Application of naphthoγ-pyrone compounds in the preparation of anti-Helicobacter pylori drugs or preventive health care products
A technology of pyrone and compound, which is applied in the field of marine fungal metabolites as new antibacterial agents, and achieves the effects of simple operation, high extraction yield and high product purity
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Embodiment 1
[0041] Example 1 Isolation of fungal DM94
[0042] The strain DM94 was isolated from the root soil of the mangrove tree. The soil samples were first placed in a sterile petri dish and air-dried in the natural environment. Then, 1 g of the air-dried soil samples were weighed and dissolved in 10 mL of 50% old seawater to obtain 10 mL of 50% old seawater. -1 solution, continue to dilute to 10 -2 , 10 -3 , to obtain three gradient sample solutions. Take 100 μL respectively and spread it on the separation medium GPY medium, Martin medium, CA medium, PDA medium, SDA medium and fungus No. 2 medium. Two separate mediums with different dilution concentrations are in parallel. Culture at 28°C for 1 to 8 weeks. The single colonies were observed and picked, and were inoculated on the fungus No. 2 medium to obtain pure cultured strains.
Embodiment 2
[0043] Example 2 Molecular identification of fungal DM94
[0044] Extraction of genomic DNA: Obtain the DM94 genomic DNA template using a nucleic acid rapid extractor. The operation is as follows: Take a sterile DNA extraction tube filled with quartz sand, add 500 μL of TE, pick an appropriate amount of mycelium into the tube, and extract by high-speed shaking 1min. Add 500 μL of phenol-chloroform solution and centrifuge to obtain genomic DNA.
[0045] Amplification of gene sequence: 12.5μL of 2x PCR mastermix was added, primers ITS1 (5'-TCC GTAGGT GAA CCT GCG G-3') and ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3'), ( 18SF is 5'-AACCTG GTT GAT CCT GCC AGT-3'; 18SR is 5'-CGA CGG GCG GTG TGT AC-3') 1 μL each, template DNA 2 μL, and double distilled water 13.5 μL. The amplification conditions were pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30S, annealing temperature at 59°C for 30S, extension temperature at 72°C for 30S, a total of 32 cycles; post-extension at 72°C ...
Embodiment 3
[0049] The small preparation of the fermentation crude extract of embodiment 3 fungus DM94
[0050] Bacteria were grown in corn solid fermentation medium (corn kernels 50g; ammonium tartrate 2.37g; yeast extract 0.86g; KH 2 PO 4 0.25g; MgSO 4 0.17g; sea salt 0.4g; distilled water 20mL, natural pH. (Hong Kui, Yang Xue, A high-yielding pseudoshell compound strain Aspergillus pyroli TKYX429 and its application, ZL201310186483.4)) 28° C., cultured for 4 weeks. First soak the fermentation product with 80% acetone for 12h, then ultrasonicate for 20min with an ultrasonic extractor, extract with ethyl acetate for 3 to 5 times, combine the extracts, evaporate to dryness, and finally dissolve in chromatographic methanol. The samples were used for antibacterial activity detection and HPLC detection.
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