The structures of the new compounds were elucidated on the basis of 1- and 2-dimensional (1D/2D) nuclear magnetic resonance (NMR) spectroscopic and mass spectrometric analysis. The absolute configurations of compounds 1, 2, 4, 5, and 7 were determined by comparison of experimental and calculated electronic circular dichroism (ECD) spectra, and the absolute configurations of C-4′ in 6 and C-9 in 7 were determined by [Rh2(OCOCF3)4]-induced ECD spectra. Compound 1 showed moderate α-glucosidase inhibitory activities with IC50 values of 741.5 ± 2.83 μM. Compounds 6 and 11 exhibited DPPH scavenging activities with IC50 values of 15.36 ± 0.24 and 3.69 ± 0.07 μM, respectively.Two new diterpenoids, hypoxyterpoids A ( Hypoxylon species are widespread in terrestrial and marine environments, and chemical investigations of some Hypoxylon spp. have afforded a variety of natural products, such as azaphilones, diterpenes, sporothriolide derivatives, and various aromatic compounds, which exhibited antibacterial, antifungal, and cytotoxic activities + at m/z 373.1995 . The infrared (IR) spectrum of 1 at 1704, 1644 cm−1 showed the presence of carbonyl and double bond groups. Analysis of the 1H and 13C nuclear magnetic resonance , two sp3 quaternary carbons (δC 44.2 and 40.9), four olefinic carbons (two protonated), three sp3 methine (one oxygenated), six sp3 methylene carbons, and three methyl carbons. These data accounted for all 1H and 13C NMR resonances except for three unobserved exchangeable protons and suggested that 1 was a bicyclic compound. Analysis of 1H-1H correlation spectroscopy (COSY) spectrum revealed three spin system: C-1/C-2/C-3, C-5/C-6/C-7, and C-9/C-11/C-12. The heteronuclear multiple bond correlations (HMBC) correlations from H2-1 to C-3 and C-5, H2-3 to C-5, H-5 to C-4, C-10 and C-18, and from H3-18 to C-3, C-4, C-5 and C-19 completed the ring A with the methyl carbon C-18 and the carboxylic carbon C-19 attached to C-4 directly. HMBC correlations from H3-20 to C-1, C-5 and C-10 established the location of the methyl group C-20 at C-10. The exchangeable proton was located at C-2 supported by the chemical shift value for C-2 (δC 62.9). While HMBC correlations from H2-6 to C-8 and C-10, H-9 to C-8, C-10 and C-20, and from H2-17 to C-7, C-8 and C-9 established the ring B, fused with ring A at C-5 and C-10. Additional HMBC correlations from H3-16 to C-12, C-13, C-14 and C-15, from H-14 to C-12 and C-15, from H2-12 to C-13 and C-14, and from H2-11 to C-13 established the C-13–C-16 subunit, with C-12 attached to C-13 of the olefin C-13/C-14. The hydroxyl groups were located at C-2, C-15, and C-19 by default supported by the chemical shift value for C-2 (δC 62.9), C-15 (δC 167.6), and C-19 (δC 178.3). Thus, The compound 1 and rel--5-[(3E)-4-carboxy-3-methylbut-3-en-1-yl]decahydro-3-hydroxy-1,4a-dimethyl-6-methylidenenaphthalene-1-carboxylic + at m/z 439.1605 , indicating the nine degrees of unsaturation. Its 1H and 13C NMR data , two quaternary carbons, four olefinic carbons with two protonated, three sp3 methine carbons with one oxygenated, four sp3 methylene carbons, and three methyl carbons (one oxygenated). These data suggested that 2 was also a bicyclic compound. Detailed comparison of the 1H and 13C NMR data of 2  in ring B of 1 were replaced by the oxygenated methine carbon C-6 (δH/C 4.93/77.7), which was confirmed by the 1H-1H COSY correlations H-5/H-6/H-7 and the HMBC correlations from H-5 to C-9 and C-10, from H-6 to C-5 and C-10, from H2-17 to C-7, C-8 and C-9, and from H-9 to C-8 and C-10. Other HMBC correlations from H2-1 to C-2, C-5 and C-10, from H-5 to C-1, C-3, C-4 and C-10 and from H3-18 to C-3, C-4, C-5 and C-19, as well as the bicyclic feature of 2 established a seven-membered ring (ring A) with the methyl carbon C-18 and the carboxylic carbon C-19 attached at C-4. Additionally, HMBC correlations from H2-1 and H-9 to C-10 and C-20 suggested the carbonyl carbon C-20 was attached to C-10 directly. While HMBC correlation from H3-21 to C-20 located the methoxyl group at C-10. On the basis of these data, the gross structure of 2 was established as shown.Hypoxyterpoid B  from the biosynthetic considerations. The absolute configuration for 2 was also proposed, by a comparison of the experimental and calculated ECD spectra, for the enantiomers -2 (2a) and -2 (2b). The result showed that the calculated spectrum of -2a agreed with the experimental one  was obtained as a yellow oil. Its molecular formula C14H16O5 was established by HRESIMS analysis (m/z 263.0925 [M − H]−), indicating seven degrees of unsaturation. The IR spectrum of 4 at 3306, 1697, 1658, 1598 cm−1 showed the presence of hydroxy, ester carbonyl and aromatic groups. Analysis of its NMR data (δH 9.63), three methyl groups (including one methoxy), two sp3 methines (one oxygenated), eight aromatic or olefinic carbons (three protonated), and one carbonyl carbon. These data accounted for all 1H and 13C NMR resonances except for one unobserved exchangeable proton. The remaining two degrees of unsaturation suggested that 4 was a bicyclic compound. The 1H-1H correlations  to C-3 and C-9, H-9 to C-3 and C-4, and from H-10 and H3-11 to C-3 indicated that C-9 was directly attached to C-3 of the trisubstituted olefin C-3/C-4. Additional HMBC correlations from H-4 to C-1, C-4a, C-8a and C-5, H-5 to C-4, C-6, C-7, and C-8a, and from H-7 to C-1, C-5, C-6 (δC 164.7), C-8 and C-8a, as well as from the methoxy group H3-13 to C-8 established the isochromenone core structure, with the methoxy group carbon C-13 located at C-8. The remaining two exchangeable protons were located at C-6 and C-10 by default. Thus, the planar structure of 4 was established as shown  at the B3LYP/6-311G  level for four stereoisomers -4 (4a), -4 (4b), -4 (4c) and -4 (4d). The overall calculated ECD spectrum of 4a–4d were generated according to Boltzmann weighting of the conformers. The experimental ECD spectrum of 4 was nearly identical to the calculated ECD spectrum for -4 (4a) -4 (4a) , indicat5) was obtained as a yellow oil. The molecular formula was determined as C14H16O5 (seven degrees of unsaturation) by HRESIMS (m/z 263.0924 [M − H]−), which is the same with that of 4. The 1H and 13C NMR data -4 (4c), unambiguously assigned its absolute configuration as 9R,10S. Thus, the structure of 5 was elucidated, as depicted in Hypoxymarin B (NMR data  and 2D N6) was determined to be C14H18O5 (six degrees of unsaturation) by HRESIMS (m/z 267.1226 [M + H]+). In the 1H NMR spectrum , 6.71  indicated the presence of a 1,2,3,6-tetrasubstituted benzene system. Analysis of its NMR data (δC 170.8). The NMR data of 6 resembled those of aspergillumarin B (10) -induced ECD experiment was applied   except tucidated . In orde applied . In the ess rule . By compess rule ,21, the 7) was isolated as a yellow oil with the molecular formula assigned as C11H12O5 on the basis of its HREIMS (m/z 223.0614 [M − H]−), indicating six degrees of unsaturation. Analysis of the 1H and 13C NMR data (δC 169.1). The 1H and 13C NMR data of 7 were almost consistent with penicimarin A (8). Further analysis of its 1H-1H COSY correlations 4 in anhydrous CH2Cl2, a negative Cotton effect at 350 nm in the ECD spectrum was observed for the complex -7a agreed with the experimental one  + .Hypoxyterpoid A (2): brown oil; + .Hypoxyterpoid B (4): Yellow oil; − .Hypoxymarin A (5): Yellow oil; – .Hypoxymarin B (6): White powder; + .Hypoxymarin C (7): Yellow oil; − .Hypoxymarin D  in 96-well microplates, according to the method of Fan et al.  complex (1.0 mg) in CDCl3 (200 μL) and was received, to CD measurements, at a concentration of 2.5 mg/mL. The first ECD spectrum was recorded immediately after mixing, and its time evolution was monitored until stationary (30 min after mixing). The inherent CD was subtracted. The absolute configurations of the secondary alcohols in 6 and 7 were determined -induced ECD spectra. Compound 1 showed moderate α-glucosidase inhibitory activities with IC50 values of 741.5 ± 2.83 μM. Compounds 6 and 11 exhibited potent DPPH scavenging activities with IC50 values of 15.36 ± 0.24 and 3.69 ± 0.07 μM, respectively. Our findings also suggest that the fungal genus Hypoxylon spp. are a rich source of bioactive secondary metabolites, and thus worthy of in-depth investigations.In conclusion, two new diterpenoids, hypoxyterpoids A and B (


H-benzimidazol-2-yl)aniline, salicylaldehyde, and 2,4-dihydroxybenzaldehyde were synthesized and characterized using different spectroscopic methods. Besides their cytotoxic activities, they were examined in human U-937 cells, primate kidney non-cancerous COS-7, and six other, different human tumor cell lines: U251, PC-3, K562, HCT-15, MCF-7, and SK-LU-1. In addition, the synthesized compounds were screened for in vitro antiparasitic activity against Leishmania braziliensis, Plasmodium falciparum, and Trypanosoma cruzi. Additionally, antibacterial activities were examined against two Gram-positive strains  and two Gram-negative strains  using the microdilution method. The lanthanide complexes generally exhibited increased biological activity compared with the free Schiff base ligands. Interactions between the tested compounds and model membranes were examined using differential scanning calorimetry (DSC), and interactions with calf thymus DNA (CT-DNA) were investigated by ultraviolet (UV) absorption. Molecular docking studies were performed using leishmanin (1LML), cruzain (4PI3), P. falciparum alpha-tubulin (GenBank sequence CAA34101 [453 aa]), and S.aureus penicillin-binding protein 2a  as the protein receptors. The results lead to the conclusion that the synthesized compounds exhibited a notable effect on model membranes imitating mammalian and bacterial membranes and rolled along DNA strands through groove interactions. Interactions between the compounds and studied receptors depended primarily on ligand structures in the molecular docking study.Metal-based drugs, including lanthanide complexes, have been extremely effective in clinical treatments against various diseases and have raised major interest in recent decades. Hence, in this work, a series of lanthanum (III) and cerium (III) complexes, including Schiff base ligands derived from (1 In medicinal chemistry, a large number of studied coordination compounds have demonstrated biological properties. One advantage of the unique electronic structures presented by transition metals is the versatility and ability to adjust the properties of a given molecule, which is not always possible for organic drugs . The appTrypanosoma cruzi, resulting in antiparasitic activity imidazol-2-yl)phenyl)imino)methyl)phenol (L1): 2-(m-aminophenyl)benzimidazole  and salicylaldehyde  were mixed and stirred in methanol at reflux. After 2 h of reaction, the yellow precipitates were washed with a cold mixture of water:methanol (1: 1) and dried in a vacuum oven for 4 h. Yield: 1.24 g, 79%. C20H15N3O (313.36 g·mol−1): Calc. C, 76.66; H, 4.83; N, 13.41. Found: C, 76.38; H, 4.77; N, 13.49%. Melting Point: 233–235 °C. IR (ATR cm−1): υ(N=CH)imine 1602; υ(N=C)imidazole 1575; υ(C=C) 1509; υ(C-O)phenolic 1258. 1H NMR (DMSO-6d) δ (ppm) 12.99 , 12.99 , 9.10 , 8.20 , 8.12 , 7.73 , 7.65 , 7.64 , 7.56 , 7.46 , 7.23 , 7.04 , 7.03 . 13C{1H} NMR (DMSO-6d) δ (ppm) 164.52 (azomethine CH=N), 160.82 (OH-C2’’), 151.18 (imidazole CH=N), 149.28–112.38 . MS : 313.2-(((3-(1H-benzo[d]imidazol-2-yl)phenyl)imino)methyl)benzene-1,3-diol (L2): It was carried out following a procedure similar to L1, using 2-(m-aminophenyl)benzimidazole  and 2,4-dihydroxybenzaldehyde . Yield: 1.42 g, 86%. C20H15N3O2 (329.36 g·mol−1): Calc. C, 72.94; H, 4.59; N, 12.76. Found: C, 72.73; H, 4.63; N, 13.00%. Melting Point: 281–283 °C. IR (ATR cm−1): υ(N=CH)imine 1605; υ(N=C)imidazole 1568; υ(C=C) 1495; υ(C-O)phenolic 1260. 1H NMR (DMSO-6d) δ (ppm) 13.47 , 12.98 , 10.34 , 8.94 , 8.14 , 8.08 , 7.69 , 7.61 , 7.56 , 7.49 , 7.23 , 6.45 , 6.35 . 13C{1H} NMR (DMSO-6d) δ (ppm) 163.69 (azomethine CH=N), 163.55 (OH-C2’’), 163.21 (OH-C4’’), 151.28 (imidazole CH=N), 149.33–102.91 . MS (DART+) m/z: 330.4-(((3-(1H-benzo[d]imidazol-2-yl)phenyl)imino)methyl)phenol (L3): It was carried out following a procedure similar to L1, using 2-(p-aminophenyl)benzimidazole  and salicylaldehyde  in ethanol. Yield: 1.28 g, 82%. C20H15N3O (313.36 g·mol−1): Calc. C, 76.66; H, 4.83; N, 13.41. Found: C, 76.26; H, 4.73; N, 13.43%. Melting Point: 245–247 °C. IR (ATR cm−1): υ(N=CH)imine 1600; υ(N=C)imidazole 1570; υ(C=C) 1495; υ(C-O)phenolic 1277. 1H NMR (DMSO-6d) δ (ppm) 13.00 , 13.00 , 9.09 , 8.29 , 7.71 , 7.63 , 7.46 , 7.24 , 7.02 , 7.01 . ). 13C{1H} NMR (DMSO-6d) δ (ppm) 164.34 (azomethine CH=N), 160.85 (OH-C2’’), 151.24 (imidazole CH=N), 149.67–117.16 . MS : 314.2-(((4-(1H-benzo[d]imidazol-2-yl)phenyl)imino)methyl)benzene-1,3-diol (L4): It was carried out following a procedure similar to L1, using 2-(p-aminophenyl)benzimidazole  and 2,4-dihydroxybenzaldehyde  in ethanol. Yield: 1.19 g, 72%. C20H15N3O2 (329.36 g·mol−1): Calc. C, 72.94; H, 4.59; N, 12.76. Found: C, 73.12; H, 4.54; N, 12.91%. Melting Point: 262–263 °C. IR (ATR cm−1): υ(N=CH)imine 1600; υ(N=C)imidazole 1566; υ(C=C) 1505; υ(C-O)phenolic 1277. 1H NMR (DMSO-6d) δ (ppm) 13.45 , 12.91 , 10.33 , 8.92 , 8.24 , 7.66 , 7.54 , 7.53 , 7.21 , 6.43 , 6.32 . ). 13C{1H} NMR (DMSO-6d) δ (ppm) 163.64 (azomethine CH=N), 163.49 (OH-C2’’), 163.28 (OH-C4’’), 151.35 (imidazole CH=N), 149.77–102.88 . MS (DART+) m/z: 330.4- to a suspension of each of the ligands in methanol at room temperature. After 2 h of constant stirring, the product was filtered, and the solution was evaporated under pressure under high vacuum. The complexes obtained as powders were recrystallized from a mixture of methanol:ethanol (1:1) and dried under vacuum for 4 h. 2(Cl)(H2O)2]·H2O (La-L1): A solution of LaCl3⋅7H2O  in MeOH (1.5 mL) was added dropwise into 20 mL of methanol solution of L1 . The final precipitate was observed as a wheat colored powder, yield: 149 mg, 79%. C40H34ClLaN6O5 (853.1 g·mol−1): Calc. C, 56.32; H, 4.02; N, 9.85; La, 16.28. Found: C, 55.99; H, 4.09; N, 10.10; La, 16.00%. IR (ATR cm−1): υ(N = CH)imine 1622; υ(N = C)imidazole 1563; υ(C = C) 1506; υ(C-O)phenolic 1347. 1H NMR (DMSO-6d) δ (ppm) 13.02 , 9.18 , 8.35 , 8.20 , 7.76 , 7.64 , 7.63 , 7.56 , 7.46 , 7.23 , 7.03 . 13C{1H} NMR (DMSO-6d) δ (ppm) 164.46 (azomethine CH = N), 160.81 (OH-C2’’), 151.23 (imidazole CH = N), 149.21–112.42 . TGA mass loss 2.10%  hydration, 4.27%  coordinated, 26.81% . MS (MALDI-TOF) m/z: 853.2. −1∙cm2∙mol−1): 0.012.[La(L1)2(Cl)(H2O)3]·2H2O (Ce-L1): A solution of CeCl3⋅7H2O  in MeOH (1.5 mL) was added dropwise into 20 mL of methanol solution of L1 . The final precipitate was observed as a golden-colored powder, yield: 145 mg, 74%. C40H38CeClN6O7 (890.3 g·mol−1): Calc. C, 53.96; H, 4.30; N, 9.44; Ce, 15.74. Found: C, 54.12; H, 4.34; N, 9.87; Ce, 16.11%. IR (ATR cm−1): υ(N=CH)imine 1624; υ(N=C)imidazole 1565; υ(C = C) 1507; υ(C-O)phenolic 1349. TGA mass loss 3.97%  hydration, 6.28%  coordinated, 30.56% . MS (MALDI-TOF) m/z: 890.7. −1∙cm2∙mol−1): 0.015.[Ce(L1)2(Cl)(H2O)2]·2H2O·2CH3OH (La-L2): The synthesis of complex La-L2 was performed using the same procedure as that for compound La-L1. The final precipitate was observed as a dark olive-green-colored powder, yield: 161 mg, 76%. C42H44ClLaN6O10 (967.2 g·mol−1): Calc. C, 52.16; H, 4.59; N, 8.69; La, 14.36. Found: C, 53.07; H, 4.59; N, 8.92; La, 14.25%. IR (ATR cm−1): υ(N = CH)imine 1624; υ(N = C)imidazole 1559; υ(C = C) 1493; υ(C-O)phenolic 1310. 1H NMR (DMSO-6d) δ (ppm) 13.48 , 10.58 , 9.01 , 8.26 , 8.14 , 7.64 , 7.63 , 7.60 , 7.47 , 7.24 , 6.49 , 6.42 . 13C{1H} NMR (DMSO-6d) δ (ppm) 165.79 (azomethine CH = N), 163.60 (OH-C2’’), 163.41 (OH-C4’’), 151.31 (imidazole CH=N), 149.22–102.98 . TGA mass loss 6.77% , 3.99%  hydration, 4.43%  coordinated, 38.49% . MS (MALDI-TOF) m/z: 967.3. −1∙cm2∙mol−1): 0.037.[La(L2)2(Cl)(H2O)2]·H2O·CH3OH (Ce-L2): The synthesis of complex Ce-L2 was performed by the same method employed for complex Ce-L1. The final precipitate was observed as a brown-colored powder, yield: 134 mg, 66%. C41H38CeClN6O8 (918.4 g·mol−1): Calc. C, 53.62; H, 4.17; N, 9.15; Ce, 15.26. Found: C, 53.83; H, 4.28; N, 9.37; Ce, 15.94%. IR (ATR cm−1): υ(N = CH)imine 1619; υ(N = C)imidazole 1555; υ(C = C) 1492; υ(C-O)phenolic 1310. TGA mass loss 3.77% , 2.22%  hydration, 4.39%  coordinated, 20.75% . MS (MALDI-TOF) m/z: 918.4. −1∙cm2∙mol−1): 0.031.[Ce(L2)2(Cl)(H2O)3]·2H2O (La-L3): Compound La-L3 was attained by the same method employed for complex La-L1. The final precipitate was observed as a pale-green-colored powder, yield: 138 mg, 71%. C40H38ClLaN6O7 (889.1 g·mol−1): Calc. C, 54.03; H, 4.31; N, 9.45; La, 15.62. Found: C, 53.88; H, 4.24; N, 9.67; La, 15.42%. IR (ATR cm−1): υ(N = CH)imine 1634; υ(N = C)imidazole 1562; υ(C = C) 1493; υ(C-O)phenolic 1308. 1H NMR (DMSO-6d) δ (ppm) 12.97 , 9.09 , 8.35 , 7.72 , 7.61 , 7.45 , 7.22 , 7.02 , 7.02 . 13C{1H} NMR (DMSO-6d) δ (ppm) 164.19 (azomethine CH = N), 160.80 (OH-C2’’), 151.25 (imidazole CH = N), 149.70–114.02 . TGA mass loss 4.16%  hydration, 6.49%  coordinated, 33.69% . MS (MALDI-TOF) m/z: 889.5. −1∙cm2∙mol−1): 0.007.[La(L3)2(Cl)(H2O)]·CH3OH (Ce-L3): Compound Ce-L3 was attained by the same method employed for complexCe-L1. The final precipitate was observed as a cream-colored powder, yield: 155 mg, 83%. C41H34CeClN6O4 (850.3 g·mol−1): Calc. C, 57.91; H, 4.03; N, 9.88; Ce, 16.48. Found: C, 57.40; H, 4.14; N, 10.04; Ce, 16.10%. IR (ATR cm−1): υ(N = CH)imine 1635; υ(N = C)imidazole 1562; υ(C = C) 1493; υ(C-O)phenolic 1310. TGA mass loss 3.82% , 2.21%  coordinated, 27.31% . MS (MALDI-TOF) m/z: 851.2. −1∙cm2∙mol−1): 0.011.[Ce(L3)2(Cl)(H2O)2]·H2O (La-L4): Compound La-L4 was attained by the same method employed for complex La-L1. The final precipitate was observed as a yellow-green-colored powder, yield: 150 mg, 77%. C40H34ClLaN6O7 (885.1 g·mol−1): Calc. C, 54.28; H, 3.87; N, 9.50; La, 15.69. Found: C, 54.51; H, 3.98; N, 9.47; La, 15.95 IR (ATR cm−1): υ(N = CH)imine 1635; υ(N = C)imidazole 1560; υ(C = C) 1494; υ(C-O)phenolic 1313. 1H NMR (DMSO-6d) δ (ppm) 13.46 , 10.60 , 8.93 , 8.31 , 7.61 , 7.54 , 7.21 , 6.48 , 6.40 . 13C{1H} NMR (DMSO-6d) δ (ppm) 165.83 (azomethine CH = N), 163.68 (OH-C2’’), 163.38 (OH-C4’’), 151.41 (imidazole CH = N), 149.77–102.88 . TGA mass loss 2.21%  hydration, 4.33%  coordinated, 21.61% . MS (MALDI-TOF) m/z: 885.3. −1∙cm2∙mol−1): 0.028.[La(L4)2(Cl)(H2O)2]·H2O (Ce-L4): Compound Ce-L4 was obtained by the same method employed for complex Ce-L1. The final precipitate was observed as a mustard-colored powder, yield: 133 mg, 68%. C40H34CeClN6O7 (886.3 g·mol−1): Calc. C, 54.21; H, 3.87; N, 9.48; Ce, 15.81. Found: C, 54.35; H, 3.97; N, 9.67; Ce, 15.62%. IR (ATR cm−1): υ(N=CH)imine 1636; υ(N = C)imidazole 1559; υ(C = C) 1492; υ(C-O)phenolic 1313. TGA mass loss 2.34%  hydration, 4.31%  coordinated, 22.14% . MS (MALDI-TOF) m/z: 886.0. −1∙cm2∙mol−1): 0.036.[Ce(L4)Cell lines culture: The antiproliferative activity studies were carried out on U251 (human glioblastoma), PC-3 (prostate cancer), K562 (myelogenous leukemia), HCT-15 , MCF-7 , and SKLU-1 (lung carcinoma) cells, which were cultured at 37 °C in RPMI-1640 medium (Gibco) enriched with 1% nonessential amino acids (Gibco), 1% penicillin:streptomycin:amphotericin B solution, 10% fetal bovine serum , and 2 mM L-glutamine (Invitrogen) in a humidified atmosphere of 5% (v/v) CO2. Trypan blue was used as indicator of cell viability. Human tumor cytotoxicity was determined under protocols approved by the NCI 1 [he NCI 1 . Antiproliferative activity: The cells were removed from the tissue culture flasks by treatment with trypsin and diluted with fresh media. Of these cell suspensions, 100 µL/well of 7.5 × 104 cell/mL (U251), 7.5 × 104 cell/mL (PC-3), 5.0 × 104 cell/mL (K562), 10.0 × 104 cell/mL (HCT-15), 5.0 × 104 cell/mL (MCF-7), 7.5 × 104 cell/mL (SK-LU-1), and 10.0 × 104 cell/mL (COS-7) of cells suspension were seeded in 96 well micro-titer plates (Costar) and incubated for 24 h at 37 °C (5% CO2) to allow for cell attachment. The compounds were prepared in stock concentration of 20 mM, solubilized in DMSO. For the concentration of 25 µM, the % of DMSO in the well was 0.025%. Subsequently, 100 µL of a 25 µM compound solution in DMSO was added to each well. The same concentration of vehicle was used as positive control; in all cases, DMSO was constant during measurements. The cancer cell lines were exposed to the tested compounds for 48 h. Cell culture medium without tumor cells and compounds was tested as a negative control. Reference drug used was cisplatin. Cytotoxic activity on U-937 cell line: Before conducting the antiparasitic activity studies, the protocol was followed to determine the cytotoxic activity of the compounds based on the viability of the human promonocytic cell line U-937 (ATCC CRL-1593.2TM) determined using the MTT enzymatic micro-method -2,5-diphenyl tetrazolium bromide) [50) [bromide) . All of de) [50) .The in vitro leishmanicidal activity of the compounds against intracellular amastigotes was evaluated using flow cytometry based on methodology described by Pulido et al. . InfecteT. cruzi cells (Tulahuen strain) for intracellular amastigotes that express the Escherichia coli beta-galactosidase gene. Compounds were added at concentrations of 20 μg/mL on U-937 cells infected by epimastigotes after incubation for 24 h. The experiments were performed in duplicate two times, and the results are expressed as the percentages of inhibition. Reference compound used was benznidazole, a compound conventionally used for the treatment of Chagas disease [In vitro trypanocidal activity of compounds was evaluated on  disease ,71.P. falciparum 3D7 strain was grown following the procedure described by Trager and Jensen [50 (effective concentration 50) [The d Jensen . Cytotoxd Jensen . The exption 50) .S. aureus ATCC® 25923, L. monocytogenes ATCC® 19115) and two Gram-negative  strains. The final concentration of the compounds was in the range 4–2000 μg/mL. Free medium with DMSO (0.01%) was used as a negative control, while bacterial medium without an inhibiting agent was used as positive control. Ciprofloxacin (CP) and silver nitrate (AgNO3) were used as control antibiotics. The bacteria were incubated with the compounds for 24 h at 37 °C to determine the minimum inhibitory concentration (MIC). To verify that the inhibition observed in the wells corresponded to the same MIC in all cases, assays were carried out in triplicate.The in vitro antibacterial activity of the compounds was determined by broth microdilution ,75 for tThe 1,2-dimyristoyl-sn-glycero-3-phosphocholine (PC) and 1,2-dimyristoyl-sn-glycero-3-phosphorylglycerol (PG) lipids were used to mimic a synthetic bacterial membrane model (PC/PG ratio: 3:1) and a mammalian membrane model (PC). The lipids were suspended in a 2:1 (v/v) chloroform/methanol mixture as suggested in the literature . The PC/From 2 mg of lipids, MLVs were obtained with ratio 1:50 (compound/lipids). The samples were placed in DSC Tzero hermetic pans and the scanning was carried out over a 10–40 °C temperature at a heating rate of 1 °C/min in DSC Q25 equipment ; HEPES buffer was used as reference solution. To obtain the temperature of phase transition (Tm) and the transition enthalpy (DH), the Trios software (TA Instruments) was used in the analysis of the thermograms.260/A280 ratio between 1.7 and 1.9, indicating that the DNA was free of RNA and proteins. The interaction studies of the compounds obtained with highly polymerized and lyophilized calf thymus DNA (CT-DNA from Sigma Aldrich) were carried out with electronic absorption experiments, following the methodology reported in the literature . The DNAAn UV-visible Evolution 220 spectrophotometer , equipped with Single Cell Peltier System for temperature control, was used to obtain the electronic spectra. The interaction studies were carried out with CT-DNA titrations at a constant concentration of the compounds (60 μM) at 25 °C, and compounds titrations at a constant concentration of CT-DNA (48 μM) at 25 °C. After the addition of each titrant, the electronic absorption spectra of the evaluated compound were measured in the range of 500–230 nm. DNA absorbance was eliminated by adding an equal amount of DNA to the sample and the standard solution ,79. http://cao.labshare.cn/cb-dock/, accessed on 7 May 2021) using the prepared ligand and protein. Discovery Studio Visualizer software  was used to analyze ligand–receptor interactions to find the amino acids that interact with the molecules. The protein preparation, the molecule preparation, the grid generation, and the molecule-protein docking were performed using Autodock software . For thehttps://chemdrawdirect.perkinelmer.cloud/js/sample/index.html, accessed on 7 May 2021). The structure was optimized using Avogadro version 1.2  with Universal Force Field (UFF) [The 3D structure of the ligands and the complexes were realized with Chemdraw online version (ld (UFF)  in orderLeishmania spp., T. cruzi, P. falciparum, and S. aureus. The proteins used as receptors for the ligands were leishmanin (1LML), cruzain (4PI3), tubulin Alpha , and PBP2A of S. aureus (5M18). For P. falciparum tubulin Alpha, the GenBank sequence CAA34101 (453 aa) was used to build the 3D model using Swiss-model ; this was validated by PROSA  and Molprobity .The receptor proteins to be evaluated belong to the following organisms: https://pubchem.ncbi.nlm.nih.gov/, accessed on 7 May 2021). Then, with Avogadro, they were converted to a 3D structure, and the structure was optimized using the GAFF force field. Then, the PDB format for docking was obtained.In order to compare the affinity results of ligands and complexes, control molecules were used: amphotericin for leishmanin, vinylsulfone for cruzain, vinblastine for alpha tubulin, and finally cefepime, ceftobiprole, and benzopenicillin for PB2A. These controls were first obtained in a pubchem SDF format . The validation of leishmanin with L2 ligand was performed through molecular dynamics. The model system was hydrated by means of Charmm-Gui solution builder. The simulation was performed with GROMACS version 2020.2. Minimization was run for 0.1 ns using the steep descent algorithm, with a Verlet cutoff scheme and coulombtype pme. Equilibration was performed for 2.5 ns, with a Verlet cutoff-scheme and coulombtype pme, using the Nosé–Hoover algorithm at 310.15 K. Finally, the molecular dynamics was run for 10 ns, also with the Nosé–Hoover algorithm at temperature 310.15 K, with a Verlet cutoff scheme and coulombtype pme. RMSD analysis, hydrogen bonds, affinity energy, and the distance between L2 ligand and leishmanin were performed. The visualization of the leishmanin surface with the L2 ligand was performed with pymol educative version (In summary, a number of novel lanthanide complexes derived from Schiff base ligands, including a benzimidazole fragment, were obtained and unequivocally characterized using different analytical techniques. Preliminary in vitro assays were conducted to evaluate their potential pharmacological applications through various bioassays, including antiproliferative, antiparasitic, and antibacterial assays. The results suggested that structural modifications in the compounds play important roles in their biological activities, revealing that ligands L1 and L2 and their metal complexes showed lower MIC values compared with ligands L3 and L4 and their complexes, which may be due to substitutions on the aminophenol ring. Similarly, the compounds appeared to influence membrane fluidity by altering the hydrophobic core of the bilayer, which might be correlated with potential pharmacological applications. These findings could become a useful reference in further attempts to develop new pharmacological agents. Additional studies examining how the modifications affect the molecular architecture are currently ongoing in our laboratory to shed further light on the mechanism of action associated with these types of compounds.


Iris tenuifolia Pall. afforded five new compounds; an unusual macrolide termed moniristenulide (1), 5-methoxy-6,7-methylenedioxy-4-O-2′-cycloflavan (2), 5,7,2′,3′-tetrahydroxyflavanone (3), 5-hydroxy-6,7-dimethoxyisoflavone-2′-O-β-d-glucopyranoside (9), 5,2′,3′-dihydroxy-6,7-dimethoxyisoflavone (10), along with seven known compounds . The structures of all purified compounds were established by analysis of 1D and 2D NMR spectroscopy and HR-ESI-MS. The antimicrobial activity of the compounds 1–3, 5, 9, and 10 was investigated using the agar diffusion method against fungi, Gram-positive and Gram-negative bacteria. In consequence, new compound 3 was found to possess the highest antibacterial activity against Enterococcus faecalis VRE and Mycobacterium vaccae. Cell proliferation and cytotoxicity tests were also applied on all isolated compounds and plant crude extract in vitro with the result of potent inhibitory effect against leukemia cells. In particular, the newly discovered isoflavone 10 was active against both of the leukemia cells K-562 and THP-1 while 4–6 of the flavanone type compounds were active against only THP-1.Phytochemical investigation of the ethanol extract of underground parts of  Iris is a genus of the family Iridaceae that consists of about 360 species of perennial herbs and sub shrubs. Iris species are widely distributed in the temperate northern hemisphere, including Euro-Asia, North Africa and North America + corresponding to a molecular formula of C21H24O11. Its 1H NMR spectrum acquired in DMSO-d6  and δH 5.95 , two olefinic protons at δH 6.94 and δH 5.75, methylene signals between δH 3.21–1.81 and number of oxygenated protons between δH 5.50–3.08 corresponding to hydroxy groups as well as oxymethines. The presence of a 2,5,7-trisubstituted chromane-4-one was identified by analysis of HMBC correlations observed for the meta-coupled aromatic doublets H-6 and H-8 as well as methylene signals H-2 and H-3 , which is characteristic downfield shift of a hydroxyl group at C-5 and a carbonyl group at C-4. In addition, the presence of a hydroxyl group at C-5 was supported by HMBC correlations from 5-OH (δH 12.04) to C-5 (δC 163.1), C-6 (δC 97.3) and C-10 (δC 103.4). HSQC, HMBC, and COSY data clearly revealed the existence of a glucose residue. Further analysis of the spin-spin couplings (J = 7.7 Hz) allowed the identification of β-d-glucose. Connectivity of sugar moiety with chromane ring was deduced through HMBC correlation from the anomeric proton H-1″ (δH 5.09) to C-7 (δC 164.1) of aglycon. A remaining spin system observed on the COSY spectrum comprised a series of protons H-2′–H-6′. HMBC correlations from olefinic protons H-2′ and H-3′ to a carbonyl carbon at δC 164.8 (C-1′) allowed for establishing 6-hydroxy-2-hexenoic acid residue. Trans configuration of the double bond has been identified by a large coupling constant of 15.6 Hz. This substructure has been linked to the aglycon on position C-2 by HMBC correlations from the H-5′ and H-6′ to a carbon at δC 76.9 (C-2) and in turn H-3 to a carbon at δC 70.9 (C-6′). Interestingly, a macrolide structure was established via ester linkage between sugar residue and carbonyl carbon of hexenoic acid by strong heteronuclear long-range correlation from H-6″ to C-1′. ROESY correlations between diastereotopic H-3eq (β) and H-2 and H-6′ indicated that these protons are on the same side of the chromane ring. In turn, H-3ax showed ROESY correlation to 6′-OH. From the above findings, relative configuration at C-2 and C-6′ has been established as β-configured , 7.20 , 6.89 , 6.87 , 6.08 , two signals of oxygenated methylene at δH 5.81 and δH 5.76 , two oxygenated methine protons at δH 5.64  and 5.25 , two signals of diastereotopic methylene protons at δH 2.27  and 2.15  and one methoxy singlet at δH 4.09 . Analysis of 13C NMR spectrum combined with HSQC allowed for identifying the existence of 17 carbon atoms, including 12 sp2 carbons, two methylene carbons at δC 26.5 and 100.8, two methine carbons at δC 67.5 and 62.4, and one methoxy group at δC 60.1  along with the respective HMBC correlations confirmed a presence of disubstituted aromatic B-ring, while the remaining sharp singlet at δH 6.08 (H-8) described a typical penta-substituted aromatic A-ring. Furthermore, the COSY experiment showed another spin system from H-2 to H-4 and indicated a CH-CH2-CH sequence. Additionally, the key HMBC correlation from H-6′ (δH 7.33) to C-2 (δC 67.5) revealed the flavan (2-phenylchromane) skeleton (H 5.64) to C-2′ (δC 153.6) and their chemical shift indicated their linkage through an oxygen bridge. All these data deduced that compound 2 has a 4-O-2′-cycloflavan as a partial structure. The remaining two doublet signals at δH 5.81 and 5.76 had one bond correlation to a carbon atom at δC 100.8, which were assigned as a methylenedioxy group. These methylenedioxy doublet was connected to a flavan core by key HMBC correlations to C-6 and C-7. The location of methoxy group (δH 4.09) at C-5 was suggested by HMBC correlations. On the basis of the above evidence, the planar structure of compound 2 was elucidated as 5-methoxy-6,7-methylenedioxy-4-O-2′-cycloflavan and considered as an unprecedented natural product. This spectroscopic data of compound 2 is somewhat similar to the literature values for the known 4-O-2′-cycloflavan core structures, possessing a different substitution pattern in aromatic A- and B-rings [Compound  δC 60.1 . The COSskeleton . The HMB3 was isolated as yellow solid. It showed a molecular ion peak at m/z 289.0714 [M + H]+ ascribable to a molecular formula of C15H12O6. The 1H NMR spectrum showed resonances for five aromatic proton signals at δH 5.88 , 5.90 , 6.69 , 6.79 , 6.88 , three aliphatic protons at δH 5.70 , 3.17 , and 2.69 , and four oxygenated protons at δH 12.12, 10.79, 9.51, and 8.71, one of which was appeared as a chelated hydroxyl group. The 13C NMR spectrum contained signals from 15 carbon atoms, which were with the full agreement of HR-MS (H 12.12) to C-5 (δC 163.5), C-6 (δC 95.8) and C-10 (δC 101.7) and from 7-OH to C-6 (δC 95.8) and C-7 (δC 166.6). In addition, a COSY correlation of three aromatic protons at δH 6.69, 6.79, 6.88 along with HMBC correlations from proton H-4′ (δH 6.79) to C-2′ (δC 142.6), C-3′ (δC 145.2) and from proton H-6′ (δH 6.88) to C-2 (δC 74.0), C-2′ (δC 142.6), and C-4′ (δC 115.2) revealed a presence of a 2,3-dihydroxyphenyl moiety (ring-B) and altogether confirmed the flavanone structure. This was further supported by key long-range heteronuclear correlations from the methine proton H-2 (δH 5.70) to C-2′ (δC 142.6). The position of the remaining two hydroxyl protons at δH 9.51 and 8.71 were assigned to C-3′ and C-2′ respectively, due to observed HMBC correlations. Spectral data of 3 possess close similarity to those for the known compounds 5 and 6 + by HR-ESI-MS. The 1H NMR spectrum revealed the presence of five aromatic protons , 7.27 , 7.35 , and 7.09 , characteristic for an ortho-substituted B-ring of aglycone  and C-7 (δC 158.3). The chelated hydroxyl group was positioned at C-5 by means of HMBC. In addition, a characteristic isoflavonoid signal for H-2 was appeared at δH 8.44. The isoflavone nature was supported by long-range correlations from H-2 (δH 8.44) to C-4 (δC 180.5), C-9 (δC 149.2), and C-1′ (δC 120.0) in the HMBC spectrum. Furthermore, two methoxyl singlet signals were apparent at δH 3.79 and 3.94, and they were located at C-6 and C-7 due to HMBC correlations between 6-OCH3 and C-6 (δC 128.3), as well as 7-OCH3 and C-7 (δC 158.3). Moreover, a series of COSY cross signals comprising six protons in the range of δH 3.0–4.0, four hydroxy protons δH 4.57–5.05, as well as a doublet at δH 4.89, revealed the presence of a glucose moiety (H-1″ to H-6″) + peak at m/z 331.0825, corresponding to the molecular formula C17H14O7. 1H NMR, 13C NMR, and HSQC data of compound 10 closely resembled to that of 9, differing only on the absence of signals corresponding to a sugar moiety and different pattern on aromatic signals of B-ring  with two doublets of doublets at δH 7.03  and 6.71 , and their HMBC correlations clearly indicated ortho-dihydroxyl substitution on B-ring. The position of the hydroxyl groups, which were appeared as two singlets in the 1H NMR spectrum at δH 8.48 and 6.09, were assigned at C-2′ and C-3′, due to the HMBC correlations  or against the fungus Sporobolomyces Salmonicolor. This is the first report on the antimicrobial activity of the tested compounds.As for the fungi, the compound 1–12) along with plant raw extract were evaluated in vitro  against the K-562, followed by compounds 2 and 3 with GI50 value at 31.5 and 32.3 µM. Compound 3, the tetrahydroxy flavanone, showed selective antiproliferative inhibition effect against the K-562 cells only. These results are in a good agreement with previous studies and supports the evidence that the ortho-dihydroxyl structural fragment in B-ring is very important for anticancer activity D25 + 44 , CD  305 nm (Δε + 1.25) and 287 nm (Δε − 3.96), UV (CH3CN/H2O): λmax 206, 228, 282, 326 nm; 1H NMR  and 13C NMR  data, see m/z 453.1409 [M + H]+ .Moniristenulide (O-2′-cycloflavan (2): colorless crystal, [α]D25 + 317 , UV (CH3CN/H2O): λmax 196, 206, 286 nm; 1H NMR  and 13C NMR  data, see m/z 299.0919 [M + H]+ .5-Methoxy-6,7-methylenedioxy-4-3): yellow powder; [α]D25 + 10 , CD  325 nm (Δε + 2.40) and 283 nm (Δε − 11.70), UV (CH3CN/H2O): λmax 202, 288 nm; 1H NMR  and 13C NMR  data, see m/z 289.0714 [M + H]+ .(2S)-5,7,2′,3′-Tetrahydroxyflavanone (4): yellow powder, UV (CH3CN/H2O): λmax 206, 228, 280 nm; 1H NMR  δH 11.86 , 9.89 , 7.43 , 7.19 , 6.88 , 6.86 , 6.31 , 6.06 , 5.72 , 3.26 , 2.74 . 13C NMR  δC 74.6 (C-2), 41.2 (C-3), 197.9 (C-4), 143.1 (C-5), 127.4 (C-6), 155.8 (C-7), 90.4 (C-8), 159.4 (C-9), 103.6 (C-10), 124.5 (C-1′), 154.4 (C-2′), 115.6 (C-3′), 129.6 (C-4′), 119.2 (C-5′), 127.1 (C-6′), 102.5 (-CH2-). HR-ESI-MS: m/z 301.0722 [M + H]+ .5,2′-Dihydroxy-6,7-methylenedioxyflavanone (5): yellow crystal, [α]D25 + 9 , UV (CH3CN/H2O): λmax 206, 244, 284 nm; 1H NMR  δH 11.88 , 9.52 , 8.74 , 6.90 , 6.80 , 6.69 , 6.30 , 6.07 , 5.73 , 3.24 , 2.73 . 13C NMR  δC 74.6 (C-2), 41.2 (C-3), 197.9 (C-4), 143.1 (C-5), 127.3 (C-6), 155.7 (C-7), 90.4 (C-8), 159.4 (C-9), 103.6 (C-10), 125.2 (C-1′), 142.6 (C-2′), 145.2 (C-3′), 115.3 (C-4′), 119.0 (C-5′), 117.1 (C-6′), 102.4 (-CH2-). HR-ESI-MS: m/z 317.0666 [M + H]+ .5,2′,3′-Trihydroxy-6,7-methylenedioxyflavanone (6): yellow crystal; UV (CH3CN/H2O): λmax 206, 226, 286 nm; 1H NMR  δH 12.09 , 9.56 , 8.74 , 6.89 , 6.79 , 6.69 , 6.11 , 6.08 , 5.73 , 3.79 , 3.23 , 2.73 . 13C NMR  δC 74.2 (C-2), 41.2 (C-3), 196.9 (C-4), 163.2 (C-5), 94.7 (C-6), 167.4 (C-7), 93.8 (C-8), 159.4 (C-9), 102.6 (C-10), 125.4 (C-1′), 143.1 (C-2′), 145.2 (C-3′), 115.2 (C-4′), 119.0 (C-5′), 117.1 (C-6′), 55.9 (7-OCH3). HR-ESI-MS: m/z 303.0877 [M + H]+ .5,2′,3′-Trihydroxy-7-methoxyflavanone (7): white powder; UV (CH3CN/H2O): λmax 206, 226, 282 nm; 1H NMR  δH 11.88 , 9.49 , 6.99 , 6.97 , 6.90 , 6.13 , 6.08 , 5.94 , 5.46 , 4.77 , 3.78 , 3.74 . 13C NMR  δC 77.7 (C-2), 70.7 (C-3), 198.4 (C-4), 163.1 (C-5), 94.9 (C-6), 167.6 (C-7), 93.8 (C-8), 162.5 (C-9), 101.4 (C-10), 130.4 (C-1′), 146.7 (C-2′), 150.2 (C-3′), 117.2 (C-4′), 123.9 (C-5′), 118.6 (C-6′), 60.5 (7-OCH3), 55.9 (2′-OCH3). HR-ESI-MS: m/z 333.0982 [M + H]+ .3,5,3′-Trihydroxy-7,2′-dimethoxyflavanone (8): yellow crystal, UV (CH3CN/H2O): λmax 196, 226, 262 nm; 1H NMR  δH 12.94 , 10.78 , 8.24 , 7.40 , 7.24 , 7.09 , 7.00 , 6.52 , 3.75 , 3.73 . 13C NMR  δC 155.3 (C-2), 119.8 (C-3), 180.1 (C-4), 152.8 (C-5), 131.5 (C-6), 157.5 (C-7), 94.0 (C-8), 153.1 (C-9), 104.7 (C-10), 120.2 (C-1′), 157.5 (C-2′), 111.3 (C-3′), 129.9 (C-4′), 120.0 (C-5′), 131.6 (C-6′), 59.9 (6-OCH3), 55.6 (7-OCH3). HR-ESI-MS: m/z 315.0888 [M + H]+ .5,7-Dihydroxy-6,2′-dimethoxyisoflavone (O-β-d-glucopyranoside (9): yellow crystal, UV (CH3CN/H2O): λmax 206, 216, 262, 336 nm; 1H NMR  and 13C NMR  data, see m/z 477.1399 [M + H]+ 5-Hydroxy-6,7-dimethoxyisoflavone-2′-10): yellow powder, UV (CH3CN/H2O): λmax 206, 222, 258, 338 nm; 1H NMR  and 13C NMR  data, see m/z 331.0825 [M + H]+ .5,2′,3′-Trihydroxy-6,7-dimethoxyisoflavone (11): yellow amorphous powder, UV (CH3CN/H2O): λmax 198, 256, 302, 344 nm; 1H NMR  δH 12.48 , 9.70 , 9.14 , 7.03 , 7.02 , 6.93 , 6.61 , 6.38 , 3.84 , 3.77 . 13C NMR  δC: 148.3 (C-2), 137.3 (C-3), 176.5 (C-4), 160.7 (C-5), 97.5 (C-6), 165.0 (C-7), 92.0 (C-8), 156.7 (C-9), 104.6 (C-10), 124.9 (C-1′), 145.9 (C-2′), 150.5 (C-3′), 123.7 (C-4′), 118.8 (C-5′), 120.9 (C-6′), 60.3 (2′-OCH3), 56.1 (7-OCH3). HR-ESI-MS: m/z 331.0835 [M + H]+ .3,5,3′-Trihydroxy-7,2′-dimethoxyflavonol (Irisflavone D) (β-Sitosterol (12): white powder, 1H NMR  δH 5.35 , 3.52 , 1.01 , 0.92 , 0.84 , 0.82 , 0.80 , 0.68 . 13C NMR  δC 37.4 (C-1), 31.8 (C-2), 71.9 (C-3), 42.5 (C-4), 140.9 (C-5), 121.9 (C-8), 29.8 (C-7), 32.0 (C-8), 50.3 (C-9), 36.6 (C-10), 21.2 (C-11), 39.9 (C-12), 42.4 (C-13), 56.9 (C-14), 25.5 (C-15), 28.4 (C-16), 56.2 (C-17), 36.3 (C-18), 19.2 (C-19), 34.1 (C-20), 26.2 (C-21), 45.9 (C-22), 23.2 (C-23), 12.1 (C-24), 29.3 (C-25), 19.9 (C-26), 19.5 (C-27), 18.9 (C-28), 12.0 (C-29).1, 2, 3, 5, 9, and 10 were tested for their antimicrobial activities against S. aureus (JMRC:STI 10760), B. subtilis (JMRC:STI 10880), S. aureus MRSA (JMRC: ST 33793) E. faecalis VRE (JMRC: ST 33700), E. coli (JMRC:ST 33699), P. aeruginosa (JMRC:ST 33771), P. aeruginosa (JMRC:ST 33772), M. vaccae (JMRC:STI 10670), S. salmonicolor (JMRC:ST 35974), C. albicans (JMRC:STI 25000), and P. notatum (JMRC:STI 50164)) using agar diffusion assay as previously published [S. salmonicolor and the filamentous ascomycete P. notatum, which were cultivated on malt agar, and the ascomycetous yeast C. albicans, which was cultivated on yeast morphology agar. After inoculation of the test organisms, a disc (9 mm in diameter) was removed from the center of the Petri dish and 50 μL of the test solution (1 mg/mL in DMSO) was added to the cavity. After 18 h of incubation, the inhibiting areola were measured and documented as diameters in mm. Ciprofloxacin (5 µg/mL in deionized water) and amphotericin B (10 µg/mL in DMSO/MeOH 1:1) were used as reference substances against bacterial and fungal strains, respectively.Compounds ublished . Strains1–12) were assayed against human umbilical vein endothelial cells (HUVEC), human chronic myeloid leukemia cells (K-562), human acute monocytic leukemia cells (THP-1), and human lung carcinoma cells (A549) for their antiproliferative effects and against human cervix carcinoma cells (HeLa) for their cytotoxic effect. The antiproliperative and cytotoxic effects were tested via CellTiter-Blue and methylene blue assay as previously described [4 cells/well. As for the antiproliferative effect of the compounds, the cells were incubated for 72 h, and GI50 values were evaluated to be defined as the concentration causing 50% inhibition of proliferation compared to the untreated control. With regard to the cytotoxic assay, HeLa cells were pre-incubated for 48 h without the test compounds. Then, the cells were exposed with different concentrations of compounds and incubated for 72 h. After that, the adherent HeLa cells were fixed by glutaraldehyde and stained with a 0.05% solutions of methylene blue (SERVA 29198) for 15 min. CC50 was evaluated to be defined as the concentration required for the death of 50% of the cell monolayer as compared to control groups. Under our experimental conditions, the optical density measured from the CellTiter-Blue reagent and methylene blue assay is proportional to the number of viable cells. In this experiment, absorbances were measured at 570 nm against the reference wavelength of 600 nm (CellTiter-Blue assay) and at 660 nm (methylene blue assay). Doxorubicin (Adriamycin®) and imatinib (Gleevec®) were used as positive controls for HUVEC, K-562, and HeLa cells. A repeat determination has been conducted in all experiments, and four replicates were assayed. The calculations of the different values of GI50 and CC50 were performed with software Magellan version 3.00 .Compounds  includes an unusual macrolide termed moniristenulide (1), together with seven known compounds . Notably, eight out of nine isolated flavonoids have a rare 2′,3′-disubstituted configuration on the B-ring, out of which the compounds bearing ortho-dihydroxyl groups in B-ring, namely 3, 5, and 10, showed the broadest antimicrobial activity. On top of that, the molecules with methoxy or methylenedioxy substitution on the A-ring together with ortho-hydroxyl groups on the B-ring showed promising antiproliferative activities against leukemia cell lines in combination with low cytotoxicity, as shown for compounds 4–6 and 10.The chemical investigation of the underground parts of


YJ191021. Their planar structures and absolute configurations were elucidated by HR-ESI-MS, 1D/2D NMR data, and time-dependent density functional theory (TDDFT)/ECD calculation. The isolated compounds were assayed for their inhibition against three agricultural pathogenic fungi, four fish pathogenic bacteria, and two agricultural pathogenic bacteria. Compound 1 exhibited moderate antifungal and antibacterial activities against Vibrioanguillarum, Xanthomonas oryzae pv. Oryzicola, and Rhizoctoniasolani with minimal inhibitory concentration (MIC) values of 8, 12.5, and 25 μg/mL, respectively. Furthermore, 1 displayed notable anti-inflammatory activity with IC50 value of 1.46 ± 0.21 μM in Propionibacteriumacnes induced human monocyte cell line (THP-1).Six new prenylated indole diketopiperazine alkaloids, asperthrins A–F ( Aspergillus sp. YJ191021 attracted our attention, not only for the characteristic indole diketopiperazine ultraviolet (UV) absorptions of the crude extracts, but also for their potent antimicrobial activities against agricultural pathogenic fungi.Endophytic fungi refer to microorganisms that spend their entire or part of their life cycle in plant tissues, animals, and environments without causing any obvious infection or visible disease to the host . EndophyAspergillus and Penicillium + ion peak at m/z 446.2071  in the HR-ESI-MS data and 13C NMR data, its molecular formula was determined as C26H27N3O4, indicating 15 degrees of unsaturation. For the NMR data , 1.55 , 1.42 , 1.41 , one methine proton signal at δH 2.31 , five olefinic protons at δH 7.80 , 6.88 , 7.05 , 7.74 , 5.96 , one exchangeable proton at δH 8.82 , as well as eight aliphatic protons at δH 1.83–3.40  attributable to four methylene groups. The 13C NMR and DEPT data disclosed 26 carbon signals, including four sp3 non-protonated carbons at δC 60.3 (C-11), 66.5 (C-17), 35.6 (C-22) [one oxygen-bearing sp3 carbon at δC 77.1 (C-27)], and eight sp2 non-protonated carbons . There were odd numbers of olefinic carbon signals in the 13C NMR spectrum, which implied that the double bond in the indole ring was connected between the carbon atom and nitrogen atom. According to the previously introduced molecular formula, there were four oxygen atoms in compound 1. Except for two carbonyl groups and one cyclic ether group, the last oxygen atom was deducted to form the imine-oxide group located at 1-N. The presence of six double bonds and two carbonyls accounted for eight of the 15 degrees of unsaturation, indicating the existence of a heptatomic ring system for 1.Asperthrin A  to C-3 (δC 132.7)/C-6 (δC 155.1)/C-8 (δC 139.6), and H-26 (δH 5.96) to C-7 (δC 111.4)/C-27 (δC 77.1)/C-28 (δC 27.9) indicated the presence of the isopentenyl-substituted indole motif (δH 2.21) to C-11 (δC 60.3)/C-17 (δC 66.5)/C-18 (δC 172.1)/C-21 (δC 46.3) and from H-21 (δH 2.31) to C-12 (δC 168.2)/C-22 (δC 35.6)/C-23 (δC 17.5). Besides, the HMBC correlations from H-10 (δH 7.05) to C-2 (δC 145.4)/C-21 (δC 46.3) and from H-23 (δH 1.22) to C-2 (δC 145.4)/C-21 (δC 46.3) proved the existence of a conjugated exo-double bond-bearing cyclohexene, which was formed by the connection between prenylated indole and diazaoctane moieties. Based on the spectral analysis, the planar structure of 1 was the same as 6-epi-avrainvillamide, isolated from A. taichungensis diazaoctane ring system + ion at m/z 486.2002  from the HR-ESI-MS and 13C NMR data was 18 amu higher than the molecular mass of 1, implying the presence of an additional hydroxy group in its structure. A careful comparison of the 13C NMR data of 2 with those of 1 (δC 75.8) and C-10 (δC 36.0), indicating a saturation of the double bond between C-3 and C-10. The HMBC correlations from H-4 (δH 7.32) to C-3 (δC 75.8) (δH 7.52) to H-23 (δH 1.34) and H-10a (δH 2.64) to 3-OH (δH 6.39)/19-NH (δH 7.52)/H-23 (δH 1.34) indicated these protons were co-facial and β-oriented. Accordingly, the ROESY correlations between H-21 (δH 2.13) and H-24 (δH 1.54) revealed that H-21 and H-24 were α-oriented. The absolute configurations of C-3, C-11, C-17, and C-21 in 2 were assigned as 3R, 11R, 17R, and 21S based on the negative Cotton effect at 225 nm in ECD spectra (Asperthrin B (ose of 1  showed sδC 75.8)  confirmeδC 75.8)  from 19- spectra  and calc spectra B.3) was isolated as white powders. Based on the [M + Na]+ ion at m/z 516.2102  in the (+)- HR-ESI-MS and 13C NMR data, the molecular formula was determined as C27H31N3O6, which was 30 amu more than 2. Comparation of the 1H NMR spectrum (3 with that of 2 indicated that there was a methoxy group in 3. The HMBC correlation from OMe-10 (δH 3.03) to C-10 (δC 76.9) (δH 7.74) and OMe-10 (δH 3.03)/H-23 (δH 1.31) indicated that these protons were co-facial and assigned as β-oriented. The ROESY correlations between 3-OH (δH 6.31) and H-10 (δH 4.72)/H-21 (δH 3.08), and between H-21 (δH 3.08) to H-24 (δH 1.32), indicated that they were in the α-orientation. Based on the negative Cotton effect at 225 nm in ECD spectra   suggesteδC 76.9)  between  spectra  and the  spectra C, the ab4) was isolated as white powders. The molecular formula was determined as C27H31N3O6 by the (+)-HRESIMS data from the [M + Na]+ ion at m/z 516.2106  as same as that of 3. Detailed analyses of the 1D NMR and 2D NMR spectra  and OMe-10 (δH 3.31) in 4 differed from those of 3, implying that the configuration of C-10 was opposite to that of 3. The relative configuration was assigned by the ROESY correlations from 19-NH (δH 7.87) to OMe-10 (δH 3.31)/H-21 (δH 3.53), 3-OH (δH 6.27) to H-21 (δH 3.53)/H-24 (δH 1.30), and H-10 (δH 4.12) to H-23 (δH 1.15)  . The abs results .5) was obtained as white powders. The molecular formula was determined as C28H31N3O6 by the [M + H]+ ion at m/z 528.2102  from HR-ESI-MS and 13C NMR data. The proton signal at δH 2.03  in 1H NMR spectrum (δH 10.69) to C-2 (δC 176.3) and H-4 (δH 6.89)/H-10 (δH 5.75)/H-23 (δH 0.52)/NH-1 (δH 10.69) to C-3 (δC 64.3) (5 was the same as that of 10-O-acetylsclerotiamide + ion at m/z 504.2103  and 13C NMR data, indicating 13 degrees of unsaturation. The 1H NMR, 13C NMR, and HSQC spectra (3-methylenes (including one oxygen-bearing methylene), two sp3-methine carbon signals (including one oxygenated carbon), five sp3 non-protonated carbons (including three oxygen-bearing carbons), four olefinic methines, and six sp2 non-protonated carbons. The 1H and 13C NMR data of 6 resembled those of gartryprostatin B + .Asperthrin A (2): White powder; + .Asperthrin B (3): White powder; + .Asperthrin C (4): White powder; + .Asperthrin D (5): White powder; + .Asperthrin E (6): White powder; + .Asperthrin F (Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola) and three agricultural fungi . Furthermore, Compound 1 was tested for antibacterial activities against four fish pathogens, Edwardsiella tarda, Vibrio anguillarum, Aeromonas hydrophilia, and Vibrio parahaemolyticus. Chloromycetin was used as a positive antibacterial control and ketoconazole was used as a positive antifungal control. The experimental procedure is detailed in the (Minimum Inhibitory Concentration (MIC) assays were used to assess antimicrobial activities of all isolated compounds against two agricultural pathogenic bacteria  were used in anti-inflammatory experiments. The P. acnes in logarithmic growth phase was used to induce inflammation in THP-1 cells. MTT method was carried out for tested compounds to determine their safe concentration to THP-1 cells. Besides, antimicrobial assays were performed to exclude false anti-inflammatory activity of these compounds raised from their inhibition to P. acnes. The inhibitory activity of the test compounds on the secretion of inflammatory factor 1L-1β by THP-1 cells was assayed by ELISA experiment [The human monocyte cell line THP-1  and periment ,25. The 1–6), along with eight known analogues (7–14), were isolated from the solid rice cultures of the marine-derived fungus Aspergillus sp. YJ191021. Asperthrin A (1) showed potent anti-bacterial, anti-fungal, and anti-inflammatory activities at micromolar level. These results expand the chemical diversity of prenylated indole alkaloids and provide a basis for further development of prenylated indole alkaloids into natural agrochemicals and drug leads.In summary, six new prenylated indole diketopiperazine alkaloids, asperthrins A-F (


Smart self-organising systems attract considerable attention in the scientific community. In order to control and stabilise the liquid crystalline behaviour, and hence the self-organisation, the polymerisation process can be effectively used. Mesogenic units incorporated into the backbones as functional side chains of weakly cross-linked macromolecules can become orientationally ordered. Several new calamitic reactive mesogens possessing the vinyl terminal group with varying flexible chain lengths and with/without lateral substitution by the methyl (methoxy) groups have been designed and studied. Depending on the molecular structure, namely, the type and position of the lateral substituents, the resulting materials form the nematic, the orthogonal SmA and the tilted SmC phases in a reasonably broad temperature range, and the structure of the mesophases was confirmed by X-ray diffraction experiments. The main objective of this work is to contribute to better understanding of the molecular structure–mesomorphic property relationship for new functional reactive mesogens, aiming at further design of smart self-assembling macromolecular materials for novel sensor systems. Functional self-assembling systems’ build-up from the organic molecules attract considerable attention from the scientific community due to their extraordinary properties that can be utilised for smart applications ,2,3. SomIn order to stabilise the liquid crystalline behaviour, and hence the self-organisation, the polymerisation process can be effectively used ,7,8,9. MUtilisation of the various types of polymeric backbones  was used as solvent and the signal of solvent served as the internal standard. Chemical shifts (δ) are given in ppm and spin-spin coupling constants (J) are given in Hz. Column chromatography was carried out using Merck Kieselgel 60 (60−100 μm). The purity of final compounds was checked by HPLC analysis  and were found to be >99.8%. All starting materials and reagents were purchased from local ditributors of Sigma-Aldrich (Merck), Acros Organics or Fluorochem. Solvents used for the syntheses were “p.a.” purity grade. 1 and hydroxy-esters 2a–d were synthesised following the procedures from the literature carbonyl}phenyl 4-hydroxybenzoate (3a)1  dissolved in toluene (20 mL) was added dropwise to the stirred mixture of 2a  and dry pyridine (8 mL) in toluene (70 mL). The reaction mixture was stirred for 6 h and then refluxed for 30 min. The resulting cooled mixture was filtered, and the filtrate was washed with diluted hydrochloric acid  and water (100 mL). The separated organic layer was dried with anhydrous magnesium sulphate. After the evaporation of the solvent, the residue was dissolved in tetrahydrofuran (50 mL) and cooled to −20 °C. To this precooled solution, concentrated aqueous ammonia  was added, with constant stirring. The reaction mixture was stirred and let warm to room temperature, and the progress of the hydrolysis was monitored by TLC. After ca. 45 min, the resulting mixture was poured into water (100 mL) and neutralised with hydrochloric acid. The organic layer was separated, and the aqueous layer was extracted with diethylether (2 × 50 mL). Combined organic layers were washed with water (50 mL) and dried over anhydrous sodium sulphate. Solvent was removed under reduced pressure and the oily residue was purified by chromatography on silica (dichloromethane:acetone (96:4)). Yield = 5.96 g (80%) of viscous liquid 3a.Benzoyl chloride 1H NMR (CDCl3): 8.12 , 8.02 , 7.28 , 6.90 , 4.88 , 4.22 , 1.77–1.94 , 1.60–1.73 , 1.21–1.35 , 0.88 .4′-{[2-(Ethoxy)-2-oxoethoxy]carbonyl}phenyl 4-hydroxybenzoate (3b)3a, starting from benzoyl chloride 1  and hydroxy-ester 2b , and the subsequent hydrolysis of the carbonate protective group, 1.35 g (88%) of hydroxy-ester 3b was obtained.Using the procedure described for 1H NMR (CDCl3) 8.12 , 8.02 , 7.28 , 6.90 , 4.88 , 4.28 , 1.36 .1-(Hexyloxy)-2-methyl-1-oxopropan-2-yl 4-[(4-hydroxybenzoyl)oxy]benzoate (3c)1  and hydroxy-ester 2c , 3.10 g (78%) of hydroxy-ester 3c was obtained by the procedure described for 3a.Starting from benzoyl chloride 1H NMR (CDCl3) 8.12 , 8.02 , 7.28 , 6.90 , 4.15 , 1.68 2C), 1.62 , 1.11–1.45 , 0.89 .4′-[(Octyloxy)carbonyl]phenyl 4-hydroxybenzoate (3d)3a, chloride 1  was reacted with hydroxy-ester 2d , and the subsequent hydrolysis of the carbonate protective group yielded 1.55 g (90%) of ester 3d.In the same procedure as that of 1H NMR (CDCl3) 8.10–8.22 , 7.28 , 6.93 , 4.32 , 1.77 , 1.10–1.55 , 0.87 .Octyl 4-({4′-[(4′′-formylbenzoyl)oxy]benzoyl}oxy)benzoate (6)3d  was dissolved in tetrahydrofuran (50 mL) and cooled to 0 °C. Dicyclohexylcarbodiimide (DCC)  and 4-pyridine (DMAP)  were added, and the mixture was stirred for 3 h at room temperature. Precipitated N,N’-dicyclohexylurea was filtered off, and the resulting filtrate was washed with HCl  and water. The organic layer was dried with anhydrous sodium sulphate. Removal of the solvent under reduced pressure yielded benzoate 6 , which was utilised in the further step without additional purification.A mixture of 4-formylbenzoic acid  and hydroxy-ester 1H NMR (CDCl3): 10.11 , 8.10–8.37 , 7.44 , 7.32 , 4.33 , 1.74–1.88 , 1.16–1.60 , 0.88 .4-{[4′-({4′′-[(Octyloxy)carbonyl]phenoxy}carbonyl)phenoxy]carbonyl}benzoic acid (7)6  in pyridine (50 mL), cooled to −10 °C by the ice-salt bath. After the last addition, the mixture was kept overnight at −20 °C and then it was slowly added to the mixture of the concentrated HCl (50 mL) in the ice-cold water (100 mL). The resulting suspension was neutralised by an additional amount of the concentrated HCl (ca. 18 mL) and filtered through the suction with a pad of celite. Filtered solid was boiled with acetone (100 mL) and filtered again. Macerate was dried with anhydrous sodium sulphate. The evaporation of acetone yielded crude 7, which was crystallised from hexane. Yield = 4.36 g (90%).The solution of potassium permanganate  in water (20 mL) at ca. 50 °C was added in two portions with a 15 min interval to the agitated solution of 4-formylbenzoate 1H NMR (CDCl3): 8.10–8.35 , 7.44 , 7.35 , 4.36 , 1.70–1.84 , 1.19–1.65 , 0.88 .4′-[(4′′-{[2-(Hexyloxy)-2-oxoethoxy]carbonyl}phenoxy)carbonyl]phenyl 3-methyl-4-(undec-10-en-1-yloxy)benzoate (UKHG)3a  and acid 4a  were dissolved in dry dichloromethane (50 mL) and cooled to 2–8 °C. Then, N,N′-dicyclohexylcarbodiimide (DCC)  and 4-pyridine (DMAP)  were added. The mixture was stirred for six hours and then filtered. The resulting filtrate was evaporated, and the residue was purified by column chromatography  and recrystallised from hexane to obtain 2.04 g (91%) of UKHG.Ester 1H NMR (CDCl3) 8.29 , 8.20 , 8.05 , 8.00 , 7.37 , 6.90 , 5.72–5.94 , 4.90–5.08 , 4.88 , 4.21 , 4.07 , 2.30 , 1.97–2.15 , 1.77–1.94 , 1.60–1.73 , 1.21–1.58 , 0.89 . 13C NMR : 167.83 (CH2COO), 165.19 (COOCH2), 164.54 (COOAr), 163.90 (C′OOAr), 162.01 (C-4), 155.68 (C-1′), 154.93 (C-1′′), 149.06 (C-3), 139.20 (CH=CH2), 132.56 (C-2), 131.88 , 131.62 , 130.27 (C-6), 127.19 (C-3), 126.87 (C-4′′), 126.25 (C-4′), 122.25 , 121.88 , 120.15 (C-1), 114.14 (CH=CH2), 110.18 (C-5), 68.27 (CH2OAr), 65.65 (COOCH2), 61.28 (CH2COO), 33.80 (CH2CH=), 31.33 (CH2CH2CH3), 29.02–29.57 , 28.91 (CH2CH2O), 28.44 (CH2CH2O), 26.04 (CH2(CH2)2O), 25.43 (CH2(CH2)2O), 22.50 (CH2CH3), 16.24 (ArCH3), 13.98 (CH2CH3). Elemental Analysis for C41H50O9 (686.83): calc. C 71.70, H 7.34; found C 71.54, H 7.20.4′-[(4′′-{[2-(Hexyloxy)-2-oxoethoxy]carbonyl}phenoxy)carbonyl]phenyl 3-methoxy-4-(undec-10-en-1-yloxy)benzoate (UVHG)4b  was reacted with ester 3a  in the presence of DCC  and DMAP  in dichloromethane. Yield = 1.91 g (87%).Analogously to the case of UKHG (see above), acid 1H NMR (CDCl3) 8.30 , 8.21 , 7.87 , 7.68 , 7.37 , 6.97 , 5.73–5.95 , 4.90–5.06 , 4.88 , 4.21 , 4.13 , 3.97 , 1.99–2.13 , 1.84–1.98 , 1.61–1.75 , 1.21–1.58 , 0.89 . 13C NMR : 167.83 (CH2COO), 165.17 (COOCH2), 163.87 (COOAr), 163.06 (C′OOAr), 155.57 (C-1′), 154.90 (C-1′′), 153.56 (C-4), 149.06 (C-3), 139.20 (CH=CH2), 131.91 , 131.62 , 126.88 (C-4′′), 126.39 (C-4′), 124.60 (C-6), 122.24 , 121.87 , 120.84 (C-1), 114.14 (CH=CH2), 112.64 (C-2), 111.43 (C-5), 69.12 (CH2OAr), 65.65 (COOCH2), 61.28 (CH2COO), 56.16 (OCH3), 33.79 (CH2CH=), 31.33 (CH2CH2CH3), 29.02–29.63 , 28.93 (CH2CH2O), 28.44 (CH2CH2O), 25.87 (CH2(CH2)2O), 25.43 (CH2(CH2)2O), 22.50 (CH2CH3), 13.98 (CH2CH3). Elemental Analysis for C41H50O10 (702.83): calc. C 70.07, H 7.17; found C 71.17, H 7.25.4′-[(4′′-{[2-(Ethoxy)-2-oxoethoxy]carbonyl}phenoxy)carbonyl]phenyl 3-methoxy-4-(undec-10-en-1-yloxy)benzoate (UVHGET)4b  was reacted with ester 3b  in the presence of DCC  and DMAP  in dichloromethane. Yield = 1.90 g (95%).Analogously to the case of UKHG, acid 1H NMR (CDCl3) 8.30 , 8.21 , 7.86 , 7.68 , 7.37 , 6.97 , 5.70–5.92 , 4.90–5.07 , 4.87 , 4.28 , 4.12 , 3.97 , 1.99–2.11 , 1.81–1.97 , 1.21–1.55 . 13C NMR : 167.73 (CH2COO), 165.17 (COOCH2), 164.39 (COOAr), 163.87 (C′OOAr), 155.57 (C-1′), 154.90 (C-1′′), 153.56 (C-4), 149.06 (C-3), 139.20 (CH=CH2), 131.91 , 131.64 , 126.87 (C-4′′), 126.39 (C-4′), 124.60 (C-6), 122.24 , 121.88 , 120.83 (C-1), 114.14 (CH=CH2), 112.64 (C-2), 111.41 (C-5), 69.10 (CH2OAr), 61.53 (CH2COO), 61.28 (COOCH2), 56.16 (OCH3), 33.79 (CH2CH=), 28.70–29.69 , 25.87 (CH2CH2O), 14.13 (CH2CH3). Elemental Analysis for C37H42O10 (646.72): calc. C 68.71, H 6.55; found C 68.59, H 6.66.4′-{[4′′-({[1-(Hexyloxy)-2-methyl-1-oxopropan-2-yl]oxy}carbonyl)phenoxy]carbonyl}phenyl 3-methyl-4-(undec-10-en-1-yloxy)benzoate (UKHM)4b  was reacted with ester 3c  in the presence of DCC  and DMAP  in dichloromethane. Yield = 1.84 g (83%).Analogously to the case of UKHG, acid 1H NMR (CDCl3) 8.29 , 8.13 , 8.05 , 8.00 , 7.39 , 7.32 , 6.90 , 5.70–5.95 , 4.88–5.08 , 4.16 , 4.07 , 2.30 , 1.98–2.13 , 1.79–1.92 , 1.71 2), 1.12–1.66 , 0.86 . 13C NMR : 172.61 (CCOO), 164.62 (COOAr), 163.92 (C′OOAr), 162.03 (C-4), 155.68 (C-1′), 154.93 (C-1′′), 149.06 (C-3), 139.20 (CH=CH2), 132.57 (C-2), 131.88 , 131.39 , 130.28 (C-6), 127.80 (C-3), 127.20 (C-4′′), 126.25 (C-4′), 122.25 , 121.73 , 120.16 (C-1), 114.15 (CH=CH2), 110.20 (C-5), 78.96 (CCOO), 68.27 (CH2OAr), 65.53 (COOCH2), 33.79 , 31.31 (CH2CH2CH3), 28.97–29.65 , 28.92 (CH2CH2O), 28.37 (CH2CH2O), 26.06 (CH2(CH2)2O), 25.47 (CH2(CH2)2O), 24.74 (C(CH3)2), 22.47 (CH2CH3), 16.26 (ArCH3), 13.97 (CH2CH3). Elemental Analysis for C43H54O9 (714.88): calc. C 72.24, H 7.61; found C 73.47, H 7.71.4′-({4′′-[(Octyloxy)carbonyl]phenoxy}carbonyl)phenyl undec-10-en-1-yl terephthalate (UTHH8)7  and undec-10-en-1-ol  was dissolved in dichloromethane (100 mL) and cooled to 2–8 °C. Dicyclohexylcarbodiimide (DCC)  and 4-pyridine (DMAP)  were added, and the mixture was stirred for two hours, during which it was warmed to room temperature. Precipitated dicyclohexylurea was filtered off, and the resulting filtrate was diluted with dichloromethane (100 mL), washed with HCl  and water. The organic layer was dried with anhydrous sodium sulphate. The solvent was removed under the reduced pressure and the crude product was purified by column chromatography on silica  Further re-crystallisation from hexane yielded 4.77 g (93%) of UTHH8 final product.A mixture of benzoic acid 1H NMR (CDCl3) 8.25–8.37 , 8.10–8.25 , 7.43 , 7.32 , 5.71–5.93 , 4.89–5.07 , 4.29–4.44 , 2.05 , 1.79–1.92 , 1.22–1.54 , 0.90 . 13C NMR : 165.63 (2 × COOCH2), 163.82 (2 × COOAr), 155.07 (C-1′), 154.35 (C-1′′), 139.18 (CH=CH2), 135.22 (C-4),132.54 (C-1), 132.00 , 131.21 , 130.21 , 129.77 , 128.26 (C-4′′), 126.91 (C-4′), 122.04 , 121.68 , 114.14 (CH=CH2), 65.79 (COOCH2), 65.32 (COOCH2), 33.79 (CH2CH=CH2), 31.79 (CH2CH2CH3), 29.01–29.72 (m), 28.90 (7 × CH2), 28.71 (CH2CH2O), 28.62 (CH2CH2O), 26.03 (CH2(CH2)2O), 26.00 (CH2(CH2)2O), 22.64 (CH2CH3), 14.10 (CH2CH3). Elemental Analysis for C41H50O8 (670.83): calc. C 72.24, H 7.61; found C 73.22, H 7.67.2) were supplied by Military University of Technology ). The cells were filled with the studied material in the isotropic phase by means of capillary action. The texture observation on the samples with homeotropic alignment, e.g., free-standing films (FSF), was also performed; while preparing the FSF, the liquid crystalline material was mechanically spread over a circular hole (diameter 3 mm) in a metal plate placed in the hot stage. The heating/cooling stage Linkam LTS E350  with a TMS 93 temperature programmer was used for the temperature control, which allows temperature stabilisation within ±0.1 K.The sequence of mesophases was determined by the observation of the characteristic textures and their changes in a polarising optical microscope (POM), Nikon Eclipse E600POL . Planar cells (bookshelf geometry) of 12 μm thickness  using a Perkin–Elmer DSC8000 calorimeter . The samples of about 4–8 mg, hermetically sealed in aluminium pans, were placed into the calorimeter chamber filled with nitrogen. The calorimetric measurements were performed on cooling/heating runs at a rate of 5 K minα radiation, λ = 1.54 Å, formed by Goebel mirror, Anton Paar DCS 350 heating stage, scintillation detector), and the temperature stability was 0.1 K. Samples were prepared in the form of thin films on a heated surface. The smectic layer thickness was determined using Bragg’s law: nλ = 2d sinθ, where n is a positive integer and θ is the angular position of the diffraction peak.X-ray diffraction (XRD) measurements were performed to determine the structural properties of the smectic mesophases. Experiments in the small diffraction angle range allowed the determination of the smectic layer spacing, d. A Bruker D8 Discover system was used , UVHG (b) and UKHG (c) reactive mesogens are presented in For the calculation of the length of the reactive mesogen molecules in the energy-optimised conformation, the MOPAC/AM1 model was used. The resulting molecular structures with the principal axis of minimum moment of inertia, e.g., the long molecular axis, are shown in ≈ L/3(S2 + 2) [The orientational order parameter, S2, can be estimated according to the relation d (S2 + 2) ,52, wher3 and CH3O, respectively), it is possible to conclude that the presence of a bulky methoxy group makes the tilted smectic phase unfavourable. However, a lateral methyl substitution in combination with a branched terminal chain (UKHM) fully suppressed the self-assembling behaviour of the resulting material with a given type of molecular core. The kinetics of the phase transitions for UKHG reactive mesogen has been studied in dependence on the heating/cooling rate of DSC runs. The resulting phase diagrams for the UKHG reactive mesogen clearly demonstrated a noticeable difference in the phase transition temperatures on the heating/cooling DSC rate. The Iso-N and N-SmA phase transition temperatures slightly decreased while increasing the cooling rate.Several new calamitic reactive mesogens, with vinyl terminal group with different length of flexible chains and with/without lateral substitution by the methyl and methoxy groups placed on the molecular core, have been designed and synthesised. Depending on the molecular structure, the reactive mesogens exhibit the nematic, orthogonal SmA and tilted SmC phases in a reasonably broad temperature range. The structure of the smectic mesophases has been confirmed by XRD measurements. The calculated length of the most extended conformer correlates well with the smectic layer spacing obtained experimentally. Lateral substitution by a bulky methoxy group on the molecule core deteriorated the arrangement of the molecules (UVHG and UVHGET), and only nematic and orthogonal SmA phases were detected. The lateral substitution by the methyl group on the molecular core had a very positive effect on the self-assembling behaviour (UKHG), and rich mesomorphic behaviour (N-SmA-SmC) was observed. While comparing the mesomorphic behaviour for UKHG and UVHG reactive mesogens differing by the substituent type only (i.e., CHThe reactive mesogens reported here with a functional vinyl terminal group can be further used as reactive mesogens, i.e., both the monomers and co-monomers, for the design of the smart self-assembling macromolecular materials, such as siloxane-based polymers, co-polymers and elastomers, exhibiting self-assembling behaviour favourable for various applications, including smart sensors ,53,54,55


The triad not only maintains the photochromic properties of BTE moiety in solution, film, and solid state but also exhibits remarkable AIE properties. Moreover, the fluorescence of BTE-2PBT PMMA film could be modulated with high contrast by alternate UV and visible light irradiation. Photoerasing, rewriting, and non-destructive readout of fluorescent images on BTE-2PBT PMMA film well demonstrate its potential application as optical memory media.A bisthienylethene-dipyrimido[2,1- Photochromic diarylethenes have been acknowledged for their excellent photochemical reactivity, thermal stability, and fatigue resistance ,2 and witrans-cyanostilbene benzothiazoles (PBTs) as atypical propeller-shaped novel AIEgens with full-color tunability, excellent solid-state fluorescence quantum yields, and high crystallizability [b]benzothiazole (BTE-2PBT) triad. The novel triad not only well maintains its photochromic properties in solution, film, and solid state but also exhibits remarkable AIE properties. More importantly, the fluorescence of BTE-2PBT triad could be reversibly modulated with high contrast in solid state by UV and visible light irradiation, making BTE-2PBT triad a promising candidate for the application as photo-erasible optical memory media.Recently, our research group discovered pyrimido were measured according to a previously reported method with bis(2-methyl-1-benzothiophen-3-yl)hexafluorocyclopentene as the reference compound. A WFH-203B black-box type UV analyzer  and an MVL-210M-visible light source  were employed for photo-induced cyclization and cycloreversion, respectively.oBTE-2PBT- and cBTE-2PBT- were recorded on an Agilent 1220 instrument equipped with a ZORBAX SIL analytical column  ).HPLC traces of W = ca. 110,000) containing BTE-2PBT (5 wt %) was prepared by spin coating on a 1 cm × 2 cm quatz glass slide. During the writing process, the film was irradiated through a photomask under a 254 nmWFH-204B UV lamp  at a distance of 5 cm for 5 min. For erasing, the film was directly subjected to an MVL-210M-visible light source  at a distance of 5 cm for 3 min. Continuous non-destructive reading was performed under a 365 nm WFH-204B UV lamp  at a distance of 5 cm for 5 min.The PMMA film (M(4-(3-(Ethoxycarbonyl)-2-methyl-4H-benzothiazolopyrimidin-4-yl)phenyl)boronic acid (1). To a mixture of (4-formylphenyl)boronic acid , ethyl acetoacetate , 2-aminobenzothiazole  was added Hf(OTf)4 . The reaction was stirred in a sealed tube at 80 °C for 6 h. Flash column chromatography  afforded 1 as a yellow solid ; mp 241–242 °C. 1H-NMR : δ 8.00 , 7.74 , 7.66 , 7.42‒7.36 , 7.29 , 7.17 , 6.45 , 4.10‒4.00 , 3.34 , 1.20  ppm; 13C-NMR : δ 165.4, 162.6, 153.9, 142.9, 137.4, 134.1 (×2), 126.7, 125.9 (×2), 123.9, 122.7 (×2), 112.2, 102.6, 59.4, 56.6, 23.1, 14.0 ppm; IR (KBr): vmax 3346, 2975, 1714, 1608, 1519, 1470, 1427, 1414, 1368, 1342, 1323, 1272, 1241, 1212, 1165, 1085, 1046, 1018, 743 cm−1; HRMS (ESI+): m/z calcd for C20H20BN2O4S [M + H]+ 395.1231; found 395.1230.Diethyl 4,4′-bis)bis)bis (BTE-2PBT). To a solution of 1  and dibromobisthienylethene 2  in THF/H2O  was added tetrakis(triphenylphosphine)palladium  and Na2CO3 . The reaction was stirred at 80 °C for 12 h under nitrogen atmosphere. After the reaction was cooled to ambient temperature, THF was removed under reduced pressure. The residual aqueous solution was extracted with CH2Cl2 (20 mL) and washed with deionized H2O (10 mL × 2). The organic phase was combined, dried over Na2SO4, and concentrated in vacuo. Flash column chromatography on silica gel afforded BTE-2PBT as a light greenish powder ; mp > 300 °C. 1H-NMR : δ 7.49‒7.39 , 7.26‒7.21 , 7.19 , 7.17‒7.07 , 6.41 , 4.25‒4.13 , 2.45 , 1.86 , 1.30  ppm; 13C-NMR : δ 166.9 (×2), 163.5 (×2), 155.2 (×2), 141.7 (×3), 141.2 (×2), 138.2 (×2), 136.2, 133.5 (×2), 132.3 (×2), 128.7 (×2), 128.0 (×4), 126.8 (×2), 126.0 (×5), 124.2 (×2), 124.0 (×2), 122.8 (×2), 122.4 (×2), 111.8 (×2), 103.0 (×2), 60.2 (×2), 57.5 (×2), 29.8 (×2), 23.9 (×2), 14.6 (×2), 14.5 (×2) ppm; 19F NMR : δ −110.03 , −131.85 ; IR (KBr): vmax 3442, 1700, 1674, 1597, 1508, 1371, 1331, 1272, 1241, 1199, 1113, 1093, 1076, 988, 742 cm−1; HRMS (ESI+): m/z calcd for C55H43F6N4O4S4 [M + H]+ 1065.2066; found 1065.2063.BTE-2PBT triad has been designed and synthesized. The triad well combines the photochromic properties of BTE moiety and AIE properties of PBT moieties. More importantly, the solid-state fluorescence of BTE-2PBT could be reversibly switched by alternate UV and visible light irradiation with ultra-high contrast. Repeated writing and erasing of fluorescent images on BTE-2PBT PMMA film with non-destructive readout indicate that this novel triad has the potential to be applied as a practical solid-state luminescent material.In summary, a photo-switchable AIE-active


Benincasa cerifera Savi. extracts are effective in treating diabetes and its complications. Benincasae Exocarpium (BE) is a fruit peel of B. cerifera that has been reported to be used for the prevention and treatment of metabolic diseases such as hyperglycemia, obesity, and hyperlipidemia. However, there are not enough studies on the compounds and bioassays to support the efficacy of BE. The inhibitory activity of the BE extracts and fractions against advanced glycation end-products (AGE) formation and α-glucosidase activity was evaluated. These assays are relevant for the treatment of type 2 diabetes and its complications. Based on these results, compounds 1–11 were isolated through bioassay-guided isolation. In addition, we developed a high-performance liquid chromatography (HPLC) method that can simultaneously analyze these 11 compounds. Activity evaluation of the compounds was also conducted, and eight compounds exhibited significant activity. Among these, flavonoid compounds showed strong activity. A quantitative evaluation of eight bioactive compounds  was conducted. In conclusion, this study demonstrated the potential of BE for prevention and treatment of type 2 diabetes and its complications.Diabetes is a chronic metabolic disease that is a constant problem. Previous studies have reported that  The number of diabetic patients is steadily increasing worldwide, and type 2 diabetes, especially among young people such as children and adolescents, is becoming a problem . The cauBenincasa cerifera Savi. belongs to the Cucurbitaceae family and is commonly called ash gourd, wax gourd, winter melon, Chinese watermelon, and donggua −, 1H-NMR  δ: 7.43–7.41 , 6.79 ; 13C-NMR  δ: 170.23 (C-7), 151.50 (C-4), 146.03 (C-3), 123.89 (C-1), 123.11 (C-6), 117.72 (C-2), 115.76 (C-5).C8H8O3; ESI-QTOF-MS/MS m/z: 151.1472 [M − H]−, 1H-NMR  δ: 9.84 , 7.44–7.42 , 6.97 , 5.78 , 3.99 , 1.63 ; 13C-NMR  δ: 191.00 (CHO), 151.76 (C-5), 146.13 (C-2), 130.68 (C-1), 124.51 (C-4), 114.09 (C-3), 110.17 (C-6), 56.18 (OCH3).C6H6O3; ESI-QTOF-MS/MS m/z: 125.0236 [M − H]−, 1H-NMR  δ: 9.58 , 7.21 , 6.51 , 4.71 ; 13C-NMR  δ: 177.06 (C-1), 160.54 (C-2), 152.36 (C-5), 122.71 (C-3), 109.96 (C-4), 57.61 (C-6).C21H20O10; ESI-QTOF-MS/MS m/z: 431.0967 [M − H]−, 1H-NMR  δ 7.92 , 6.91 , 6.77 , 6.50 , 4.87 , 4.57 , 4.03 , 3.67 , 3.23–3.09 ; 13C-NMR  δ: 181.96 (C-4), 163.52 (C-2), 163.32 (C-7), 161.18 (C-4′), 160.67 (C-5), 156.23 (C-9), 128.48 , 121.11 (C-1′), 115.99 , 108.89 (C-6), 103.39 (C-10), 102.79 (C-3), 93.63 (C-8), 81.58 (C-5″), 78.94 (C-3″), 73.05 (C-1″), 70.61 (C-2″), 70.22 (C-4″), 61.48 (C-6″).C21H20O10; ESI-QTOF-MS/MS m/z: 431.0211 [M − H]−, 1H-NMR  δ: 7.70, 6.84 , 6.47 , 6.30 , 4.74 , 4.04 , 3.69 , 3.64 , 3.55 , 3.53 , 3.51 ; 13C-NMR  δ: 181.42 (C-4), 164.11 (C-2), 163.42 (C-7), 161.95 (C-4′), 159.14 (C-5), 157.21 (C-9), 128..74 , 121.42 (C-1′), 115.24 , 107.61 (C-6), 103.25 (C-10), 102.88 (C-3), 94.27 (C-8), 80.45 (C-5″), 78.64 (C-3″), 74.41 (C-1″), 71.11 (C-2″), 69.10 (C-4″), 60.00 (C-6″).C21H20O11; ESI-QTOF-MS/MS m/z: 447.1042 [M − H]−, 1H-NMR  δ: 13.07 , 10.45 , 10.01 , 9.01 , 7.50 , 7.44 , 6.84 , 6.60 , 6.21 , 4.62 , 3.7–3.6 , 3.54 , 3.49–3.21 ; 13C-NMR  δ: 180.87 (C-4), 163.94 (C-2), 161.11 (C-7) 160.74 (C-5), 154.74 (C-8a), 149.11 (C-4′), 144.45 (C-3′), 120.78 (C-1′), 118.29 (C-6′), 114.44 (C-5′), 114.14 (C-2′), 104.14 (C-8), 103.45 (C-4a), 101.14 (C-3), 97.74 (C-6), 80.44 (C-5″), 77.64 (C-3″), 72.27 (C-1″), 70.67 (C-2″), 70.64 (C-4″), 60.12 (C-6″).C7H6O5; ESI-QTOF-MS/MS m/z: 169.0134 [M − H]−, 1H-NMR  δ: 7.05 ; 13C-NMR  δ: 170.41 (C=O), 146.36 , 139.57 (C-4), 121.96 (C-1), 110.32 .C9H8O4; ESI-QTOF-MS/MS m/z: 179.0342 [M − H]−, 1H-NMR  δ: 7.40 , 7.01 , 6.95 , 6.74 , 6.16 ; 13C-NMR  δ: 167.95 (C-9), 148.16 (C-4), 145.58 (C-7), 144.63 (C-3), 125.73 (C-1), 121.20 (C-6), 115.77 (C-5), 115.14 (C-8), 114.64 (C-2).C27H30O14; ESI-QTOF-MS/MS m/z: 577.0651 [M − H]−, 1H-NMR  δ: 8.06 ; 6.89 , 6.78 , 6.25 , 4.97 , 4.75 , 4.63 , 4.41 , 4.04 , 3.56 , 3.09–2.91 , 2.10 , 1.89 , 0.46 ; 13C-NMR  δ: 182.09 (C-4), 163.97 (C-2), 162.36 (C-7), 161.19 (C-5), 160.65 (C-4′), 155.81 (C-9), 128.99 (C-6′), 121.58 (C-1′), 115.87 (C-5′), 104.45 (C-10), 104.19 (C-8), 102.42 (C-3), 100.30 (C-1′′′), 98.27 (C-6), 81.80 (C-5″), 79.89 (C-3″), 75.03 (C-2″), 71.65 (C-1″), 71.45 (C-4′′′), 70.64 (C-2′′′), 70.44 (C-3′′′), 70.22 (C-4″), 68.21 (C-5′′′), 61.13 (C-6″), 21.11 (C-1), 17.71 (C-6′′′).C27H30O15; ESI-QTOF-MS/MS m/z: 593.1464 [M − H]−, 1H-NMR  δ: 7.98 , 6.94 , 6.60 , 6.24 , 5.13 , 5.05 , 4.36–3.11 , 1.95 ; 13C-NMR  δ: 165.19 (C-2), 161.44 (C-7), 161.30 (C-5), 128.78 (C-3′), 128.35 (C-5′), 122.28 (C-1′), 115.69 (C-4′), 104.77 (C-10), 104.47 (C-8), 102.45 (C-3), 98.12 (C-6), 80.31 (C-5″), 78.88 (C-3″), 75.76 (C-2″), 74.47 (C-2′′′), 72.27 (C-4′′′), 70.71 (C-3′′′), 69.90 (C-4″), 69.72 (C-5′′′), 61.56 (C-6′′′).C50 values of 684.12 ± 2.82, 628.05 ± 1.73, and 419.31 ± 1.28 µg/mL, respectively). In the BSA-MGO system, the EA and BuOH fractions were the most potent AGE formation inhibitors . The extract mildly inhibited AGE formation in BSA-MGO . On the other hand, the Hx and water fractions showed no or slight inhibition of AGE formation in the two systems.Advanced glycation end-products (AGEs) produced by hyperglycemia and oxidative stress are major causes of diabetic complications ,34. This50 values of 212.20 ± 4.15 µg/mL). The Hx, BuOH, and water fractions mildly inhibited α-glucosidase  showed higher inhibitory activity than AMG as the positive control , as well as compounds 2 and 9 . Compound 8 had an IC50 value of 191.73 ± 2.15 µM, which is similar to AMG. In contrast, compounds 1, 3, and 4 showed no activity. In the BSA-MGO system, as in the BSA-glucose system, five flavonoids  were more potent inhibitors of AGE formation compared to the other compounds . Compounds 2 and 9 mildly inhibited AGE formation . In contrast, compounds 1, 3, 4, and 8 showed no or slight inhibitory activity.Compounds 10 showed the highest inhibitory activity against α-glucosidase  compared to acarbose as a positive control . Including compound 10, flavonoid compounds  and compound 2 exhibited IC50 values like the positive control . Compound 8’s inhibitory activity was also lower than that of the other compounds, but the value was significant . In contrast, compounds 1, 3, 4, and 9 showed no, or slight, inhibitory activity. The results are shown in Compound p-hydroxybenzoic acid (255 nm), protocatechuic acid (260 nm), isovanillin (231 nm), 5-hydroxymethylfurfural (282 nm), isovitexin (334 nm), vitexin (268 nm), orientin (226 nm), gallic acid (213 nm), caffeic acid (325 nm), vitexin-2″-O-rhamnoside (268 nm), and vitexin-4″-O-glucoside (268 nm). The maximum absorption wavelength of each compound and the peak area at 280 nm were compared and it was confirmed that there was no significant difference between the two cases. Therefore, the detection wavelength was determined to be 280 nm. Subsequently, an analysis method validation was performed. The specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), intra-day and inter-day precision, and accuracy of the analysis method were confirmed. Additionally, the recovery and robustness were also confirmed  of all compounds was greater than 0.999, and the results are shown in The linearity of the 11 compounds was measured at five concentrations between 0.5–50 μg/mL. In all calibration curves, the correlation coefficient  out of the 11 compounds were selected based on the activity assay. The eight bioactive compounds showed strong activity in the anti-glycation (AGE formation inhibitory) and anti-diabetic (α-glucosidase inhibitory) assays. HPLC-PDA analysis of the BE extracts was performed to quantitatively evaluate the bioactive compounds  > Compound 10  > Compound 2  > Compound 7  > Compound 11  > Compound 9  > Compound 6  > Compound 8 . Therefore, we proposed that the content criteria of BE be 0.113% for compound 2, 0.432% for compound 5, 0.069% for compound 6, 0.101% for Compound 7, 0.051% for Compound 8, 0.071% for Compound 9, 0.340% for Compound 10, and 0.081% for Compound 11.The compounds of BE showed high contents in the following order: Compound B. cerifera samples were collected from Cheonan, Korea. They were authenticated by Professor, Wan Kyunn Whang .Benincasae Exocarpium was purchased from the Kyung-dong market in Seoul, Korea. In addition, n-hexane (Hx), ethyl acetate (EtOAc), n-butanol (BuOH) and distilled water were used for extraction, fractionation, and open column chromatography. An octadecylsilane (ODS) gel (75 µm) , Sephadex LH-20 (25–100 µm)  and MCI CHP 20P gel  were used for open column chromatography.Methanol (MeOH), ethanol (EtOH), 1H- and 13C-nuclear magnetic resonance (NMR) spectra were recorded at 600 and 150 MHz, respectively, using a JEOL spectrometer . Chemical shifts are presented as parts per million (ppm) on the δ scale, and the coupling constants (J) are shown in Hertz. Chloroform-d (CDCl3-d), dimethyl sulfoxide-6d (DMSO-6d), and methanol-4d (CD3OD-4d) were purchased from Sigma-Aldrich Co.  as solvents for NMR analysis.18 column . HPLC-grade solvents  were obtained from Thermo Fisher Scientific .Electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS/MS) was performed using Bruker compact data analysis software version 4.3, a Bruker compact . High-performance liquid chromatography-photodiode array detector (HPLC-PDA) was analyzed using Empower Pro 2.0, a Waters 2695 system pump equipped with a Waters 2489 photodiode array detector . The HPLC column was a Fortis C® F200 PRO spectrophotometer  was used for the AGE formation inhibitory activity assay in the BSA-glucose and BSA-MGO systems. In addition, the α-glucosidase inhibitory activity assay was performed using Biotek EPOCH2 . Regarding the activity assays, sodium azide, sodium phosphate dibasic, sodium phosphate monobasic, aminoguanidine hydrochloride (AMG), bovine Serum Albumin (BSA), DMSO, D-(−)-fructose, D-(+)-glucose, methylglyoxal (MGO), α-glucosidase from Saccharomyces cerevisiae (Saccharomycetaceae), 4-nitrophenyl α-D-glucopyranoside (p-NPG) and acarbose were purchased from Sigma-Aldrich, Co. .Among the anti-diabetes activity assays, the Infinitein vacuo, suspended in water (H2O), and then partitioned using Hx, EtOAc, and BuOH. The results yielded Hx (100.46 g), EtOAc (21.15 g), BuOH (35.74 g), and water (154.22 g) fractions. Among these four fractions, the EtOAc and BuOH fractions were the most potent in the three anti-diabetes activity assays. In addition, in the fraction analysis using HPLC-PDA, various polar components were confirmed in the two fractions. Therefore, repeated open column chromatography of both active fractions was performed.The BE (4.0 kg) was dried, powdered, and then extracted in methanol (20 L × 3) at room temperature. The filtrate was concentrated to dryness (406.03 g) 3:MeOH:water (70:30:4→50:50:4), and four sub-fractions were obtained. Sub-fraction EtOAc 1 was isolated using MCI gel column chromatography with 50% MeOH to obtain sub-fraction EtOAc 1-3. Sub-fraction EtOAc 1-3 was separated using a Sephadex LH-20 column and eluted with 20% MeOH to obtain sub-fraction EtOAc 1-3-2. Sub-fraction EtOAc 1-3-2 was subjected to MCI gel column chromatography using 30% EtOH to obtain compound 1. To increase the purity of compound 1, Sephadex LH-20 column with EtOH 50% was performed. Sub-fraction EtOAc 2 was applied to a Sephadex LH-20 open column and eluted with 20% to 70% MeOH to obtain two sub-fractions (sub-fraction EtOAc2-1 to EtOAc2-2). Sub-fraction EtOAc 2-1 was applied to Sephadex LH-20 and eluted with 30% EtOH to obtain compound 2. Sub-fraction EtOAc 2-2 was subjected to ODS column chromatography with 10% EtOH to obtain compound 3. Sub-fraction EtOAc 3 was subjected to ODS column chromatography with 30% MeOH to obtain sub-fraction EtOAc 3-2. Sub-fraction EtOAc 3-2 was applied to Sephadex LH-20 column with 50% MeOH to obtain compound 4. Sub-fraction EtOAc 4 was applied to a Sephadex LH-20 column with 50% MeOH to obtain two sub-fractions (sub-fraction EtOAc 4-2 and EtOAc 4-3). Sub-fraction EtOAc 4-2 was subjected to MCI gel column chromatography with 30% EtOH to obtain compound 5. Sub-fraction EtOAc 4-3 was subjected to MCI gel column chromatography with 10% MeOH to sub-fraction obtain EtOAc 4-3-1 and EtOAc 4-3-2. Sub-fraction EtOAc 4-3-1 was applied to a Sephadex LH-20 column with 30% EtOH to obtain compound 6. Sub-fraction EtOAc 4-3-2 was subjected to ODS column chromatography with 10% MeOH to obtain compound 7.The EtOAc fraction (21.15 g) was subjected to silica gel open column chromatography with CHCl3:MeOH (90:10→30:60). Two sub-fractions were obtained . Sub-fraction BuOH 1 was subjected to MCI gel column chromatography with 40% MeOH and two sub-fractions (sub-fraction BuOH 1-3 and BuOH 1-5) were obtained. The BuOH 1-3 sub-fractions was applied to a Sephadex LH-20 column with 10% EtOH to yield compound 8. In addition, sub-fraction BuOH 1-5 was subjected to ODS column chromatography with 5% MeOH to obtain compound 9. Sub-fraction BuOH 2 was applied to a Sephadex LH-20 column with 20% MeOH to obtain sub-fractions BuOH 2-1 and BuOH 2-3. Sub-fraction BuOH 2-1 was subjected to ODS column chromatography with 20% MeOH and yielded the sub-fraction BuOH 2-1-3. Sub-fraction BuOH 2-1-3 was applied to a Sephadex LH-20 column with 10% EtOH to obtain compound 10. Finally, sub-fraction BuOH 2-3 was applied to a Sephadex LH-20 column with 50% MeOH to obtain sub-fraction BuOH 2-3-1. Sub-fraction BuOH 2-3-1 was subjected an MCI gel column with 10% MeOH to obtain sub-fraction BuOH 2-3-2. Sub-fraction BuOH 2-3-2-1 was applied to Sephadex LH-20 with 10% EtOH to obtain compound 11.The BuOH fraction (35.7 g) was subjected to silica gel open column chromatography with CHCl1–11) was weighed (8 mg) and dissolved in 800 µL of each solvent . The optimal 1H and 13C NMR spectroscopic analytical conditions were as follows: 1H NMR: scans-30, relaxation delay-10, and gain-optimal selection; 13C NMR: scans-1800, relaxation delay-30, gain-optimal selection.Each isolated compound (1–11 were determined using ESI-QTOF-MS/MS. Isolated compounds (1 mg) were dissolved in 10 mL of MeOH and filtered using a 0.45 µm syringe filter. The prepared compounds were directly injected into a Bruker Compact system.The molecular weights of the isolated compounds v/v) and solvent B . Solvent B was increased from 10% to 50% for 5 min and then from 50% to 100% for 10 min, maintained for 3 min, decreased from 100% to 10%, and maintained for 5 min. The flow rate of the mobile phase was 0.2 mL/min, and the injection volume was 30 µL. The optimal ESI-QTOF-MS/MS analysis conditions were as follows: source type electrospray ionization, ion polarity-negative, scan-50–800 m/z, set capillary-4500 V, set endplate offset-500 V, set nebulizer-1.2 bar, set dry heater-200 °C, and set dry gas-10.0 L/min.The mobile phase was run on a gradient schedule with solvent A , a Fortis C18 Column  was used. Mobile phases A and B consisted of water containing 1% acetic acid and acetonitrile, respectively, and were run on the following gradient schedule. Mobile phase A was maintained for 24 min and increased from 10% to 27%. The flow rate of the mobile phase was 1.0 mL/min, and the injection volume was 10 µL. Column temperature was maintained at 25 °C. Detection was performed at a UV absorbance wavelength of 280 nm. This analysis method is validated by various evaluation sections including specificity, linearity, LOD and LOQ, and intra- and inter-day precision and accuracy [1–11 were dissolved in 1 mL of MeOH and filtered through a 0.45 μm syringe filter. The compounds (1–11) were mixed in the same ratio to make a standard solution. They were diluted to concentrations between 2.5–50 ug/mL and used for HPLC-PDA analysis to validate the analysis conditions.To analyze the isolated compounds (accuracy . The sam50) values of triplicate measurements of the samples and AMG were calculated and expressed as the mean ± SD.The AGE formation inhibitory assay of BSA-glucose was conducted using a spectrophotometric method developed in a previous study . All tesThe AGE formation inhibitory assay of BSA-MGO was conducted using spectrophotometry according to a previously described method with modification ,39. All α-Glucosidase inhibitory activity was measured spectrophotometrically according to the previously described method . All samp < 0.05, p < 0.01, and p < 0.001 indicated statistical significance.Statistical significance was measured using one-way analysis of variance (ANOVA) and multiple comparisons with 1H, 13C-NMR, and ESI-QTOF-MS/MS. AGE formation and α-glucosidase inhibitory activity were confirmed for compounds 1–11. Among the isolated compounds, five flavonoids  showed stronger inhibitory activity against AGE formation in BSA-glucose and BSA-MGO systems than in positive controls. In addition, these compounds showed similar or high α-glucosidase inhibitory activity to the positive control group. Compound 2 showed high inhibitory activity against alpha-glucosidase and significant inhibition of AGE formation in BSA-MGO and BSA-glucose systems. Compounds 8 and 9 also showed significant inhibition of AGE formation in the BSA-glucose system. Compounds 1, 3, and 4 showed no or slight inhibitory activity. HPLC-PDA simultaneous analysis was developed for separated compounds 1–11, and validation was performed. Using the developed analysis method, the content of eight bioactive compounds (2 and 5) among compounds 1–11 was confirmed. The optimized extraction conditions for the eight compounds were 70% EtOH and 60 min of extraction. In addition, by presenting content standards for eight compounds, standards for quality management were prepared.In this study, we confirmed the inhibitory activity of extracts and fractions of BE against AGE formation and α-glucosidase. Since EA and BuOH fractions exhibited the strongest inhibitory activities, we isolated 11 compounds (seven from EA fractions and four from BuOH fractions) in accordance with bioassay-guided isolation. They were identified by In summary, a systematic experiment  was conducted to confirm that BE has an anti-diabetic effect. We confirmed anti-diabetic activity of the BE extract and its fractions. The various compounds were separated by open column chromatography, and anti-diabetic activity assays of the active ingredients were performed. In addition, the content standard of the BE was set through a multi-component simultaneous content analysis method using HPLC-PDA.Based on these results, the possibility of BE as a functional material for the treatment of type 2 diabetes and diabetes complications was confirmed. Additional studies, including in vivo studies of BE, need to be conducted to evaluate whether efficacy is sufficient for clinical application and the drug effects of isolated compounds. Furthermore, compounds isolated from BE may be valuable functional agents against other diseases.