Application of cyclic lipopeptide Fengycin in preparation of antineoplastic drug
An anti-tumor drug, a cyclic lipopeptide technology, applied in the directions of anti-tumor drugs, cyclic peptide components, drug combinations, etc., can solve the problems of staying anti-fungal activity, and the anti-tumor activity of cyclo lipopeptide Fengycin has not been reported, and achieves low cellularity. toxic effects
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Embodiment 1
[0032] Example 1 The Fengycin used in the examples of the present invention is derived from Bacillus amyloliquefaciens fmb-50 (Bacillus amyloliquefaciens fmb-50, CGMCC No.6249, preserved in the Enzyme Engineering Laboratory of the School of Food Science and Technology, Nanjing Agricultural University), and fermented at 32°C at 150r / min ( Fermentation medium: glucose 20.0g, L-sodium glutamate 5.0g, MgSO 4 0.5g, KCl0.5g, KH 2 PO 4 1.0g, FeS0 4 ·7H 2 O0.15mg, MnSO 4 5.0mg, CuS0 4 ·5H 2 (00.16mg, pH7.0, replenish water to 1L), after 36h, collect fermented liquid, extract with methanol, extract liquid is through silica gel column chromatography, discard with chloroform / methanol (2 / 1) eluting, replace with chloroform / methanol / water (65 / 25 / 4) elution, and collect the eluate, reverse phase chromatography (RP-HPLC) purification, freeze-drying.
[0033] The spectral data of structural identification is shown in Figure 8, and its The ESI mass is consistent with the literature: Bi...
Embodiment 2
[0034] Example 2 Cytotoxicity study of cyclic lipopeptide Fengycin on 95D, A-549, H460, SGC-7901, HCT-116, MCF-7 and normal cells L02 and HELF
[0035] The experimental method is the internationally accepted MTT method: according to the growth rate of the cells, the cells in the logarithmic phase are seeded in a 96-well plate at 180 μl / well, and 20 μl / well is added after 24 hours of adherent growth. Set up three replicate wells for each concentration, and set the corresponding concentration of PBS vehicle control and no cells to zero empty. cells at 37°C, 5% CO 2 Cultivate under conditioned conditions for 72 hours, then add 20 μl, 5 mg / ml MTT, at 37°C, 5% CO 2 Incubate under the conditions for 4 hours, discard the supernatant, add 150 μl DMSO to each well, shake for 10 minutes, detect the OD value with a microplate reader at a wavelength of 570 nm, and calculate the inhibition rate.
[0036] The specific operation is: take logarithmic phase tumor cells (95D, A-549, H460, SGC...
Embodiment 3
[0037] Example 3 Effect of Cyclolipopeptide Fengycin on 95D Cell Cycle and Apoptosis-Related Proteins
[0038] The analysis of cell cycle adopts the method of PI single staining. Take 95D cells in logarithmic phase, adjust the cell concentration to 5×105 / ml, inoculate in six-well plates, and stimulate them with 200 μg / ml Fengycin for 6 hours, 12 hours, and 24 hours after culturing for 24 hours. The cells were collected by centrifugation, washed twice with PBS, resuspended with 75% cold ethanol and placed at -20°C overnight. The cells were centrifuged and washed twice with PBS, the cells were resuspended in PBS and 20 mg / ml RNaseA was added and incubated at 37°C for 30 min, then the cells were added 50 mg / ml PI and incubated for 30 min at room temperature in the dark, and then detected by flow cytometry. The results showed that Fengycin could cause G0 / G1 phase arrest in 95D cells. see results figure 2 (A).
[0039] 95D cells in logarithmic phase were inoculated into six-we...
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