume of ethyl acetate. The ethyl acetate extracts were combined, concentrated and dried under reduced pressure to obtain an ethyl acetate phase.

[0044] (3) Preparation of compounds

[0045] The ethyl acetate phase was packed with YMC ODS C18 (50 μm particle size, 12 nm pore size) and separated by gradient elution (B: 10%-70% B, 0-40 min; 70%-100% B, 40-60 min) with water (A) and acetonitrile (B) at a flow rate of 200 mL / min. Fractions were collected every 200 mL, for a total of 59 fractions. These fractions were numbered in the order received and analyzed by thin-layer chromatography. Similar fractions were combined to yield components Fr.1-Fr.15.

[0046] According to the liquid phase detection results, component Fr.15 is rich in the 210nm ultraviolet characteristic absorption peak of unsaturated fatty acid compounds, so Fr.15 is further separated and purified.

[0047] A Zorbax Eclipse Plus C18 column (5 μm, 4.6 mm × 250 mm) was used for semi-preparative chromatography. Gradient elution was performed with water (phase A) and acetonitrile (phase B) (0-45 min, B: 65%-90%; 45-65 min, B: 90%-90%), and detection was performed at a wavelength of 210 nm. Figure 1 ). The chromatographic peak with RT in the range of 16-20 min was collected to obtain Fr.15-1.

[0048] A Zorbax Eclipse Plus C18 column (5 μm, 4.6 mm × 250 mm) was used for semi-preparative chromatography to further separate Fr.15-1. Gradient elution was performed with water (phase A) and acetonitrile (phase B) (0-45 min, B: 65%-70%), and detection was performed at a wavelength of 210 nm. Figure 2 The chromatographic peak at 28.062 min was collected to obtain compound 1 (9.7 mg) ( Figure 3 ); Collect the chromatographic peak at 37.082 min to obtain compound 2 (21.6 mg) ( Figure 4 ).

[0049] (4) Compound structure identification

[0050] Using mass spectrometry, optical rotation, ultraviolet, infrared, and nuclear magnetic resonance detection, the structures of two compounds obtained from the mycelium of Flammulina velutipes were analyzed and their precise chemical structures were determined. The specific compound information is as follows:

[0051] Compound 1: (4S,10S,2E)-4,10-dihydroxy-2-octadecenoic acid

[0052] The molecular formula is C 18 H 34 O4, white amorphous powder, (c 0.400,MeOH); UV(MeOH)λ max (logε)206(0.649);IR(ATR)v max 3148,2952,2927,2853,1741,1261,1094,1028cm -1 ;HREIMSm / z:314.2450M + (C 18 H 34 O4, calculated value 314.2452). 1 H-NMR(CDCl3,500MHz)δ:7.44(1H,dd,J=1.5,5.7Hz,H-3),6.11(1H,dd,J=2.0,5.7Hz,H-2),5.03(1H,m,H-4),3.58(1H,m,H-10),1.76(1H,m, H-5),1.67(1H,m,H-5),1.44(7H,m,H-6,8,9,11,12),1.31(3H,m,H-6, 8,12),1.28(12H,m,H-7,13,14,15,16,17),0.88(3H,t,J=6.9,H-18); 13C-NMR(CDCl3,125MHz)δ:173.3(C-1),156.4(C-3),121.7(C-2),83.6(C-4),72.1(C-10),37.7(C-9),37.5(C-11),33.3(C -5),32.0(C-16),29.8(C-7),29.6(C-13),29.4(C-14,15),25.8(C-12),25.6(C-8),25.1(C-6),22.8(C-17),14.2(C-18).

[0053] Compound 2: (4S,2E)-4-hydroxy-15-oxoicos-2-enoic acid

[0054] The molecular formula is C 20 H 36 O4, light yellow oil, (c 0.400,MeOH); UV(MeOH)λ max (logε)205(0.424);IR(ATR)v max 3260,2952,2929,2855,1737,1703,1379,1030cm -1 ;HRFIMSm / z:341.2691[M+H] + (C 20 H 37 O4, calculated value 341.2686). 1 H-NMR(CDCl3,500MHz)δ:7.44(1H,dd,J=1.5,5.7Hz,H-3),6.11(1H,dd,J=2.0,5.7Hz,H-2),5.03(1H,m,H-4),2.38 (4H,m,H-14,16),1.55(4H,m,H-13,17),1.27(20H,m,H-5,6,7,8,9,10,11,12,18,19),0.88(3H,t,J=6.9Hz,H-20); 13C-NMR(CDCl3,125MHz)δ:211.7(C-15),168.5(C-1),156.4(C-3),121.7(C-2),83.5(C-4),43.0(C-16),42.8(C-14),33.2(C-5),32.1(C-18) ,31.8(C-7),29.8(C-8),29.5(C-9),29.4(C-10),29.2(C-11),29.1(C -12),24.9(C-6),24.0(C-13),23.7(C-17),22.7(C-19),14.2(C-20).

[0055] After structural analysis, it was determined that the two compounds were new compounds, both of which were unsaturated fatty acids. Their names and structural formulas are shown below:

[0056] Compound 1: (4S,10S,2E)-4,10-dihydroxy-2-octadecenoic acid

[0057]

[0058] Compound 2: (4S,2E)-4-hydroxy-15-oxoicos-2-enoic acid

[0059]

[0060] Example 2 Anti-inflammatory activity detection of compounds 1 and 2

[0061] Take RAW264.7 cells in the logarithmic phase and dilute them to 1×10 5 A single cell suspension of 100 μg / mL was inoculated into a 96-well plate, 194 μL of cell suspension was added to each well, and after culturing at 37 ° C in a CO2 incubator for 24 hours, 1 μL of compound 1, compound 2 and positive control dexamethasone were added respectively. The compound and positive control were set to three concentration gradients of 5, 2, and 1 μmol / mL, and three parallels were set for each concentration. After the cells with the compound or positive control were incubated for another 1 hour, 5 μL of LPS at a concentration of 100 μg / mL was added to induce damage. The experiment also retained the LPS group (only 5 μL of LPS was added) and the blank control group (no addition). After 48 hours of culture, the cell culture supernatant was aspirated for the following two tests:

[0062] (1) Effects of compounds 1 and 2 on NO release from LPS-stimulated RAW264.7 cells:

[0063] Pipette 100 μL of cell culture supernatant into a microplate. Add 50 μL of Griess reagent to each well. Incubate at room temperature in the dark for 10 minutes. Measure the OD value at 543 nm using a microplate reader. Calculate the NO concentration in the cell culture supernatant of each group and the inhibition rate of NO release using a NaNO2 standard curve. Calculate the inhibition rate of the sample according to Publication 1.

[0064]

[0065] The half inhibition rate was calculated based on the inhibition rate under different concentration gradients. The results are shown in Table 1 below:

[0066] Table 1 IC of compounds inhibiting NO release from RAW264.7 cells 50 value

[0067]

[0068] The experimental results showed that compounds 1 and 2 had strong activity in inhibiting NO production, and their effects were better than the positive control dexamethasone.

[0069] (2) Effects of compounds on LPS-induced inflammatory cytokine expression in RAW264.7 cells:

[0070] The cell culture supernatant was aspirated and the TNF-α and IL-6 levels in the cell supernatant were detected according to the ELISA method instructions.

[0071] Experimental results ( Figure 5 、 6 ) showed that TNF-α and IL-6 levels were significantly elevated in the Compound 1 and 2 groups, indicating that both Compound 1 and Compound 2 significantly inhibited the release of TNF-α and IL-6. Specifically, at a concentration of 25 nmol / mL, Compound 1 reduced TNF-α levels by 59.95% and IL-6 levels by 26.13%. At a concentration of 25 nmol / mL, Compound 2 reduced TNF-α levels by 16.32% and IL-6 levels by 24.22%.

Claims

1. An unsaturated fatty acid in the monokaryotic mycelium of Flammulina velutipes, which is a compound of structural formula 1 or 2 or a pharmaceutically acceptable salt thereof:

2. The method for preparing unsaturated fatty acids from monokaryotic mycelia of Flammulina velutipes according to claim 1, comprising the following steps: (1) Fermentation of monokaryotic strains of Flammulina velutipes The monokaryotic strain of Flammulina velutipes was inoculated into a first-stage shake flask and cultured in the dark at 150 rpm and 25°C for 7 days; then transferred to a second-stage shake flask at a volume percentage of 10% and cultured in the dark at 150 rpm and 25°C for 4 days; then transferred to a static culture for 30 days; The culture medium formula in the first-stage shake flask and the second-stage shake flask is as follows: 20% potato, 2% glucose, natural pH; (2) Extraction and extraction of mycelium The freeze-dried mycelia are soaked and extracted with 80-95% by volume ethanol aqueous solution at room temperature for 1-4 times, each time for 24 hours, and the extracts are combined and concentrated to obtain an ethanol extract of Flammulina velutipes mycelia; The ethanol extract of the Flammulina velutipes mycelia was further extracted with an equal volume of petroleum ether for 1-4 times, the petroleum ether phase was discarded, and then extracted with an equal volume of ethyl acetate for 1-4 times, the ethyl acetate extracts were combined, and concentrated under reduced pressure to dryness to obtain an ethyl acetate phase; (3) Preparation of compounds The ethyl acetate phase was packed with YMC ODS C18, particle size 50 μm, pore size 12 nm, and gradient elution was performed with water as phase A and acetonitrile as phase B, 0-40 min, B: 10%-70%; 40-60 min, B: 70%-100%, separation, flow rate 200 mL / min; collect one fraction every 200 mL, and combine similar fractions; The last group of fractions containing the characteristic ultraviolet absorption peak of unsaturated fatty acids at 210 nm was further separated using a Zorbax Eclipse Plus C18 column, 5 μm, 4.6 mm × 250 mm, with water as phase A and acetonitrile as phase B, and gradient elution was performed: 0-45 min, B: 65%-90%; 45-65 min, B: 90%-90%, with detection at a wavelength of 210 nm; components with a RT range of 16-20 min were collected and further separated using a Zorbax Eclipse Plus C18 semi-preparative column, with water as phase A and acetonitrile as phase B, gradient elution: 0-45 min, B: 65%-70%, with detection at a wavelength of 210 nm, and the chromatographic peak at 28.062 min was collected to obtain compound 1; the chromatographic peak at 37.082 min was collected to obtain compound 2.

3. Use of the unsaturated fatty acids in the monokaryotic mycelium of Flammulina velutipes according to claim 1 as a drug for preventing or treating inflammation.

4. A composition for preventing and / or treating inflammation or playing an auxiliary role during or after the treatment of inflammation, comprising one or more unsaturated fatty acids in the monokaryotic mycelium of Flammulina velutipes according to claim 1.

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