Triterpenoid compounds and application thereof in diabetes treatment drugs

[0047] Example 1 Preparation method of compound 1 and 2

[0048] 1. Preparation of Inonotus obliquus fermented product by fermentation

[0049] 1) Bacteria:

[0050] The classification of Inonotus obliquus is named Fuscoporia obliqua (Pers: Fr.) Aoshima, which is preserved by the China Agricultural Microbial Culture Collection and Management Center, and the strain number is 1511C0001ACCC51184.

[0051] 2) Cultivation and preservation of seed strains

[0052] Solid medium: glucose 3g, potato extract powder 0.5g, KH2PO40.2g, soy peptone 0.5g, agar 1.5g, add water to 100mL, adjust pH to 6.0.

[0053] Solid culture method: Strains are inoculated on the slope of solid culture medium and cultivated at 28°C for 7-10 days.

[0054] After the solid culture is completed, place it on a slope at 4-10°C and refrigerate for later use.

[0055] 3), shake flask seed culture

[0056] Medium: glucose 3g, yeast extract powder 1.0g, soy peptone 1.0g, KH2PO40.2g, add water to 100mL, adjust pH to 6.0.

[0057] Liquid volume: 150mL medium in a 500mL triangular flask

[0058] Inoculation volume: 30% glycerol frozen hyphae 3ml

[0059] Culture temperature: 28℃

[0060] Training time: 7 days

[0061] Shaker speed: 150rpm

[0062] 4) Seed culture medium for seed tank

[0063] Medium: glucose 1.2kg, soybean powder 1kg, KH2PO4 80g, add water to 40L, adjust pH to 6.0.

[0064] Packing quantity: 40L of medium in a 100L seed tank, sterilized at 123°C for 30min.

[0065] Seed pot cultivation method: cultured 1500ml shake flask seed liquid is connected to the seed pot at 28°C for 7 days. During the cultivation process, the tank pressure is controlled: 0.05MPa, and the ventilation volume is 1:1 (V/V).

[0066] After culturing, microscopically check that the hyphae are strong, deep stained, free of bacteria, and bacteria concentration ≥15%

[0067] 5), fermentation tank culture

[0068] Medium: glucose 12kg, soybean powder 10kg, KH2PO4 0.8kg, L-glutamic acid 0.8kg, add water to 400L, adjust pH 5.5.

[0069] Capacity: 400L of culture medium in 1 ton fermenter

[0070] Fermentation tank culture method: The seed liquid on the cultivated 40L tank is connected to the fermentation tank at 28°C for 8 days. Control tank pressure during cultivation: 0.05 MPa, stirring speed 160 rpm, ventilation volume 1: 1.3 (V/V).

[0071] Judgment of the fermentation end point: the mycelium has deep staining, more vacuoles, and bacteria concentration ≥30%.

[0072] 2. Extraction and separation to obtain compounds 1 and 2

[0073] The fermentation product was filtered and dried to obtain a total of 6000 grams of mycelium of the fermentation product.

[0074] The fermentation broth of Inonotus obliquus was filtered to obtain mycelium. After the mycelium was dried (6Kg), it was extracted three times with 95% ethanol (30 liters each time). The extracts were combined and concentrated under reduced pressure (606g) . The above extract was suspended in 5 liters of water, extracted with petroleum ether 5 times (1 liter each time), and concentrated under reduced pressure to obtain a petroleum ether extract (160 g). The petroleum ether extract was passed through a silica gel column (silica gel 200-300 mesh, 1.5Kg), and an organic solvent was used for gradient elution (ie petroleum ether-acetone 20:1, petroleum ether-acetone 10:1, petroleum ether-acetone 5:1 , Petroleum ether-acetone 2:1, each solvent eluted 2 column volumes), collect petroleum ether-acetone (20:1) fraction (6g). The above part was purified by a reversed-phase C-18 column, and the elution conditions were (methanol-water 5:5, methanol-water 6:4, methanol-water 7:3, methanol-water 8:2, methanol-water 9: 1. Each solvent is eluted by 2 column volumes), the same parts are combined by thin-layer detection, and the same parts are combined and concentrated under reduced pressure to obtain compound 1 (240 mg) and compound 2 (350 mg).

[0075] Example 2: Physicochemical properties and spectral data of compound 1

[0076]

[0077] Compound 1: White amorphous powder; molecular formula is C 31 H 46 O 3;Optical rotation [α] 20 D +70.5(c0.088,CHCl 3 ); infrared (KBr)ν max 3567, 2964, 2928, 1714, 1185, 1125cm -1; Hydrogen spectrum and carbon spectrum (CDCl 3 ,500MHz) See Table 1 Mass spectrum (ESI) m/z: 467[M+H] +.

[0078] Example 3: Physicochemical properties and spectral data of compound 2

[0079]

[0080] Compound 2: White amorphous powder; molecular formula is C 31 H 48 O 3;Optical rotation [α] 20 D +100.0(c0.19,CHCl 3 ); infrared (KBr)ν max 3418,2934,1662,1595,1517,1458,1417,1189,1042,675cm -1; Hydrogen spectrum and carbon spectrum (CDCl 3 ,500MHz) See Table 1 Mass spectrum (ESI) m/z: 469[M+H] +.

[0081] Table 1 NMR data of compounds 1 and 2

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[0083]