Cyathin compounds, preparation method and application of cyathin compounds to neuroinflammation resisting

[0090] The ten compounds of the present invention, nestomycin 1-10, are isolated from the fermented liquid of Cyathus africanus. The Nestella africanum was purchased from the China General Microorganism Culture Collection and Management Center, number: CGMCC 5.1163; the strain was preserved in the Natural Medicinal Chemistry Research Center, School of Chemistry and Pharmacy, Northwest A&F University, number: (No.CA20150728).

[0091] The storage and activation culture conditions of the N. africanus are as follows: the preserved strain adopts a PDA slant medium. To activate the strain, inoculate the strain on a PDA plate, activate and cultivate it at 28°C for 7 days, until the plate is covered with mycelium. PDA plate medium: Each liter medium contains 200g potatoes, 20g glucose, and 16g agar powder.

[0092] One, the extraction method of the compound of the present invention, identification and anti-neurinflammation assay and application:

[0093] 1. Experimental materials

[0094] Medium:

[0095] Yeast peptone medium: glucose 20g, yeast extract 2g, peptone 5g, MgSO 4.7H 2 O 0.5g, KH 2 PO 4 1g, water 1000ml, pH=6.5;

[0096] Reagents and instruments:

[0097] Commonly used organic solvents: chloroform, methanol, ethyl acetate, petroleum ether, acetone, etc. are industrial reagents, which are used after redistillation. Organic solvents: chromatographic methanol, chromatographic acetonitrile, etc., use analytical or chromatographic pure reagents depending on the actual use. Unless otherwise specified, the amounts of reagents used below are volume ratios.

[0098] Commonly used instruments: polarimeter Rudolph AutopolⅢ; high performance liquid chromatography: Waters 1525; ultraviolet spectrometer: Thermo Evolution-300; nuclear magnetic resonance: Bruker AvanceⅢ500 (TMS internal standard); low-resolution mass spectrometer: Thermo Fisher LTQ Fleet. Rotary evaporator: Büchi Rotavapor R-101, R-3HB type; low-temperature coolant circulation pump: DLSB-10/20 type (Zhengzhou Great Wall Technology Industry and Trade Co., Ltd.); circulating water multi-purpose vacuum pump: SHB-Ⅲ type (Zhengzhou Great Wall Science, Industry and Trade Co., Ltd.); ultra-clean workbench: SW-OJ-2F type (Sujing Group Suzhou Antai Air Technology Co., Ltd.); vertical steam sterilizer (Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory). Column chromatography silica gel (100-200 mesh, 200-300 mesh and 300-400 mesh) and thin-layer chromatography silica gel (silica gel H) are all produced by Qingdao Ocean Chemical Factory; liquid chromatography column: Hypersil BDS 5μm C18 (250× 4.6and 250×10; Thermo); Hydroxypropyl Sephadex LH-20 and RP-C18 reverse silica gel are both produced by Merck.

[0099] 1.1 Fermentation and cultivation of N. africanii:

[0100] Fermentation culture conditions include: get the activated good N. 2 ) was inoculated on 200mL yeast peptone liquid medium, and 200mL yeast peptone liquid medium was placed in a 500mL Erlenmeyer flask for cultivation; the culture conditions were 28°C and 130rpm for 28 days; fermentation obtained a total of 40L fermentation broth.

[0101] The method for activating the N. africanii bacteria cake: inoculate the N. africanii species on a PDA plate (PDA plate medium), and activate and cultivate it at 28° C. for 7 days until the plate is covered with mycelium. PDA plate medium: Each liter medium contains 200g potatoes, 20g glucose, and 16g agar powder.

[0102] 1.2 Compound extraction and separation:

[0103]After the fermentation broth obtained in 1.1 is concentrated, fraction A, fraction B, fraction C, fraction D, fraction E and fraction F are obtained through organic solvent extraction and column chromatography gradient elution successively. The specific organic solvent can be ethyl acetate Ester, the chromatographic column is RP-18, and the gradient elution condition is methanol: water (10% → 100%); fraction E obtains fraction E1, fraction E2, fraction E3, fraction E4 and fraction E5 after silica gel column chromatography ; The fraction E2 was purified to obtain the antocin compound 1, and the fraction E3 was purified to obtain the antocin compound 7. The purification method can be gel column LH-20 and semi-preparative HPLC;

[0104] Fraction E4 was eluted by gel column LH-20, and then eluted by silica gel column chromatography to finally obtain anotomycin compound 8 and anoderin compound 6;

[0105] Fraction D was sequentially eluted by gel column LH-20 and silica gel column chromatography to obtain fractions D1, D2, D3, D4 and D5, and fraction D3 was purified to obtain nestocin compound 10. The purification method was sequentially passing through gel column LH-20 and silica gel column chromatography are further processed with semi-preparative HPLC after elution;

[0106] Fraction C is separated by silica gel chromatographic column, chloroform:methanol (50:1 → 10:1), obtains fractions C1, C2, C3, C4 and C5 successively, and fraction C2 obtains fractions C2.1 and C2.1 after passing through gel column LH-20 Fraction C2.2 and Fraction C2.1 were purified to obtain the nestomycin compound 3, and the purification method was semi-preparative HPLC;

[0107] Fraction C2.2 was eluted by silica gel column chromatography to obtain anoderin compound 2 and anoderin compound 5;

[0108] Fraction B is separated by silica gel chromatographic column, chloroform:methanol (50:1 → 10:1), and fractions B1, B2, B3, B4 and B5 are obtained successively, and the adenestin compound 9 obtained after fraction B3 is purified, the purification method is as follows: After passing through the gel column LH-20, it was treated with preparative thin-layer chromatography;

[0109] Fraction B4 was sequentially eluted by gel column LH-20 and silica gel column chromatography to obtain the nestomycin compound 4;

[0110] The extraction method of concrete nestomycin compound comprises:

[0111] Concentrate the 40L fermentation broth obtained in 1.1 to 5L, add ethyl acetate in an equal volume ratio and extract 3 times to obtain 30.2g of the crude extract, segment with a RP-18 column, and wash with methanol:water (10%→100%) gradient Take off, obtain 6 fractions A-F successively (see for details figure 1 ). Fraction E is separated by silica gel chromatographic column, chloroform:methanol (volume ratio 50:1 → 10:1), obtains 5 fractions (E1-E5) successively (see for details figure 2 ), fraction E2 is further purified by gel column LH-20 (solvent is methyl alcohol) and semi-preparative HPLC (55%, methanol-water, 2ml/min) successively to obtain the nestomycin compound 1 (t R = 13.0 min, 200.0 mg). Fraction E3 was further purified by gel column LH-20 (solvent is methanol) and semi-preparative HPLC (90%, methanol-water, 2ml/min) in turn, and the obtained anotin compound 7 (t R = 9.0 min, 8.0 mg). Fraction E4 was successively eluted by gel column LH-20 (solvent is methanol) and silica gel column chromatography (chloroform:methanol, 20:1) to obtain anotomycin compound 8 (5.5 mg) and anotomycin compound 6 ( 7.2mg) (see image 3 ). Fraction D is separated by silica gel chromatographic column, chloroform:methanol (50:1 → 10:1), obtains 5 fractions (D1-D5) successively (see for details Figure 4 ), the fraction D3 passed through the gel column LH-20 (solvent is methanol) successively, and was eluted by silica gel column chromatography (chloroform:methanol, 20:1) and further used semi-preparative HPLC (39%, methanol-water, 2ml /min) purification to obtain the nestomycin compound 10 (t R = 30.0 min, 6.0 mg). Fraction C is separated by silica gel chromatographic column, chloroform:methanol (50:1 → 10:1), obtains 5 fractions (C1-C5) successively (see for details Figure 5 ), fraction C2 is divided into 2 parts after passing through gel column LH-20 (solvent is methanol), C2.1 and C2.2 (see Image 6 ). Fraction C2.1 was purified by semi-preparative HPLC (40%, methanol-water, 2ml/min) to give the nestocin compound 3(t R = 13.5 min, 4.2 mg). Fraction C2.2 was eluted by silica gel column chromatography (chloroform:methanol, 40:1) to obtain anoderin compound 2 (7.3mg) and anoderin compound 5 (6.0mg) (see Figure 7 ). Fraction B is separated by silica gel chromatographic column, chloroform:methanol (50:1 → 10:1), obtains 5 fractions (B1-B5) successively (see for details Figure 8 ), fraction B3 was further purified by preparative thin-layer chromatography (chloroform:methanol, 20:1) after being passed through gel column LH-20 (solvent is methanol), and the obtained nestocin compound 9 (8.3mg) was obtained. Fraction B4 was sequentially eluted by gel column LH-20 (solvent: methanol) and silica gel column chromatography (chloroform:methanol, 25:1) to obtain anotomycin compound 4 (5.4 mg).

[0112] 1.3. The physical and chemical properties of the ten nestomycin compounds are:

[0113] Table 1 Neticin Compounds 1-10 1 H NMR data

[0114]

[0115]

[0116] Table 2 Neticin Compounds 1-10 13 C NMR data

[0117]

[0118]

[0119] The physical and chemical properties of nidusin compound 1 of the present invention are:

[0120] C 20 h 30 o 4 ,White powder.

[0121] [α] D 25 =-82.0 (c=0.17, MeOH);

[0122] UV(MeOH):λ max (logε):230(3.06)nm;

[0123] IR(KBr):ν max =3451,2958,2866,2082,1640,1050cm -1;

[0124] HR-ESI-MS: m/z 357.2032[M+Na] +;

[0125] 1 H-NMR spectrum and 13 C-NMR spectrum data are shown in Table 1 and Table 2; Figure 9.

[0126] The physical and chemical properties of nidusin compound 2 of the present invention are:

[0127] C 20 h 28 o 5 ,White powder.

[0128] [α] D 25 =-77.9 (c=0.19, MeOH);

[0129] UV(MeOH):λ max (logε):214(3.10)nm;

[0130] IR(KBr):ν max =3374,2939,2872,1696,1384,1031cm -1;

[0131] HR-ESI-MS: m/z 371.1825[M+Na] +;

[0132] 1 H-NMR spectrum and 13 See Table 1 and Table 2 for C-NMR spectral data, and see Figure 10.

[0133] The physical and chemical properties of nidusin compound 3 of the present invention are:

[0134] C 20 h 30 o 5 ,White powder.

[0135] [α] D 25 =-66.5 (c=0.18, MeOH);

[0136] UV(MeOH):λ max (logε):230(2.85)nm;

[0137] IR(KBr):ν max =3369,2933,2870,1648,1046,982cm -1;

[0138] HR-ESI-MS: m/z 373.1982[M+Na] +;

[0139] 1 H-NMR spectrum and 13 C-NMR spectrum data are shown in Table 1 and Table 2; Figure 11.

[0140] The physical and chemical properties of nidusin compound 4 of the present invention are:

[0141] C 20 h 28 o 5 , yellow oil.

[0142] [α] D 25 =-105.7 (c=0.09, MeOH);

[0143] UV(MeOH):λ max (logε):230(3.36)nm;

[0144] IR(KBr):ν max =3417,2962,2874,1689,1593,1383cm -1;

[0145] HR-ESI-MS: m/z 371.1825[M+Na] +;

[0146] 1 H-NMR spectrum and 13 C-NMR spectrum data are shown in Table 1 and Table 2; Figure 12.

[0147] The physical and chemical properties of nidusin compound 5 of the present invention are:

[0148] C 20 h 30 o 5 , white solid. [α]D 25 =-47.1 (c=0.09, MeOH); UV(MeOH):λ max (logε):231(3.30)nm; IR(KBr):ν max =2961,2384,2310,1738,1366,1218cm -1; HR-ESI-MS: m/z 373.1985 [M+Na] +; 1 H-NMR spectrum and 13 See Table 1 and Table 2 for C-NMR spectral data, and see Figure 13. The physical and chemical properties of nidusin compound 6 of the present invention are:

[0149] C 20 h 30 o 5 ,White powder. [α] D 25 =-68.4 (c=0.07, MeOH);

[0150] UV(MeOH):λ max (logε):230(3.46)nm; IR(KBr):ν max =2935,1735,1687,1606,1369,1217cm -1; HR-ESI-MS: m/z 373.1985 [M+Na] +; 1 H-NMR spectrum and 13 C-NMR spectrum data are shown in Table 1 and Table 2; hydrogen spectrum, carbon spectrum and infrared spectrum are shown in Figure 14 , 15 and 16.

[0151] The physical and chemical properties of the nestomycin compound 7 of the present invention are:

[0152] C 20 h 30 o 4 ,White powder. [α] D 25 =-157.6 (c=0.11, MeOH); UV(MeOH):λ max (logε):230(3.25)nm; ESI-MS(positive)m/z:357.30[M+Na] +; 1 H-NMR spectrum and 13 C-NMR spectrum data are shown in Table 1 and Table 2; Figure 17.

[0153] The physical and chemical properties of the nestomycin compound 8 of the present invention are:

[0154] C 20 h 32 o 4 ,White powder. [α] D 25 =-5.88 (c=0.55, MeOH); UV(MeOH):λ max (logε):213(2.23)nm; ESI-MS(positive)m/z:359.30[M+Na] +; 1 H-NMR spectrum and 13 C-NMR spectrum data are shown in Table 1 and Table 2, and mass spectrograms are shown in Figure 18.

[0155] The physical and chemical properties of the nestomycin compound 9 of the present invention are:

[0156] C 20 h 30 o 5 , yellow solid; [α] D 25 =-28.6 (c=0.46, MeOH); UV(MeOH):λ max (logε):230(2.72)nm; ESI-MS(positive)m/z:701.58[2M+H] +; 1 H-NMR spectrum and 13 C-NMR spectrum data are shown in Table 1 and Table 2, and mass spectrograms are shown in Figure 19.

[0157] The physical and chemical properties of nidusin compound 10 of the present invention are:

[0158] C 20 h 28 o 4 , yellow oil. [α] D 25 =-157.8 (c=0.09, MeOH); UV(MeOH):λ max (logε):230(3.34)nm; ESI-MS(negative)m/z:663.53[2M-H] -; 1 H-NMR spectrum and 13 C-NMR spectral data are shown in Table 1 and Table 2; mass spectrograms are shown in Figure 20.

[0159] 1.4 Western Blot Analysis