ophenone as beginning supplies (Scheme S1) [31]. Isobavachalcone (four) was synthesized via Claisen-Schmidt condensation working with 4-hydroxybenzaldehyde with 2 ,4 -dihydroxyacetophenone as starting components (Scheme S2) [32]. Structures in the final solutions three and four have been confirmed by comparing their spectroscopic information with those reported in the literatures [33,34]. Echinatin (three): 1 H-NMR (CD3 OD, 400 MHz, in ppm, J in Hz) eight.03 (1H, d, J = 15.six, H-), 7.97 (2H, d, J = eight.8, H-2 ,6 ), 7.62 (1H, d, J = 15.six, H-), 7.61 (1H, d, J = eight.five, H-6), six.89 (2H, d, J = 8.8, H-3 ,5 ), six.47 (1H, d, J = two.two, H-3), 6.44 (1H, dd, J = eight.5, 2.two, H-5), three.89 (3H, s, 2-OMe). Isobavachalcone (4): 1 H-NMR (CD3 OD, 400 MHz, in ppm, J in Hz) 7.84 (1H, d, J = 8.9, H-6 ), 7.78 (1H, s, J = 15.four, H-), 7.64 (1H, d, J = 15.four, H-), 7.62 (2H, d, J = eight.6, H-2,six), 6.85 (2H, d, J= 8.six, H-3,five), 6.43 (1H, d, J = eight.9, H-5 ), five.23 (1H, m, H-2 ), 3.33 (2H, overlapped, H-1 ), 1.78 (3H, s, H-4 ), 1.66 (3H, s, H-5 ). three.five. Microorganisms and Screening for Biostransformation All of the microorganisms had been obtained from the Korean Collection for Kind Cultures (KCTC, Daejeon, Korea) and Korean Culture Center of Microorganisms (KCCM, Seoul, Korea). The strains made use of for preliminary screening are as follows: Absidia coerulea KCTC 6936, Aspergillus niger KCCM 60332, Aspergillus oryzae KCCM 60345, Hormoconis resinae KCTC 6966, Mortierella ramanniana var. angulispora KCTC 6137, Penicillium chrysogenum KCTC 6933, Pichia pastoris KCTC 7190, Tremella mesenterica KCTC 7131. IL-12 Modulator Formulation fermentation experiments were performed in three sorts of media. A. coerulea, A. niger, A. oryzae, P. chrysogenum were incubated on malt medium (malt extract 20 g/L, D-glucose 20 g/L, peptone 1 g/L). H. resinae, M. ramanniana var. angulispora, P. pastoris have been Bcl-2 Antagonist Biological Activity cultured on potato sucrose medium (potato dextrose 24 g/L and sucrose 20 g/L). T. mesenterica was cultured on yeast-malt medium (D-glucose 10 g/L, peptone five g/L, malt extract three g/L, and yeast extract three g/L). Biotransformation was carried out in accordance with the two-stage procedure [35]. Inside the screening research, the actively growing microbial cultures have been incubated in 250 mL flasks containing 50 mL of media with gentle agitation (200 rpm) at 25 C in a temperaturecontrolled shaking incubator. Ethanol solution (20 mg/mL, 50 ) of the substrate 1, 2, three, or four was added to each flask 24 h after inoculation. And further incubation was performed beneath exactly the same situation for six days. Two controls were made use of for biotransformation within this study, i.e., culture controls consisting of microorganisms expanding within the culture media with out substrates, and substrate controls consisting of culture media and substrates incubated without the need of microorganisms. Basic sampling and TLC monitoring have been performed on Merck silica gel F254 -precoated glass plates. A. niger was identified because the most potent strain to metabolize 1 and hence chosen for scale-up fermentation. 3.six. Scale-up Fermentation, Extraction, and Isolation of Metabolites 51 For scale-up fermentation, A. niger was incubated in 500 mL Erlenmeyer flasks containing 150 mL of media. Following a additional 24 h incubation, the ethanol remedy (20 mg/mL, 150 ) of every single substrate (1, two, three, or four) was evenly distributed to each flask containing stage II cultures (Table five).Int. J. Mol. Sci. 2021, 22,11 ofTable 5. Scale-up fermentation of substrates with a. niger. Substrate 1 two three 4 Substrate Quantity (mg/Flask) three 3 3 three Variety of Flasks eight 13 15 36 Total E
