H-benzo[d]imidazole derivatives 1a,b were reacted with a β-ketoester such as acetylacetone in the presence of sodium ethoxide to obtain the desired molecules 2a,b. The latter acted as a key molecule for the synthesis of new carbazone derivatives 4a,b that were submitted to react with 2-oxo-N-phenyl-2-(phenylamino)acetohydrazonoyl chloride to obtain the target thiadiazole derivatives 6a,b. The structures of the newly synthesized compounds were inferred from correct spectral and microanalytical data. Moreover, the newly prepared compounds were subjected to molecular docking studies with DNA gyrase B and exhibited binding energy that extended from −9.8 to −6.4 kcal/mol, which confirmed their excellent potency. The compounds 6a,b were found to be with the minimum binding energy  as compared to the standard drug ciprofloxacin  against the target enzyme DNA gyrase B. In addition, the newly synthesized compounds were also examined and screened for their in vitro antimicrobial activity against pathogenic microorganisms Staphylococcus aureus, E. coli, Pseudomonas aeruginosa, Aspergillus niger, and Candida albicans. Among the newly synthesized molecules, significant antimicrobial activity against all the tested microorganisms was obtained for the compounds 6a,b. The in silico and in vitro findings showed that compounds 6a,b were the most active against bacterial strains, and could serve as potential antimicrobial agents.2-azido-1 Nitrogen-containing heterocyclic analogues have received great interest in drug discovery because of their well-known activity in pharmaceutical and medicinal fields ,2,3,4. BA wide range of antibacterial properties have been gained in hybrid molecules containing benzimidazole and triazole moieties, as shown in On the other hand, DNA gyrase is considered an important bacterial enzyme that is involved in the control of topological transitions of DNA ,17. In aIn light of the potential therapeutic properties of these heterocyclic compounds and with the contribution of our research work ,29,30,31H-benzo[d]imidazol-2-yl)-1H-1,2,3-triazol-4-yl) ethan-1-one 2a,b, were obtained by the reaction of benzo[d]imidazoles 1a and/or 1b with β-ketoester acetylacetone in the presence of sodium ethoxide in ethanol under reflux aceto hydrazonoyl chloride in ethanol containing a catalytic amount of TEA (2–3 drops) to obtain the corresponding 1,3,4-thiadiazole derivatives 6a,b  were docked to the target enzyme DNA gyrase subunit b to check their binding profile. The docking results exhibited that the compounds (1a–6b) were fitted well in the active site pockets of the target and possessed good binding energy values ΔG , as summarized in 1a and 1b formed arene- σ with the target enzyme through the amino acid residue Asn46 at distances of 3.91 and 3.93 Å, respectively. Moreover, 1a docked to the target through one HB with the residue Val71 at 1.87 Å. Compounds 2a and 2b showed the same interactions with the target enzyme as they formed three HB and one arene–cation interactions with the residues Ala47, Thr165, and Arg76. Compound 4a formed two HB interactions with Thr34 at 2.98 and 3.00 Å. On the other hand, derivative 4b exhibited one HB interaction with Asp49 at 2.20 Å. Compound 6a showed one HB with the target through Thr164 at 2.45. Finally, compound 6b, with the highest binding affinity of −9.8 kcal/mol, docked to the target DNA gyrase B through one HB with the residue Asp49 at 2.40 Å. The 2D and 3D representations of the intermolecular interactions of the best docked compounds 6a and 6b with the target enzyme are shown in Based on the bacterial activity results, the compounds , and showed three HB interactions with the residues Arg76, Thr165, and Val43 at 3.00, 2.74, and 2.04 Å, respectively .6a,b firmly docked to the active site pockets of the target enzyme of DNA gyrase subunit B, which functionally participates in DNA inhibitions.The results justified that the compounds 1a. Moreover, the molecules were negative in the AMES toxicity and carcinogenicity test, which suggests that they are non-mutagenic. In addition, the physiochemical properties results exhibited acceptable values as the compounds had M.W (molecular weight) (<500 g/mol), except compounds 6a,b. The partition coefficients of the compounds were <5. The TPSA  were found to be in the favorable range (<140). Further, the HBA (H-bond acceptors) and HBD (donors) in the compounds comply with Lipinski′s rule of five and were found to be in the acceptable range. Overall, we can conclude that they showed good absorption, distribution, and oral bioavailability within the body by not violating Lipinski′s rule of five more than once.With the help of free accessible servers such as admetSAR, mol inspiration, and SwissADME, the pharmacokinetics, physicochemical, and drug-likeness of the newly prepared compounds are summarized in Aspergillus niger, candida albicans, Pseudomonas aeruginosa, Staphylococcus aureus, and E coli using the agar diffusion method. The inhibition zone diameters were measured and compared with that of the standard drugs, as tabulated in 6a and 6b had strong activity against all the tested pathogenic microbes. In addition, compounds 2a and 4a only showed effects against the Gram-negative and Gram-positive bacteria and had no effect on the fungi tested. On the other hand, compound 2b showed strong effects against the fungi tested and no effect against the bacteria tested. Finally, compound 4b revealed moderate activity against all the microorganisms tested, except Staphylococcus aureus.All the newly synthesized molecules were screened for their antimicrobial activity against the pathogenic microorganisms 1H-NMR and 13C-NMR spectra were recorded in (CD3)2SO solutions on a BRUKER500 FT-NMR system spectrometer, and chemical shifts are expressed in ppm units using TMS as an internal reference. Mass spectra were recorded on a GC-MS QP1000 EX Shimadzu. Elemental analyses were carried out at the Microanalytical Center of Cairo University.All melting points were determined on an electrothermal apparatus and are uncorrected. IR spectra were recorded (KBr discs) on a Shimadzu FT-IR 8201 PC spectrophotometer. H-benzo[d]imidazole 1a and/or 2-azido-7-chloro-1H-benzo[d]imidazole 1b (10 mmol) were reacted with acetylacetone  under reflux in ethanol containing sodium ethoxide  for 5 h. The resulting solid that formed after cooling was collected and recrystallized from ethanol to afford 2a and 2b, respectively.2-azido-1H-benzo[d]imidazol-2-yl)-5-methyl-1H-1,2,3-triazol-4-yl)ethan-1-one derivatives 2a1-(1-(1−1): v 3461(NH), 2919, 2852 (CH), 1715(C=O), 1618 (C=N); 1600(C=C); 1H-NMR : δ 2.10 , 2.42 , 7.21 , 7.54 , 11.52 ; 13C-NMR : δ 9.5 (CH3), 27.4 (CH3), 115.2, 123.4, 133.1, 138.3, 138.7, 141.5, 195.9 (C=O); ESI-MS: m/z(%): 241 [M+] (11), 225 (12), 212 (13), 177 (50), 166 (18), 77 (91), 65 (100). Anal. Calcd. for C12H11N5O (241): C, 59.74; H, 4.60; N, 29.03% found: C, 59.78; H, 4.52; N, 29.01%.White crystals, m.p. 218–220 °C, yield: 58%; FT-IR -5-methyl-1H-1,2,3-triazol-4-yl) ethan-1-one 2b1-(1-(7-chloro-1−1): v 3450 (NH), 2921, 2858 (CH), 1705 (C=O), 1622 (C=N); 1600 (C=C); 1H-NMR : δ 2.16 , 2.47 , 7.21–7.25 , 11.58 ; 13C-NMR : δ 9.5 (CH3), 27.4 (CH3), 113.5, 120.2, 124.4, 133.1, 138.3, 140.7, 141.5, 194.8 (C=O); ESI-MS: m/z(%): 275 [M+] (15), 273 (17), 265 (18), 238 (12), 222 (14), 170 (100), 166 (28), 65 (50). Anal. Calcd. for C12H10ClN5O (275): C, 52.28; H, 3.66; N, 25.40% found: C, 52.34; H, 3.62; N, 25.37%.White crystals, m.p. 228–230 °C, yield: 52%; FT-IR  in 2-propanol (20 mL), methyl hydrazine carbodithioate 3  was added. The mixture was stirred at RT for 2 h. The solid product was filtered off, recrystallized from Acetic acid to afford the target molecules 4a and 4b, respectively. To a solution of H-benzo[d]imidazol-2-yl)-5-methyl-1H-1,2,3-triazol-4-yl) ethylidene) hydrazine-1-carbodithioate 4aMethyl-2-(1-(1-(1−1): v 3417 , 1622 (C=N); 1600 (C=C); 1H-NMR : δ 2.16 , 2.36 , 2.47 , 7.35–7.85 , 8.95 , 11.58 ; 13C-NMR : δ 10.0 (CH3), 16.9 (CH3), 18.7 (CH3), 115.2, 123.2, 138.7, 141.5, 145.6 (C=N), 200.0 (C=S); ESI-MS: m/z(%): 345 [M+] (42), 325 (18), 293 (10), 271 (8), 240 (12), 222 (14), 197 (15), 181 (20), 166 (28), 77 (14), 50 (10). Anal. Calcd. for C14H15N7S2 (345): C, 48.68; H, 4.38; N, 28.38% found: C, 48.57; H, 4.32; N, 28.31%.Yellow crystals, m.p. 240–242 °C, yield: 75%; FT-IR -5-methyl-1H-1,2,3-triazol-4-yl) ethylidene) hydrazine-1-carbodithioate 4bMethyl-2-(1-(1-(7-chloro-1−1): v 3419 , 1628 (C=N); 1612 (C=C); 1H-NMR : δ 2.18 , 2.32 , 2.42 , 7.14–7.42 , 8.98 , 11.47 ; 13C-NMR : δ 9.1 (CH3), 16.9 (CH3), 19.7 (CH3), 117.2, 124.2, 139.7, 142.5, 142.6, 143.5 (C=N), 197.1 (C=S); ESI-MS: m/z(%): 379 [M+] (12), 352 (17), 287 (51), 252 (18), 250 (12), 227 (14), 170 (15), 165 (20), 70 (14), 65 (100). Anal. Calcd. for C14H14ClN7S2 (379): C, 44.26; H, 3.71; N, 25.81% found: C, 44.32; H, 3.65; N, 25.73%.Yellow crystals, m.p. 263–265 °C, yield: 75%; FT-IR  and the appropriate 2-oxo-N-phenyl-2-(phenylamino) aceto hydrazonoyl chloride  in ethanol (20 mL), TEA (0.5 mL) was added; the mixture was stirred at RT for 2–4 h (monitored with TLC). The resulting solid was collected and recrystallized from the proper solvent to obtain the desired 1,3,4-thiadiazolines 6a and 6b, respectively.To a mixture of methyl carbodithioate H-benzo[d]imidazol-2-yl)-5-methyl-1H-1,2,3-triazol-4-yl)ethylidene) hydrazono)-N,4-diphenyl-4,5-dihydro-1,3,4-thiadiazole-2-carboxamide 6a5-(1-(1-(1-−1): v 3435 , 1685 (C=O), 1600 (C=N), 1590 (C=C); 1H-NMR : δ 2.38 , 2.46 , 7.32–7.91 , 9.81 , 11.52 ; 13C-NMR : δ 10.1 (CH3), 20.5 (CH3), 115.20, 122.40, 123.06, 129.60, 133.7, 138.30, 138.90, 141.50 (C=N), 158.20 (C=N), 160.00 (C=O), 164.60 (C=N); ESI-MS: m/z(%): 534 [M+] (12), 520 (16), 492 (26), 478 (14), 425 (17), 357 (17), 312 (10), 287 (51%), 252 (18), 251 (12), 236 (14), 172 (15), 167 (21), 77 (12), 50 (100). Anal. Calcd. for C27H22N10OS (534): C, 60.66; H, 4.15; N, 26.20% found: C, 60.72; H, 4.11; N, 26.15%.Yellow crystals, m.p. 251–253 °C, yield: 67%; FT-IR -5-methyl-1H-1,2,3-triazol-4-yl) ethylidene)hydrazono)-N,4-diphenyl-4,5-dihydro-1,3,4-thiadiazole-2-carboxamide 6b5-((1-(1-(7-chloro-1−1): v 3447 , 1692 (C=O), 1611 (C=N), 1595 (C=C); 1H-NMR : δ 2.32 , 2.48 , 7.37–7.81 , 9.82 , 11.57 ; 13C-NMR : δ 10.1 (CH3), 20.5 (CH3), 115.2, 122.4, 123.0, 129.6, 133.7, 138.3, 138.9, 148.5 (C=N), 156.2 (C=N), 162.0 (C=O), 165.6 (C=N); ESI-MS: m/z(%): 570 [M + 2] (15) 568 [M+] (14), 530 (12), 467 (30), 420 (16), 397 (12), 342 (18), 278 (61), 251 (11), 245 (17), 226 (14), 167 (15), 165 (22), 77 (17), 50 (100). Anal. Calcd. for C27H21ClN10OS (568): C, 56.99; H, 3.72; N, 24.61% found: C, 56.92; H, 3.65; N, 24.58%.Yellow crystals, m.p. 282–284 °C, yield: 65; FT-IR  with resolution 2.3 Å . In addiAspergillus niger and candida albicans (NRRL Y-477) using sabouraud dextrose agar medium and Pseudomonas aeruginosa , E. coli (E. coli O157), and Staphylococcus aureus  using nutrient agar medium.The antimicrobial activities of the tested compounds were tested against the pathogenic microorganisms The synthesized compounds were tested for their antimicrobial activity using the agar diffusion method  . 1H-NMR, 13C-NMR, ESI-MS, and elemental analysis. All the compounds were evaluated for their antimicrobial activity against different strains of bacteria and fungi. Looking at the biological activity, we can conclude that all the compounds showed good to excellent results. In addition, the in vitro findings of the compounds were validated with the help of the molecular docking study with the target enzyme DNA gyrase B by investigating its binding modes of interaction. The molecular docking study revealed that all the synthesized compounds exhibited good binding energy toward the target enzyme. The in silico and in vitro findings showed that compounds 6a,b were the most active against bacterial strains. The results suggested that the compounds 6a,b could be used as promising scaffolds for the design and development of potential antimicrobial inhibitors. Herein, in the present study, we reported the synthesis of a novel set of benzimidazole-triazole hybrid analogues, and the compounds were characterized by means of FT-IR,


Monascus species are asexually or sexually reproduced homothallic fungi that can produce a red colorant, specifically the so-called red yeast rice or Anka, which is used as a food ingredient in Asia. Traditional experiences of using Monascus for treating indigestion, enhancing blood circulation, and health remedies motivate us to investigate and repurpose Monascus-fermented products. Here, two new 5H-cyclopenta[c]pyridine type azaphilones, 5S,6S-monaspurpyridine A (1) and 5R,6R-monaspurpyridine A (2), two new xanthonoids, monasxanthones A and B (3 and 4), one new naphthalenone, monasnaphthalenone (5), and one new azaphilone, monapurpurin (6), along with two known compounds were isolated from the 70% EtOH extract of a citrinin-free domesticated strain M. purpureus BCRC 38110. The phytochemical properties of the xanthonoid and naphthalenone components were first identified from Monascus sp. differently from the representative ingredients of polyketide-derived azaphilones. UVB-induced cell viability loss and reactive oxygen species (ROS) overproduction in human keratinocytes were attenuated by monascuspirolide B (7) and ergosterol peroxide (8), indicating their photoprotective potentials. Ergosterol peroxide (8) decreased the melanin contents and tyrosinase activities of mouse melanocytes, depending on the concentration, suggesting their anti-melanogenic effects. In conclusion, six new and two known compounds were isolated from M. purpureus BCRC 38110, and two of them exhibited dermal protective activities. The results revealed the novel potential of M. purpureus for developing natural cosmeceutics against skin photoaging. Monascus sp. fermentation products, also called red yeast rice, red koji rice, or Anka, are well-known microbial natural products which have been extensively used for more than 1000 years. The Monascus-fermented products have been extensively used for preserving meat and fish, food coloration, and rice wine brewing. Aside from that, the medicinal properties of Monascus-fermented products have been described in the ancient Chinese pharmacopeia for easing digestion, smoothing pain, and blood sweeping -4′(3′H)-one derivatives were also found +, C15H19NO6Na, Calcd.: C15H19NO6Na, 332.11046. 1H NMR  δ: 1.07 , 1.24 , 1.28 , 3.00 , 4.07 , 4.22 , 7.58 , 8.87 . 13C NMR  δ: 14.1 (C-14), 21.8 (C-11), 23.5 (C-10), 48.6 (C-8), 63.5 (C-13), 68.4 (C-9), 84.8 (C-5), 87.1 (C-6), 120.8 (C-4), 129.8 (C-7a), 145.6 (C-1), 159.5 (C-4a), 167.6 (C-3), 171.8 (C-12), 203.5 (C-7).Yellowish syrup. c 0.04, MeOH). UV (MeOH) λmax (log ε) 204 (4.18), 252 sh (3.82), 272 sh (3.46) nm. IR νmax (ATR) 3375 (OH), 1728 (C=O), 1604 (C=C) cm−1. CD (MeOH) λext 203 (Δε +0.4), 215 (Δε +8.8), 228 (Δε +9.0), 248 (Δε −6.5), 343 (Δε +2.2), 396 (Δε −0.6) nm. HRESIMS m/z: 332.11020 [M + Na]+, C15H19NO6Na, Calcd.: C15H19NO6Na, 332.11046. 1H NMR  δ: 1.07 , 1.24 , 1.28 , 3.00 , 4.07 , 4.22 , 7.58 , 8.87 . 13C NMR  δ: 14.1 (C-14), 21.8 (C-11), 23.5 (C-10), 48.6 (C-8), 63.5 (C-13), 68.4 (C-9), 84.8 (C-5), 87.1 (C-6), 120.8 (C-4), 129.8 (C-7a), 145.6 (C-1), 159.5 (C-4a), 167.6 (C-3), 171.8 (C-12), 203.5 (C-7).Yellowish syrup. c 0.045, CHCl3). UV (MeOH) λmax (log ε) 254 (4.04), 278 sh (4.02), 360 (3.96) nm. UV (MeOH + KOH) λmax (log ε) 254 sh (3.95), 332 (3.96) nm. IR νmax (ATR) 3437 (OH), 1713 (C=O), 1672 (C=O), 1620, 1572, 1486 (aromatic ring) cm−1. HRESIMS m/z: 409.12561 [M + Na]+, C21H22O7Na, Calcd.: C21H22O7Na, 409.12577. For 1H NMR  and 13C NMR  data, see Yellowish solid. c 0.072, CHCl3). UV (MeOH) λmax (log ε) 270 (4.27), 360 (4.12) nm. UV (MeOH + KOH) λmax (log ε) 268 (4.14), 330 (4.18), 430 (4.10) nm. IR νmax (ATR) 3429 (OH), 1710 (C=O), 1674 (C=O), 1620, 1573 (aromatic ring) cm−1. HRESIMS m/z:425.12047 [M + Na]+, C21H22O8Na, Calcd.: C21H22O8Na, 425.12069. For 1H NMR  and 13C NMR  data, see Yellowish solid. c 0.09, MeOH). IR νmax (ATR) 3422 (OH), 1707, 1674 (C=O), 1620, 1576 (aromatic ring) cm−1. HRESIMS m/z: 355.11392 [M + Na]+, C18H20O6Na, Calcd.: C18H20O6Na, 355.11576. 1H NMR  δ: 1.18 , 1.60 , 2.48 , 2.67 , 2.74 , 3.91 , 4.10 , 4.24 , 5.98 , 6.87 , 7.37 . 13C NMR  δ: 24.6 (C-16), 32.7 (C-11), 33.4 (C-10), 49.8 (C-12), 52.8 (C-14), 65.4 (C-15), 79.3 (C-8), 115.7 (C-3), 122.9 (C-6), 123.2 (C-4a), 129.7 (C-4), 131.9 (C-1), 144.7 (C-8a), 152.2 (C-5), 156.9 (C-2), 204.5 (C-10), 204.6 (C-7), 206.8 (C-13).Yellowish solid. c 0.05, MeOH). UV (MeOH) λmax (log ε) 210 (4.36), 236 sh (4.08), 290 (3.62) nm. IR νmax (ATR) 3399 (OH), 1739 (C=O), 1627, 1560, 1540 (aromatic ring) cm−1. HRESIMS m/z: 297.07357 [M + Na]+, C15H14O5Na, Calcd.: C15H14O5Na, 297.07334. 1H NMR  δ: 1.26 , 1.68 , 2.65 , 2.70 , 3.97 , 4.21 , 5.78, , 6.69, , 7.78 , 8.51 . 13C NMR  δ: 21.1 (C-10), 24.4 (C-13), 45.0 (C-11), 66.2 (C-12), 79.2 (C-1), 106.1 (C-8), 120.6 (C-9), 122.7 (C-4a), 126.7 (C-3a), 128.4 (C-4), 143.9 (C-8a), 157.9 (C-9a), 160.3 (C-7), 162.8 (C-5), 169.7 (C-3).Yellowish solid. 2 and 95% air. The cell culture medium was replaced every 2–3 days, and confluent cells were passed in a split ratio of 1:3 to 1:5 every 3–5 days with trypsinization.Mouse melanoma B16-F10 cells (BCRC 60031) were purchased from BCRC in Taiwan. A human keratinocyte HaCaT line (Cellosaurus accession number: CVCL_0038) is a spontaneously transformed human keratinocyte line established from the histologically normal skin of a 62-year-old adult , which m® (resazurin) fluorescent dye. Briefly, the cells were plated onto 96-well plates for 24 h, and the treatments were applied with the indicated concentrations and incubation times. After treatment, 10 μL of alamarBlue® Cell Viability Reagent  was added into the culture medium, and then the plates were incubated for 2 h at 37 °C in a cell culture incubator. Fluorescence with an excitation wavelength of 560 nm and emission of 590 nm, recorded using a fluorescent microplate reader , which was used to calculate the cell viability.The cell viability of the HaCaT and B16-F10 cells was determined with alamarBlueAlpha-MSH-stimulated tyrosinase activities in B16-F10 cells were used as an in vitro model for evaluating the anti-melanogenic potentials of bioactive products ,60,65. BBriefly, for determining the melanin content, the cell pellets were then suspended in 2.0 N NaOH and incubated at 95 °C for 15 min. The absorbance at 405 nm was measured using a microplate reader . The cellular melanin content was calculated as follows: Melanin content (%) =  × 100.Briefly, for determining the tyrosinase activity, the cell pellets were lysed with 150 µL PBS containing 1% Triton X-100 and 0.1 mM phenylmethylsulfonyl fluoride prior to centrifugation. The supernatants were mixed with L-DOPA  for 3 h at 37 °C. The absorbance at 490 nm was measured using a microplate reader . The tyrosinase activity was calculated as follows: tyrosinase activity (%) =  × 100.2) using a CL-1000M UV crosslinker  with a UV peak at 302 nm. After UVB exposure, the cells were then incubated with a fresh medium containing vehicle or testing compounds at 37 °C for the indicated time. The control group without treatment of the testing compounds was processed in the same way, except for the UVB irradiation.The HaCaT cell line has been a widely used keratinocyte monolayer culture model for investigating photodamage, photoprotection, and therapeutic interventions for skin diseases ,68. The 2). The fluorescence with an excitation wavelength of 495 nm and emission of 520 nm was recorded using a fluorescent microplate reader .The intracellular ROS was detected using the cell-permeable 2′,7′-dichlorodihydrofluorescein diacetate , which was cleaved by intracellular esterases and converted to the fluorescent product 2′,7′-dichlorofluorescein (DCF) in the presence of ROS. Briefly, the HaCaT cells were pretreated as described above. In the final 30 min of treatment, the cells were loaded with 10 μM H2DCF-DA for 30 min at 37 °C to allow cellular incorporation. The cells were then incubated with PBS for exposure to UVB . A value of p < 0.05 was considered statistically significant.All data are presented as the mean ± standard error of the mean (SEM), derived from at least three independent experiments in triplicate for each treatment group. Statistical significance was analyzed using the Student’s S,6S-monaspurpyridine A (1) and 5R,6R-monaspurpyridine A (2) were calculated using Gaussian16 software . The density functional theory (DFT) was applied at the B3LYP/6-311G level with IEFPCM in MeOH. The final ECD spectra were generated using GaussSum 3.0 software , two pairs of methylenes [δH 3.00 /4.07 ], one oxymethine [δH 4.22 ], and two singlet pyridine protons [δH 7.58 (s)/8.87 (s)] were observed. The 13C NMR and DEPT spectra indicated the presence of three methyl carbons , one methylene (δC 63.5), two oxygenated quaternary carbons (δC 84.8 and δC 87.1), one ester group (δC 171.8), one α,β-unsaturated C=O group on the cyclopentanone at δC 203.5, and the pyridine signals . Based on HMBC correlation  level with IEFPCM in MeOH , one chelated hydroxyl [δH 13.3 ], and two aliphatic alcohols [δH 4.26 /4.27 ]). In the 13C NMR and DEPT spectra, 11 quaternary carbons, 3 primary carbons, 3 secondary carbons, and 4 tertiary carbons could be observed. The low field shift of the carbon signals could also be characterized as three carbonyl groups (δC 198.2 (C-7), 203.2 (C-10), and 205.1 (C-14)) (δH 13.3) was at C-4 and the acetyl group was at C-3. Moreover, the key HMBC correlations of H-9/C-4a, C-5, C-8a, and C-8b verified the junction of the aromatic ring and 6-mehylcyclohex-2-en-1-one at C-9. The above elucidations constructed the chemical skeleton of 1 with 10 IHDs. The last IHD was afforded by the cyclization between C-8a and C-8b through the ether linkage. Therefore, 3 was determined to be a new natural xanthonoid and named monasxanthone A.Compound s, H-11) . It also (C-14)) . The pen (C-14)) . The HMB4 was obtained as an optically yellowish solid. The NMR /H-16 (δH 4.29)/H-17 (δH 1.25), and the HMBC correlation . Based on the information, the entire structure of 5 was suggested and named monasnaphthalenone.Compound elations  between 6 was isolated as an optically yellowish solid, and the molecular formula was determined to be C15H14O5 based on the results from HRESIMS. The 1H and 13C NMR spectra of 6 represented the characteristic skeleton as Monascus azaphilone, monascodilone -4′(3′H)-one derivative, monascuspirolide B (7) [8) [By comparing the spectroscopic data ([α]de B (7) , and one (7) pyridine type azaphilones bearing the N atom. Only one research work has discussed the sensitivity of a related skeleton compound for specific subtypes of nicotinic acetylcholine receptors [6-C1-C6 type xanthonoids (monasxanthones A and B (3 and 4)) and naphthalen-2-one ) were rarely reported in Monascus sp. [5) is rarely seen. Previous studies have identified a broad spectrum of biological activities of naphthalenes, including anti-inflammatory, antiprotozoal, cytotoxic, anti-oxidant, anti-microbial, and anti-platelet effects, supporting the potential for further investigation and applictaions of naphthalenes [Monascus sp. To the best of our knowledge, this study demonstrates the first findings on xanthonoids (3 and 4) and naphthalenone (5) from Monascus sp. Monapurpurin (6) is a linear azaphilone bearing furanone and pyranone, and it is rarely seen in naturally occurring compounds. The most similar compound to 6 is monascodilone, which was also isolated from M. purpureus. However, its bioactivity was not examined [M. purpureus but also uncovered different types of compounds from the natural source. Moreover, our results from two different models of skin disorders also demonstrated the dermoprotective potential of M. purpureus BCRC 38110 for the development of cosmeceutical products. In particular, monascuspirolide B (7) possesses photoprotective activity, and ergosterol peroxide (8) exhibits photoprotective and anti-melanogenic activities. This study provides substantive evidence for developing dietary supplements or functional foods of red yeast rice for skin photoaging and paves the way for repurposing the ancient wisdom of Monascus-fermented products.In this study, six new compounds along with two known compounds were isolated from scus sp. ,83,84. Xscus sp. . Our finthalenes . Usuallyexamined . The phy


Certain macrolide antibiotics, azithromycin included, possess anti-inflammatory properties that are considered fundamental for their efficacy in the treatment of chronic inflammatory diseases, such as diffuse pan-bronchiolitis and cystic fibrosis. In this study, we disclose a novel azithromycin analog obtained via Barton–McCombie oxidation during which an unprecedented epimerization on the cladinose sugar occurs. Its structure was thoroughly investigated using NMR spectroscopy and compared to the natural epimer, revealing how the change in configuration of one single stereocenter (out of 16) profoundly diminished the antimicrobial activity through spatial manipulation of ribosome binding epitopes. At the same time, the anti-inflammatory properties of parent macrolide were retained, as demonstrated by inhibition of LPS- and cigarette-smoke-induced pulmonary inflammation. Not surprisingly, the compound has promising developable properties including good oral bioavailability and a half-life that supports once-daily dosing. This novel anti-inflammatory candidate has significant potential to fill the gap in existing anti-inflammatory agents and broaden treatment possibilities. Many studies have demonstrated that certain macrolide antibiotics are also effective in the treatment of various chronic inflammatory disorders ,2,3. HowBuilding upon our knowledge of macrolide ribosome interactions in solution ,15, as wThe significance of desosamine originates from the analysis of crystal structures and identification of only one hydrogen bond between the macrolide and ribosome, connecting the 2′-hydroxyl group of desosamine and N1 of the A2058Ec of the ribosome ,17,18,19The importance of cladinose, on the other hand, was demonstrated in solution with NMR spectroscopy, through a drastic decrease in STD NMR enhancements and intensity of trNOESY signals observed for compounds without this moiety .In order to transform the well-known antibiotic azithromycin into a full anti-inflammatory drug without any trace of antimicrobial activity, we intended to completely abolish its ability to bind to the bacterial ribosome. Since modifications of the macrolactone methyl group 15 could not be easily achieved through conventional chemistry approaches, we contemplated modification of the azithromycin at the two other key areas important for ribosome binding: the 2′-hydroxyl group of desosamine and cladinose.Therefore, here, we present a novel scaffold, obtainable in a one-pot two-step procedure starting from commercially available azithromycin via a Barton–McCombie radical deoxygenation reaction ,21,22, i1) with O-phenyl chlorothionoformate produced 2′-phenylthiocarbonate derivative 2  coupling constants between H-4” and H-5″. As a comparison, in 3 this constant is 9.8 Hz. Consequentially, and witnessed by the axial position of H-1” , cladinose in 4 does not seem to be stabilized by the anomeric effect.Although cladinose assumes the usual conformation in ed chair . Typical4 affects the mutual arrangement of the sugars, as well as their orientation with respect to the lactone ring. Unlike in both 3 and azithromycin, the sugars become more spatially removed which is noticeable by the disappearance of nOe cross-peaks between the two moieties. Consequentially, the lactone ring exhibits outward folding of the C-3 to C-5 region into “folded-out” conformation, characterized by a large 3JH-2,H-3 = 11 Hz and very strong nOe interaction between protons H-4 and H-11.This “new” conformation of cladinose in 3:4 to afford only 3 using a larger number of equivalents of tributyltin hydride, we then wished to address the question of overturning the ratio to afford predominantly 4. Exchanging tributyltin hydride with tris(trimethylsilyl)silane favored the formation of epimer 4 (entry 3). The formation of 4 is favored still further by decreasing the concentration of the reaction mixture (entry 4). Moreover, exchange of the AIBN ) with less toxic ACCN ) further increased the ratio of 4 (entry 5). After further optimization of the tris(trimethylsilyl)silane/ACCN reaction conditions, it was found that the best ratio of 4 was obtained if 0.5 eq of ACCN was used and when the reaction temperature was increased to 115 °C (entry 6).Having shown that we could overturn the initial ratio of 2.5:1 4 proceeds via 2′-5″ hydrogen transfer and the subsequent attack of the hydride reagent at C-5″ from the less hindered side  compared to those with adenine at the same position +.To a stirred solution of 2′-dehydroxy azithromycin (3):2  in toluene (10 mL) under argon was added tributyltin hydride  and AIBN . The reaction was heated to 90 °C for 3 h and then cooled to RT. The reaction mixture was concentrated in vacuo and the residue purified by Biotage 40 + M using a gradient of 0 to 12% of (10% 0.880 NH3 in MeOH)/DCM to afford title product  as a white foam.To a stirred solution of 38H73N2O11 (M+H+) 733.5214, found 733.5208.HRMS (ES) calc for C1H NMR  [δ/ppm] 8.98 , 5.11 , 4.68 , 4.67 , 4.52 , 4.28 , 4.05 , 3.66 , 3.62 , 3.42 , 3.31 , 3.04 , 2.92 , 2.76 , 2.69 , 2.53 , 2.42 , 2.35 , 2.32 , 2.28 (3′-N(CH3)2, s), 2.04 , 2.03 , 2.02 , 1.93 , 1.89 , 1.75 , 1.68 , 1.59 , 1.47 , 1.35 , 1.33 , 1.27 , 1.25 , 1.24 , 1.23 , 1.19 , 1.13 , 1.09 , 1.08 , 0.91 , 0.90 , 0.89 .13C NMR  [δ/ppm] 178.8 (C-1), 101.0 (C-1′), 95.0 (C-1″), 84.6 (C-5), 78.3 (C-3), 78.1 (C-4″), 77.5 (C-13), 74.2 (C-12), 74.0 (C-11), 73.5 (C-6), 73.0 (C-3″), 70.1 (C-9), 68.7 (C-5′), 65.5 (C-5″), 62.3 (C-10), 60.5 (C-3′), 49.3 (3″-OCH3), 45.3 (C-2), 42.2 (C-7), 41.8 (C-4), 41.2 (3′-N(CH3)2), 36.3 (9a-CH3), 34.8 (C-2″), 34.3 (C-4′), 33.9 (C-2′), 27.4 (6-CH3), 26.7 (C-8), 21.6 (5′-CH3), 21.9 (8-CH3), 21.6 (3″-CH3), 21.2 (C-14), 18.2 (5″-CH3), 16.2 (12-CH3), 15.0 (2-CH3), 11.2 (C-15), 9.0 (4-CH3), 7.4 (10-CH3).2′-dehydroxy-5″-epi-azithromycin (4):1  in toluene (200 mL) sodium hydrogen carbonate  was added. Then O-phenyl chlorothionoformate  was added in 4 portions (every 20 min). The reaction mixture was heated to 60 oC and stirred for 2 h. Sodium hydrogen carbonate was filtered off and filtrate diluted with fresh toluene (180 mL). Toluene solution was purged with nitrogen (1 h), ACCN  was added, and the reaction mixture heated to 115 °C. Then tris(trimethylsilyl)silane  and stirring at 115 °C continued for 2 h. Toluene was evaporated, the residue dissolved in water (200 mL)/ethyl acetate (100 mL) mixture, and pH adjusted to 5.5. The layers were separated and extraction with ethyl acetate repeated one more time at pH 5.5 and two times at pH 6.5. The water layer was then extracted with dichloromethane at pH 9.7. Dichloromethane layer was dried over anhydrous Na2SO4 and evaporated. The residue was precipitated from acetonitrile to afford 4.3 g of title product as a white foam.To a stirred solution of 38H73N2O11 (M+H+) 733.5214, found 733.5211.HRMS (ES) calc for C1H NMR  [δ/ppm] 7.16 , 4.76 , 4.61 , 4.47 , 4.07 , 3.89 , 3.68 , 3.67 , 3.58 , 3.50 , 3.23 , 3.19 , 2.89 , 2.70 , 2.63 , 2.61 , 2.53 , 2.45 , 2.33 , 2.24 (3′-N(CH3)2, s), 2.04 , 2.01 , 1.98 , 1.89 , 1.87 , 1.80 , 1.70 , 1.63 , 1.60 , 1.52 , 1.36 , 1.30 , 1.21 , 1.20 , 1.20 , 1.16 , 1.15 , 1.10 , 1.07 , 1.02 , 0.87 , 0.86 , 0.86 .13C NMR  [δ/ppm] 178.1 (C-1), 102.1 (C-1′), 100.2 (C-1″), 87.3 (C-5), 83.3 (C-3), 78.2 (C-13), 76.2 (C-11), 75.7 (C-3″), 74.4 (C-12), 73.6 (C-6), 72.1 (C-4″), 71.1 (C-9), 70.0 (C-5″), 68.7 (C-5′), 62.3 (C-10), 60.3 (C-3′), 48.7 (3″-OCH3), 44.4 (C-2), 41.6 (C-7), 41.4 (3′-N(CH3)2), 37.3 (C-4), 37.1 (C-2″), 36.9 (9a-CH3), 35.0 (C-4′), 33.8 (C-2′), 26.6 (C-8), 26.4 (6-CH3), 21.9 (5′-CH3), 21.7 (8-CH3), 20.9 (C-14), 19.8 (3″-CH3), 17.1 (5″-CH3), 16.3 (2-CH3), 16.2 (12-CH3), 11.3 (C-15), 9.7 (4-CH3), 7.6 (10-CH3).Escherichia coli (ATCC25922), Staphylococcus aureus (ATCC13709), Streptococcus pneumoniae (ATCC49619), Streptococcus pyogenes (ATCC700294), Moraxella catarrhalis (ATCC23246) and Haemophilus influenzae (ATCC49247). The minimum inhibitory concentration (MIC) is determined as the lowest concentration of compound that inhibited visible growth.The Clinical and Laboratory Standards (CLSI) recommended procedure  by broth microdilution was used to determine the antimicrobial activity of compounds against E. coli 0111:B4  and incubated overnight. Concentration of IL-6 was determined in cell supernatants by sandwich ELISA using capture and detection antibodies  according to the manufacturer’s recommendations.Splenocytes were isolated from spleens of BALB/cJ mice  resuspended in DMEM supplemented with 1% FBS and seeded in a 24-well plate. Cells were pre-incubated with the test compounds for 2 h, stimulated with 1 μg/mL LPS from 4 were administered intraperitoneally at a dose of 200 mg/kg (b.w.) to 10-week-old male BALB/cJ mice  2 h before intranasal challenge with 2 μg of LPS from E. coli. Then, 24 h after LPS application animals were euthanized and bronchoalveolar lavage was performed. Total number of cells in BALF was counted with a hematological analyzer . Percentages of neutrophils were determined by morphological examination of at least 200 cells on smears prepared by cytocentrifugation  and stained with Kwik-Diff staining set .Vehicle, azithromycin, and compound Compounds were dosed by oral gavage, 2 h prior to the first cigarette each day of the smoke challenge. Then, 2 h after compound administration, mice were placed into a plexiglass box . Cigarette smoke was introduced into the box via a peristaltic pump  set at 40 mL/min. Breathing air was also introduced at a rate of 0.4 L/min. A total of three cigarettes  were given back-to-back with one-minute period between each one when the mice breathed fresh air for one minute. Then, 2 h after the first group of cigarettes, two additional cigarettes were given, again allowing 1 min between each cigarette. This procedure was performed for 2 days. On the third day, mice were euthanized and bronchoalveolar lavage was performed. Slides made on a cytospin were stained with Kwik-Diff for differential cell counts. Total cell counts were performed with a hemocytometer.p < 0.05 in all cases.All values are presented as means ± S.E.M. To define statistically significant differences in cell numbers among vehicle-treated and macrolide-treated mice the data were subjected to one-way ANOVA followed by Dunnett’s Multiple Comparison Test using GraphPad Prism version 5.00 for Windows . The level of significance was set at 50 values for each compound.Test compounds were incubated using a concentration range from 0.1 to 100 µM with recombinant human CYP1A2, 2C9, 2C19, 2D6 and 3A4 (Cypex). The compounds were preincubated at 37 °C for 5 or 10 min with the CYP enzyme isoforms, in the presence of 50 mM phosphate buffer (pH 7.4) and fluorescent probes. The incubations were started by addition of a cofactor solution containing NADP, glucose 6-phosphate and glucose 6-phosphate dehydrogenase. The plates were incubated for 10 min at 37 °C reading the fluorescence signal on a plate reader throughout. The fluorescence data were then used to calculate the ICPharmacokinetic profiles were determined in male Balb/c mice and CD Sprague-Dawley rats , following single oral (p.o.) and single intravenous (i.v.) bolus administration. The compound was administered at a dose of 2 mg/mg i.v. and 10 mg/kg p.o. Blood was obtained by serial blood sampling from the tail vein over a range of time points up to 30 h postdose. Samples were extracted by protein precipitation and subjected to quantitative analysis by LC-MS/MS using compound-specific mass transitions. Drug concentration−time profiles were generated and noncompartmental PK analysis used to generate estimates of half-life, clearance, volume of distribution, and oral bioavailability.


Saponaria officinalis L., commonly known as “Soapwort”, is a rich source of triterpene glycosides; however, the chemical constituents of S. officinalis seeds have not been fully identified. In this study, we conducted a systematic phytochemical investigation of the seeds of S. officinalis and obtained 17 oleanane-type triterpene glycosides (1–17), including seven new glycosides (1–7). The structures of 1–7 were determined based on a detailed analysis of NMR spectroscopic data and chromatographic and spectroscopic analyses following specific chemical transformation. The cytotoxicities of the isolated compounds were evaluated against HL-60 human promyelocytic leukemia cells, A549 human adenocarcinoma lung cancer cells, and SBC-3 human small-cell lung cancer cells. The cytotoxicities of 1, 4, and 10 toward HL-60 cells and SBC-3 cells were nearly as potent as that of cisplatin. Compound 1, a bisdesmosidic triterpene glycoside obtained in good yield, arrested the cell cycle of SBC-3 cells at the G2/M phase, and induced apoptosis through an intrinsic pathway, accompanied by ROS generation. As a result of the mitochondrial dysfunction induced by 1, mitochondria selective autophagy, termed mitophagy, occurred in SBC-3 cells. Dolichos lablab -β-d-glucuronopyranosyl)oxy]-16α-hydroxy-23-oxo-olean-12-en-28-oic acid (8) [O-β-d-galactopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-β-d-glucuronopyranosyl)oxy]-16α-hydroxy-23-oxo-olean-12-en-28-oic acid O-β-d-xylopyranosyl-(1→4)-O-α-l-rhamnopyranosyl-(1→2)-O-[α-l-rhamnopyranosyl-(1→3)]-4-O-acetyl-β-d-fucopyranosyl ester (9) [O-β-d-galactopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-β-d-glucuronopyranosyl)oxy]-16α-hydroxy-23-oxo-olean-12-en-28-oic acid O-β-d-xylopyranosyl-(1→3)-O-β-d-xylopyranosyl-(1→4)-O-α-l-rhamnopyranosyl-(1→2)-O-[4-O-acetyl-β-d-quinovopyranosyl-(1→4)]-β-d-fucopyranosyl ester (saponarioside B) (10) [O-β-d-glucopyranosyl-(1→2)-O-β-d-galactopyranosyl-(1→3)-O-[β-d-glucopyranosyl-(1→6)]-β-d-galactopyranosyl ester (11) [O-β-d-glucopyranosyl-(1→2)-O-β-d-glucopyranosyl-(1→6)-O-[β-d-glucopyranosyl-(1→3)]-β-d-glucopyranosyl ester (vaccaroid A) (12) [O-β-d-glucopyranosyl-(1→2)-O-(6-O-(3-hydroxy-3-methylglutaryl)-β-d-glucopyranosyl)-(1→6)-O-[β-d-glucopyranosyl-(1→3)]-β-d-glucopyranosyl ester (vaccaroside B) (13) [O-β-d-glucopyranosyl-(1→2)-O-β-d-glucopyranosyl-(1→6)-O-[β-d-glucopyranosyl-(1→3)]-β-d-glucopyranosyl ester (segetoside K) (14) [d-xylopyranosyl)oxy]-olean-12-en-23,28-dioic acid 28-O-β-d-glucopyranosyl-(1→2)-O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranosyl ester (saponarioside M) (15) [d-xylopyranosyl)oxy]-olean-12-en-23,28-dioic acid 28-O-β-d-glucopyranosyl-(1→2)-O-β-d-glucopyranosyl-(1→6)-O-[β-d-glucopyranosyl-(1→3)]-β-d-glucopyranosyl ester (saponarioside D) (16) [d-xylopyranosyl)oxy]olean-12-en-23,28-dioic acid 28-O-β-d-glucopyranosyl-(1→2)-O-β-d-glucopyranosyl-(1→6)-O-[β-d-glucopyranosyl-(1→3)]-β-d-glucopyranosyl ester (saponarioside F) (17) +  using high-resolution electrospray ionization time-of-flight mass spectroscopy (HR-ESI-TOF-MS) and 13C-NMR spectral data. In the 1H- and 13C-NMR spectra of 1, the following signals were observed: six tertiary methyl groups , two oxygenated methine groups , an olefinic group , an aldehyde group , an ester carbonyl carbon [δC 175.9 (C-28)], six quaternary carbons , and eight anomeric protons/carbons . Treatment of 1 with 1 M KOH in MeOH yielded 8 and the sugar fraction, and subsequent acid hydrolysis of the sugar fraction using 1 M HCl in 1,4-dioxane/H2O (1:1) afforded d-fucose, d-glucose, d-quinovose, l-rhamnose, and d-xylose. The monosaccharides were identified based on HPLC analysis using a combination of optical rotation and refractive index detectors. As the sugar moieties of 1 were composed of eight monosaccharides, their numerous proton signals overlapped severely, precluding their assignment using conventional NMR analysis. To address this issue, one-dimensional total correlation spectroscopy (1D-TOCSY) and heteronuclear single quantum coherence (HSQC)-TOCSY spectroscopy in addition to 1H-1H correlation spectroscopy (COSY) and HSQC spectroscopy were applied. The 1H-NMR subspectra of individual glycosyl units were obtained by selective irradiation of each anomeric proton signal and other non-overlapping proton signals. Subsequent analysis of the 1H-1H COSY spectrum allowed for the sequential assignment of the proton resonances for the pentaglycosyl moiety attached to C-28 of the aglycone, enabling us to identify their multiplet patterns and coupling constants. The HSQC and HSQC-TOCSY spectra correlated the proton signals with the corresponding one-bond-coupled carbon shifts. The assigned 1H- and 13C-NMR signals indicated that the C-28 pentaglycosyl moiety consisted of a 2,4-disubstituted β-d-fucopyranosyl unit , a 3,4-disubstituted α-l-rhamnopyranosyl unit , a terminal β-d-glucopyranosyl unit , a terminal β-d-xylopyranosyl unit , and a terminal β-d-quinovopyranosyl unit . The β-orientations of the anomeric centers of Fuc, Glc, Xyl, and Qui were determined based on the relatively large 3JH-1,H-2 values. The large 1JH-1,C-1 value (171 Hz) of Rha suggested that the anomeric configuration of Rha was α-oriented -β-d-glucuronopyranosyl)oxy]-16α-hydroxy-23-oxo-olean-12-en-28-oic acid O-β-d-glucopyranosyl-(1→3)-O-[β-d-xylopyranosyl-(1→4)]-O-α-l-rhamnopyranosyl-(1→2)-O-[β-d-quinovopyranosyl-(1→4)]-β-d-fucopyranosyl ester. Although 1 has been described as a possible putative model compound of cell-targeting binding molecules in a patent  were observed in the 1H- and 13C-NMR spectra of 2. These data suggest that 2 is a monoacetylated derivative of 1. The HMBC spectrum of 2 exhibited long-range correlations from H2-6’’’’’’ of Glc (δH 4.91 and 4.65) to the carbonyl carbon of the acetyl moiety (δC 170.9). Comparing the 13C-NMR spectrum of 2 with that of 1, the signal attributable to C-6’’’’’’ of Glc was shifted downfield by 1.8 ppm. The above data indicate that the acetyl group is attached to C-6’’’’’’ of Glc. Therefore, 2 was identified as 3β-[-O-[β-d-xylopyranosyl-(1→3)]-β-d-glucuronopyranosyl)oxy]-16α-hydroxy-23-oxo-olean-12-en-28-oic acid O-(6-O-acetyl-β-d-glucopyranosyl)-(1→3)-O-[β-d-xylopyranosyl-(1→4)]-O-α-l-rhamnopyranosyl-(1→2)-O-[β-d-quinovopyranosyl-(1→4)]-β-d-fucopyranosyl ester.The 3 was obtained as an amorphous solid, and its molecular formula was determined to be C76H120O41 based on the accurate sodium adduct ion at m/z 1711.7191 [M + Na]+  in the HR-ESI-TOF-MS as well as the 13C-NMR spectral data. The 1H- and 13C-NMR spectral features of 3 resembled those of 10, except for those of the sugar moiety attached to C-28 of the aglycone. The 1H-1H COSY, 1D-TOCSY, HSQC, and HSQC-TOCSY spectra indicated that the sugar moiety attached to C-28 of the aglycone consisted of a 2,4-disubstituted β-d-fucopyranosyl unit , a 4-substituted α-l-rhamnopyranosyl unit , a 3-substituted β-d-xylopyranosyl unit , a terminal β-d-glucopyranosyl unit , and a terminal β-d-quinovopyranosyl unit . HMBC correlations were observed between H-1’’’’’’’ of Glc and C-3’’’’’’ of Xyl (II), H-1’’’’’’ of Xyl (II) and C-4’’’’’ of Rha, H-1’’’’’ of Rha and C-2’’’’ of Fuc, H-1’’’’’’’’ of Qui and C-4’’’’ of Fuc, and between H-1’’’’ of Fuc and C-28 (δC 176.0) of the aglycone. Therefore, 3 was identified as 3β-[-O-[β-d-xylopyranosyl-(1→3)]-β-d-glucuronopyranosyl)oxy]-16α-hydroxy-23-oxo-olean-12-en-28-oic acid O-β-d-glucopyranosyl-(1→3)-O-β-d-xylopyranosyl-(1→4)-O-α-l-rhamnopyranosyl-(1→2)-O-[β-d-quinovopyranosyl-(1→4)]-β-d-fucopyranosyl ester.Compound 1H- and 13C-NMR spectra of 4 (C80H124O43) with those of 3 exhibited considerable structural similarity. However, the molecular formula of 4 contained an additional C4H4O2, and signals arising from two acetyl groups were observed in the 1H- and 13C-NMR spectra of 4 . These data suggest that 4 is a diacetylated analog of 3. In the HMBC spectrum of 4, long-range correlations were observed from H-3’’’’’’’’ of Qui (δH 5.60) to an acetyl carbonyl carbon (δC 170.4), and from H-4’’’’’’’’ of Qui (δH 5.05) to another acetyl carbonyl carbon (δC 170.1). Accordingly, 4 was determined to be 3β-[-O-[β-d-xylopyranosyl-(1→3)]-β-d-glucuronopyranosyl)oxy]-16α-hydroxy-23-oxo-olean-12-en-28-oic acid O-β-d-glucopyranosyl-(1→3)-O-β-d-xylopyranosyl-(1→4)-O-α-l-rhamnopyranosyl-(1→2)-O--β-d-fucopyranosyl ester.Comparison of the 1H- and 13C-NMR spectral data of 5 (C53H84O25) suggested that 5 was analogous to 15, including the sugar moieties attached to C-3 and C-28 of the aglycone. The molecular formula of 5 contained an additional oxygen atom in comparison to that of 15 (C53H84O24). Comparing the 13C-NMR spectrum of 5 with that of 15, the methylene carbon signal (δC 21.1) attributed to C-16 in 15 was displaced by the oxygenated methine carbon signal (δC 74.1) in 5. Furthermore, H-16 (δH 5.23) showed a spin-coupling correlation with H2-15 (δH 2.48 and 1.69) in the 1H-1H COSY spectrum of 5, and long-range correlations were observed between H-18 (δH 3.47)/H2-22 (δH 2.26 and 2.01) and C-16 in the HMBC spectrum. The above spectral data indicate the presence of a hydroxy group at C-16 in 5. The configuration of the C-16 hydroxy group was determined to be α based on the nuclear Overhauser effect (NOE) correlations between H-16 and H-15α/H-15β/H-22α in the nuclear Overhauser and exchange spectroscopy (NOESY) spectrum of 5 (5 was deduced to be 16α-hydroxy-3β-[(β-d-xylopyranosyl)oxy]olean-12-en-23,28-dioic acid 28-O-β-d-glucopyranosyl-(1→2)-O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranosyl ester.The rum of 5 . Thus, 56 (C65H102O33) was shown to be essentially analogous to 16, including the sugar moieties attached to C-3 and C-28 of the aglycone, based on the 1H- and 13C-NMR spectral data. However, the molecular formula of 6 contained an additional C6H8O4 compared to that of 16 (C59H94O29), and the 13C-NMR spectra of 6 indicated the presence of a six-atom substituent, which was composed of an ester carbonyl carbon , two methylene carbons , a quaternary carbon nearing a hydroxy group , a carbonyl carbon of a carboxy group , and a methyl carbon . The signals in the 1H-NMR spectrum that could be assigned to this substituent involved two isolated spin systems consisting of two methyl groups  and a deshielded methyl group [δH 1.75 ]. These spectroscopic data indicated that the substituent was 3-hydroxy-3-methylglutaryl (HMG). In the HMBC spectrum of 6, a long-range correlation was observed between H2-6’’’’ (δH 4.92 and 4.68) of Glc (III) and C-1’’’’’’ of the HMG moiety. Accordingly, 6 was identified as 3β-[(β-d-xylopyranosyl)oxy]-olean-12-en-23,28-dioic acid 28-O-β-d-glucopyranosyl-(1→2)-O-(6-O-3-hydroxy-3-methylglutaryl-β-d-glucopyranosyl)-(1→6)-O-[β-d-glucopyranosyl-(1→3)]-β-d-glucopyranosyl ester.Compound 1H- and 13C-NMR spectra of 7 (C65H102O34) with those of 6 suggested that 7 was closely related to 6, and that they both shared the same sugar moieties at C-3 and C-28 of the aglycone. The molecular formula of 7 contained an additional oxygen atom compared to that of 6, and the methylene carbon signal (δC 23.1) assigned to C-16 in 6 was displaced by the oxygenated methine carbon signal (δC 74.0) in 7. Furthermore, H-16 (δH 5.20) exhibited a spin-coupling correlation with H2-15 (δH 2.38 and 1.65) in the 1H-1H COSY spectrum of 7, and long-range correlations were observed between H-18 (δH 3.46)/H2-22 (δH 2.33 and 2.08) and C-16 in the HMBC spectrum of 7. Thus, the presence of a hydroxy group at C-16 of the aglycone, as in 5, was evident. In the NOESY spectrum of 7, NOE correlations were observed between H-16 and H-15α/H-15β/H-22α. Therefore, the configuration at the C-16 hydroxy group was ascertained to be α. Compound 7 was elucidated as 16α-hydroxy-3β-[(β-d-xylopyranosyl)oxy]olean-12-en-23,28-dioic acid 28-O-β- d-glucopyranosyl-(1→2)-O-(6-O-3-hydroxy-3-methylglutaryl-β-d-glucopyranosyl)-(1→6)-O-[β-d-glucopyranosyl-(1→3)]-β-d-glucopyranosyl ester. The configurations of the asymmetric centers of the HMG moiety of 6 and 7 could not be determined presently, owing to insufficient compound quantities.Comparison of the 1–17 against HL-60 cells, A549 cells, and SBC-3 cells were evaluated using the modified 3--2,5-diphenyl-2-tetrazolium bromide (MTT) assay  assay . As show1 exhibited potent cytotoxic activity against SBC-3 cells and was obtained in a good yield. Thus, the apoptosis-inducing activity of 1 in SBC-3 cells was evaluated. Prior to assessing the apoptosis-inducing activity, SBC-3 cells were exposed to either 1 or cisplatin for 24 h to obtain IC50 values. The IC50 values of 1 and cisplatin were calculated to be 7.3 and 8.6 μM, respectively, based on the dose–response curves , and the apoptotic cell ratio was analyzed using flow cytometry. The percentage of early (Q4 area) and late (Q2 area) apoptotic cell populations increased significantly to 10 ± 0.32% and 33 ± 1.5% for 1 compared to 2.0 ± 0.058% and 2.2 ± 0.12%, respectively, for the vehicle control  increased to 35 ± 0.45% from 23 ± 0.48%, which was the value obtained after treatment with the vehicle control (1 phase (P2 area) population of SBC-3 cells treated with 1 for 24 h was 28 ± 0.23%, while that after treatment with the vehicle control was 4.1 ± 0.15%  and executioner caspases  . During observed .1 was confirmed, the mitochondrial membrane potential was evaluated employing the JC-1 assay. When cells are stained with JC-1 dye, at low mitochondrial membrane potentials, the concentration of JC-1 is low and it exists predominantly as a monomer, exhibiting green fluorescence with emission; at high mitochondrial membrane potentials, the dye accumulates in the mitochondria and the dye aggregates yield a red to red-colored emission. SBC-3 cells were treated with 1 for 24 h, and then analyzed using a flow cytometer. As shown in 1, the expression level of Blc-2 was remarkably diminished, and the ratio of Bcl-2/Bax was lower than that of the vehicle control , 100 μM of tert-butyl hydroperoxide , or 1 for 24 h, and then analyzed using a flow cytometer. As depicted in 1-treated groups moved to the right side. Based on these results it can be concluded that ROS generation occurred in SBC-3 cells treated with 1.Reactive oxygen species (ROS) exhibit beneficial or harmful effects on cells and tissues. ROS have been reported to be associated with intrinsic and extrinsic apoptotic pathways ,30. SBC-1 induced depolarization of the mitochondrial membrane potential and ROS generation, we investigated whether mitophagy occurred in SBC-3 cells. SBC-3 cells were treated with 1 or 7.5 μM of carbonyl cyanide m-chlorophenylhydrazone  for 24 h, and then stained with Mtphagy Dye and Lyso Dye. Mtphagy Dye is a fluorescent dye that binds mitochondria and emits red fluorescence, the intensity of which increases under acidic conditions when the mitochondria are fused with lysosomes. Lyso Dye stains lysosomes and emits green fluorescence. SBC-3 cells incubated with 1 emitted intense red fluorescence and exhibited co-localization with lysosomes, while those treated with the control did not  spectrometer  and JNM-ECZ600R/M1  spectrometer  at 300 K. Chemical shifts are recorded as δ values and referenced to tetramethylsilane (TMS) as the internal standard. HR-ESI-TOF-MS data were collected by a Waters Micromass LCT mass spectrometer . Diaion HP-20 porous polymer polystyrene resin , silica gel Chromatorex BW-300 , and ODS silica gel COSMOSIL 75C18-OPN  were adopted for CC. Thin layer chromatography (TLC) analysis was conducted using precoated silica gel 60F254 or RP18 F254S plates . The sample spots were visualized by spraying the TLC plates with H2SO4/H2O (1:9), and then heating. The preparative HPLC system consisted of an LC-20AD pump , a RID-10A detector (Shimadzu), a Rheodyne injection port , and a TSKgel ODS-100Z column . The purities of the isolated compounds were confirmed by NMR spectra and TLC. The following materials and reagents were recruited for the cell culture and cytotoxic assays: SH-1300 Lab microplate reader ; 96-well flat-bottomed and 6-well flat-bottomed plates , Countess II FL automated cell counter (Thermo Fisher Scientific); MCO-170AIC-PJ CO2 incubator ; 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) solution, fetal-bovine serum (FBS), RPMI-1640 medium, minimum-essential medium (MEM), cisplatin, and MTT ; penicillin G sodium salt and streptomycin sulfate, and TrypLE Select (1×) ; paraformaldehyde and phosphate-buffered saline (PBS) ; HL-60 cells (JCRB0085), A549 cells (JCRB0076), and SBC-3 cells (JCRB0818) .Optical rotations were measured using a P-1030 automatic digital polarimeter . IR spectral data were obtained on a Fourier-transform infrared (FT-IR) 620 spectrometer (JASCO). NMR spectral data were collected using a Bruker AVIIIHD-600  in 2010. A voucher specimen was maintained at the herbarium of the Tokyo University of Pharmacy and Life Sciences (KS-2010-003).S. officinalis (1.0 kg) were extracted with MeOH , and then the solvent was removed under reduced pressure using an evaporator. The MeOH extract (50 g) was loaded on Diaion HP-20 column and successively eluted with MeOH/H2O , EtOH (3 L), and EtOAc (3 L). The EtOH eluted fraction (10 g) was subjected to silica gel CC eluted with a stepwise gradient mixture of CHCl3/MeOH  to obtain four fractions [Fractions (Frs.) A–D]. Fr. A was divided by ODS silica gel CC eluted with MeCN/H2O (2:5) and MeOH/H2O (3:2), and preparative ODS HPLC using MeCN/H2O (7:13) to afford 13 (3.0 mg) and 15 (26 mg). Fr. B was subjected to silica gel CC eluted with CHCl3/MeOH/H2O (7:4:1), ODS silica gel CC eluted with MeOH/H2O (7:3) and MeCN/H2O , and preparative ODS HPLC using MeOH/H2O  and MeCN/H2O (3:7) to yield 5 (7.1 mg), 6 (2.5 mg), and 12 (4.1 mg). Fr. C was separated by silica gel CC eluted with CHCl3/MeOH/H2O (7:4:1), ODS silica gel CC eluted with MeOH/H2O (13:7) and MeCN/H2O , and preparative ODS HPLC using MeOH/H2O (13:7) and MeCN/H2O (3:7) to furnish 7 (3.2 mg), 10 (84 mg), 11 (4.9 mg), and 14 (2.0 mg). Fr. D was chromatographed on silica gel eluted with CHCl3/MeOH/H2O (7:4:1), and ODS silica gel eluted with MeOH/H2O  and MeCN/H2O  to obtain 1 (184 mg), 2 (38 mg), 3 (4.6 mg), 4 (15 mg), 8 (19 mg), 9 (49 mg), 16 (23 mg), and 17 (55 mg).The seeds of 1: Amorphous solid; [α]D25 –4.9 ; IR (film) νmax: 3398 (OH), 2929 (CH), 1730 (C = O) cm−1; HR-ESI-TOF-MS m/z: 1711.7161 [M + Na]+ . 1H-NMR spectral data for the aglycone moiety : δH 9.91 , 5.50 , 5.11 , 4.06 , 1.73 , 1.46 , 1.10 , 0.93 , 0.92 , 0.84 . 1H-NMR spectral data for the sugar moiety, see 13C-NMR spectral data, see Compound 2: Amorphous solid; [α]D25 –16.7 ; IR (film) νmax: 3397 (OH), 2933 (CH), 1730 (C = O) cm−1; HR-ESI-TOF-MS m/z: 1753.7300 [M + Na]+ . 1H-NMR spectral data for the aglycone moiety : δH 9.92 , 5.53 , 5.12 , 4.09 , 1.72 , 1.49 , 1.17 , 0.93 , 0.90 , 0.89 . 1H-NMR spectral data for the sugar and acyl moieties, see 13C-NMR spectral data, see Compound 3: Amorphous solid; [α]D25 –5.8 ; IR (film) νmax: 3398 (OH), 2925 (CH), 1730 (C = O) cm−1; HR-ESI-TOF-MS m/z: 1711.7191 [M + Na]+ . 1H-NMR spectral data for the aglycone moiety : δH 9.91 , 5.53 , 5.15 , 4.05 , 1.73 , 1.44 , 1.03 , 0.96 , 0.93 , 0.81 . 1H-NMR spectral data for the sugar moiety, see 13C-NMR spectral data, see Compound 4: Amorphous solid; [α]D25 + 3.6 ; IR (film) νmax: 3398 (OH), 2929 (CH), 1726 (C = O) cm−1; HR-ESI-TOF-MS m/z: 1771.7439 [M–H]− . 1H-NMR spectral data for the aglycone moiety : δH 9.91 , 5.53 , 5.12 , 4.05 , 1.74 , 1.73 , 1.44 , 0.97 , 0.93 , 0.81 . 1H-NMR spectral data for the sugar and acyl moieties, see 13C-NMR spectral data, see Compound 5: Amorphous solid; [α]D25 –76.5 ; IR (film) νmax: 3358 (OH), 2925 (CH), 1712 (C = O) cm−1; HR-ESI-TOF-MS m/z: 1143.5189 [M + Na]+ . 1H-NMR spectral data for the aglycone moiety : δH 5.59 , 5.23 , 4.64 , 1.76 , 1.56 , 1.11 , 0.99 , 0.98 , 0.92 . 1H-NMR spectral data for the sugar moiety, see 13C-NMR spectral data, see Compound 6: Amorphous solid; [α]D25 + 8.5 ; IR (film) νmax: 3377 (OH), 2925 (CH), 1717 (C = O) cm−1; HR-ESI-TOF-MS m/z: 1433.6193 [M + Na]+ . 1H-NMR spectral data for the aglycone moiety : δH 5.39 , 4.60 , 1.55 , 1.17 , 1.04 , 0.95 , 0.91 , 0.85 . 1H-NMR spectral data for the sugar and acyl moieties, see 13C-NMR spectral data, see Compound 7: Amorphous solid; [α]D25 + 4.4 ; IR (film) νmax: 3377 (OH), 2925 (CH), 1730 (C = O) cm−1; HR-ESI-TOF-MS m/z: 1449.6165 [M + Na]+ . 1H-NMR spectral data for the aglycone moiety : δH 5.57 , 5.20 , 4.64 , 1.75 , 1.56 , 1.08 , 1.05 , 0.99 , 0.93 . 1H-NMR spectral data for the sugar and acyl moieties, see 13C-NMR spectral data, see Compound 1 followed acid hydrolysis: Compound 1 (11 mg) was treated with 1 M KOH in MeOH at 80 °C for 90 min. The reaction solution was neutralized by passing through an Amberlite 120B  column eluted with EtOH, and was separated by ODS silica gel CC eluted with MeCN/H2O (2:3) to obtain 8 (5.8 mg) and a residue. The residue was dealt with 1 M HCl  at 95 °C for 2 h. The reaction mixture was neutralized by passing through an Amberlite IRA96SB (Organo) column eluted with EtOH to afford a sugar fraction. The sugar fraction was analyzed by HPLC under the following conditions: solvent, MeCN/H2O (17:3); flow rate, 0.5 mL/min; column temperature, 40 °C; pump, Tosoh DP-8020; column, Capcell Pak NH2 ; detector, Shodex OR2 . d-fucose, d-quinovose, d-xylose, d-glucose, and l-rhamnose were identified by comparing their retention times (tR) and optical rotations with those of authentic samples: d-fucose , d-quinovose , d-xylose , d-glucose , and l-rhamnose .Alkaline treatment of l-glutamine, 100 unit/mL penicillin G sodium salt, and 100 μg/mL streptomycin sulfate. The cells were incubated at 37 °C in a 5% CO2/air atmosphere. HL-60 cells (4 × 104 cells/mL), A549 cells (1 × 104 cells/mL), and SBC-3 cells (2 × 104 cells/mL) were seeded in a 96-well flat-bottomed plate. After pre-incubation for 24 h, 4 μL of EtOH/H2O (1:1) solution containing each test sample was added and incubated for 72 h; 4 μL of EtOH/H2O (1:1) solution was added to the control cells. Cell viability was evaluated using the modified MTT reduction assay method. After terminating the cell culture, 10 μL of MTT solution (5 mg/mL in PBS) was added to each well, and the plate was further incubated. After 4 h of incubation, MTT formazan was dissolved in dimethyl sulfoxide (DMSO) and the absorbance was measured at 405 nm. A dose–response curve was plotted for 1–5, 9, and 10, which inhibited cell growth by more than 50% at sample concentrations of 50 μM, and the concentrations at which 50% inhibition (IC50) of cell growth occurred were calculated. The cell growth inhibition of SBC-3 cells (5 × 104 cells/mL) treated with 1 for 24 h was evaluated using the same method as above.HL-60 cells were maintained in RPMI-1640 medium, and A549 cells and SBC-3 cells in MEM containing 10% heat-inactivated FBS supplemented with 5 cells/mL) were harvested in a 6-well flat-bottomed plate and pre-incubated for 24 h. Then, SBC-3 cells were treated with either EtOH/H2O (1:1) (control), 10 μM of cisplatin, or 10 μM of 1. After 24 h of incubation, SBC-3 cells were detached using TrypLE Select and stained with 1 × Annexin V binding buffer containing Annexin V-FITC and PI at 28 °C for 15 min according to the manufacturer’s protocol . Flow cytometry analysis was conducted using a BD FACSCelesta flow cytometer .SBC-3 cells (5 × 105 cells/mL) were cultured in a 6-well flat-bottomed plate and treated with either EtOH/H2O (1:1) (control), 10 μM of cisplatin, or 10 μM of 1. After 12 h or 24 h of incubation, SBC-3 cells were detached using TrypLE Select and fixed with EtOH/PBS (7:3) at −20 °C overnight. After removal of EtOH/PBS (7:3), the cells were stained with FxCycle PI/RNase Staining Solution (Thermo Fisher Scientific) at 28 °C for 15 min. Cell cycle distribution was analyzed using a BD FACSCelesta flow cytometer.SBC-3 cells (5 × 105 cells/mL) were seeded into a 6-well flat-bottomed plate and treated with either EtOH/H2O (1:1) (control), 10 μM of cisplatin, or 10 μM of 1. After 24 h of incubation, SBC-3 cells were detached using TrypLE Select. The SBC-3 cell proteins were extracted on ice for 30 min using RIPA buffer  containing Pierce Protease and Phosphatase inhibitor Mini Tablet (Thermo Fisher Scientific). Then, SBC-3 cells were centrifuged at 10,000× g for 30 min, and the supernatant protein solution was collected. Protein quantification was performed using the TaKaRa BCA Protein Assay Kit . Then, sample protein solutions were loaded on NuPAGE 4–12% Bis-Tris Gel (Thermo Fisher Scientific), and electrophoresis was performed using a WSE-1165 RapidasMinislab electrophoresis tank . Proteins were transferred from the gel to a polyvinylidene difluoride (PVDF) membrane using a Protein Transfer Kit for Semidry  and a WSE-4025HorizeBLOT 2M (ATTO). The PVDF membrane was then blocked using a Blocking One solution  at 28 °C. After 30 min, the PVDF membrane was reacted with the following primary antibodies dissolved in Can Get Signal Immunoreaction Enhancer Solution 1  at 4°C overnight: β-actin , Caspase-3 , Caspase-8 , Caspase-9 , PARP , Bax , and Bcl-2 . The PVDF membrane was rinsed with TBS-T buffer (1 ×), and treated with secondary antibodies  dissolved in Can Get Signal Immunoreaction Enhancer Solution 2 (TOYOBO) at 28 °C for 1 h. Finally, the PVDF membrane was reacted with ECL Prime Western Blotting Detection Reagents , and detected using the LAS-3000 luminescent image analyzer .SBC-3 cells (5 × 105 cells/mL) were seeded into a 6-well flat-bottomed plate. SBC-3 cells were treated with either EtOH/H2O (1:1) (control), 10 μM of cisplatin, or 10 μM of 1. After 24 h of incubation, SBC-3 cells were detached using TrypLE Select and stained with JC-1 dye at 37 °C for 45 min according to the manufacturer’s protocol. Flow cytometry analysis was performed using a BD FACSCelesta flow cytometer.Mitochondrial membrane potential was assayed using the JC-1 MitoMP Detection Kit . SBC-3 cells (5 × 105 cells/mL) were cultured in a 6-well flat-bottomed plate and preincubated for 24 h. Then, SBC-3 cells were treated with either EtOH/H2O (1:1) (control), 2.5 mM of NAC, 100 μM of TBHP, or 10 μM of 1 for 24 h. SBC-3 cells were detached using TrypLE Select and stained with CellROX Green reagent at 37 °C for 1 h according to the manufacturer’s protocol. ROS levels were measured using a BD FACSCelesta flow cytometer.The ROS generation assay was performed using the CellROX Green Flow Cytometer Assay Kit (Thermo Fisher Scientific). SBC-3 cells (5 × 105 cells/mL) were seeded into a 6-well flat-bottomed plate. After pre-incubation, SBC-3 cells were washed with Hank’s balanced salt solution  and treated with 100 nM Mtphagy Dye at 37 °C for 30 min. Then, SBC-3 cells were rinsed with HBSS and treated with either EtOH/H2O (1:1) (control), 7.5 μM of CCCP, or 10 μM of 1 for 24 h. After the supernatant was removed and SBC-3 cells were washed with HBSS, the cells were incubated with 1 μM Lyso Dye at 37 °C for 30 min and observed using a BZ-X710 All-in-One Fluorescence Microscope .Mitophagy detection was performed using the Mitophagy Detection Kit (DOJINDO). SBC-3 cells (5 × 10p) value of less than 0.001 or 0.01 was distinguished to represent a statistically significant difference.Statistical analysis was conducted using a one-way analysis of variance (ANOVA) followed by Dunnett’s test. A probability , including seven (1–7) previously unreported compounds, were isolated and identified. Compounds 1–5, 9, and 10 showed cytotoxicity against HL-60 cells, A549 cells, and SBC-3 cells with IC50 values in the range of 0.57–21 μM. The cytotoxicities of 1, 4, and 10 toward HL-60 cells and SBC-3 cells were almost as potent as that of cisplatin. Compound 1, a bisdesmosidic triterpene glycoside obtained in good yield, arrested the cell cycle of SBC-3 cells at the G2/M phase, and induced apoptosis through an intrinsic pathway; activation of caspase-8, -9, and -3, cleavage of PARP, reduction in the Bcl-2/Bax ratio, depolarization of mitochondrial membrane potential, and ROS generation were observed after treatment of SBC-3 cells with 1. As a result of mitochondrial dysfunction induced by 1, mitochondrial selective autophagy, termed mitophagy, occurred in SBC-3 cells. Compound 1 is a potential hit compound for the development of drugs against small-cell lung cancer.In this study, the chemical constituents of


Breonadia salicina (Vahl) Hepper and J.R.I. Wood is widely used in South Africa and some other African countries for treatment of various infectious diseases such as diarrhea, fevers, cancer, diabetes and malaria. However, little is known about the active constituents associated with the biological activities. This study is aimed at exploring the metabolomics profile and antioxidant constituents of B. salicina. The chemical profiles of the leaf, stem bark and root of B. salicina were comprehensively characterized using proton nuclear magnetic resonance (1H-NMR) spectroscopy and ultra-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). The antioxidant activities of the crude extracts, fractions and pure compounds were determined using the DPPH  free radical scavenging and reducing power assays. A total of 25 compounds were tentatively identified using the UPLC-QTOF-MS. Furthermore, the 1H-NMR fingerprint revealed that the different parts of plant had differences and similarities among the different crude extracts and fractions. The crude extracts and fractions of the root, stem bark and leaf showed the presence of α-glucose, β-glucose, glucose and fructose. However, catechin was not found in the stem bark crude extracts but was found in the fractions of the stem bark. Lupeol was present only in the root crude extract and fractions of the stem bark. Furthermore, 5-O-caffeoylquinic acid was identified in the methanol leaf extract and its respective fractions, while the crude extracts and fractions from the root and dichloromethane leaf revealed the presence of hexadecane. Column chromatography and preparative thin-layer chromatography were used to isolate kaempferol 3-O--glucuronide, lupeol, d-galactopyranose, bodinioside Q, 5-O-caffeoylquinic acid, sucrose, hexadecane and palmitic acid. The crude methanol stem bark showed the highest antioxidant activity in the DPPH  free radical scavenging activity with an IC50 value of 41.7263 ± 7.6401 μg/mL, whereas the root crude extract had the highest reducing power activity with an IC0.5 value of 0.1481 ± 0.1441 μg/mL. Furthermore, the 1H-NMR and UPLC-QTOF-MS profiles showed the presence of hydroxycinnamic acids, polyphenols and flavonoids. According to a literature survey, these phytochemicals have been reported to display antioxidant activities. Therefore, the identified hydroxycinnamic acid (caffeic acid), polyphenol (ellagic acid) and flavonoids (catechin and (epi) gallocatechin) significantly contribute to the antioxidant activity of the different parts of plant of B. salicina. The results obtained in this study provides information about the phytochemistry and phytochemical compositions of Breonadia salicina, confirming that the species is promising in obtaining constituents with medicinal potential primarily antioxidant potential. Breonadia salicina (Vahl) Hepper and J.R.I. Wood is a plant species belonging to the family Rubiaceae. The Rubiaceae family consists of ~13,500 species in ~620 genera and is one of the largest of the angiosperm family. It is divided into four subfamilies: Cinchonoideae, Ixoroideae, Antirheoideae and Rubioideae −), peak 3 (m/z 300.99929 [M-H]−), peak 4 (m/z 447.05763 [M-H]−) and peak 5 (m/z 289.07225 [M-H]−) were unambiguously confirmed as 4′-O-methyellagic acid-3-O-α-L-rhamnopyranoside, ellagic acid, ellagic acid-rhamnopyranoside isomer I and catechin, respectively. Furthermore, peaks 2–5 showed fragment ions at m/z 315, 299; m/z 242, 174; m/z 300.99898 and m/z 245.08155, respectively [m/z 485.32825 [M-H]−), peak 7 (m/z 458.33667 [M-H]−), peak 8 (m/z 499.34385 [M-H]−), peak 9 (m/z 453.33834 [M-H]−) and peak 10 (m/z 499.37155 [M-H]−) were identified as hydroxyglycyrrhetinic acid (C30H46O5), neotigogenin acetate (C29H46O4), 25-hydroxy-3-epi-dehydrotumulosic acid (C31H48O5), micromeric acid (C30H46O3) and 3-acetylursolic acid (C32H50O4), respectively. The MS-MS spectrum of peaks 6, 7, 8 and 10 yielded fragment ions at m/z 485, 441; m/z 503, 457; m/z 455.35408; and m/z 497.36498, respectively. A data comparison with the literature confirmed the identification of these compounds [m/z 305.06698 [M-H]−), 4-O-methylgallic acid (m/z 183.02975 [M-H]−) and myricetin 3-O-glucoside (m/z 479.08529 [M-H]−), respectively. The MS-MS spectrum of peaks 12 and 13 revealed fragment ions at m/z 184.03304 and m/z 480.08721, respectively [m/z 455.35397 [M-H]−) and peak 15 (m/z 487.33699 [M-H]−) were identified as ursolic acid (C30H48O3) and asiatic acid (C30H48O5), respectively, whereas peak 15 showed a fragment ion at m/z 485.32825 [19H14O12) as ellagic acid pentoside (m/z 433.04194 [M-H]−) and peak 17  as gallic acid (m/z 170.01742 [M-H]−) [− ion at m/z 533.17341 (19H34O17 [m/z 353.08839 [M-H]−) was assigned to chlorogenic acid due to the presence of characteristic product ion at m/z 191.05585 [quinic acid-H]− − ion peak at m/z 613.08129 matched with the molecular formula of C28H22O16. The 1H-NMR spectrum showed six aromatic protons signals at δH 8.25 ppm , δH 6.70 ppm , δH 6.39 ppm , and δH 6.25 ppm , respectively. Total assignment was done by a close examination of the 1D-NMR (1H-NMR and 13C-NMR), HRMS and literature data.Kaempferol 3-13C-NMR spectrum of lupeol (2) revealed the presence of 30 carbon atom signals. Furthermore, the carbon atoms at δc 108.7 ppm and δc 150.5 ppm assigned to carbons at positions 29 and 20, respectively, has showed the presence of C=C group. Moreover, the 13C-NMR spectrum displayed that the carbon signal at δc 78.2 ppm has been attributed to C-3. The 1H-NMR spectrum revealed the presence of seven tertiary methyl protons at δH 0.74 ppm , δH 0.77 ppm , δH 0.87 ppm , δH 0.96 ppm , δH 0.98 ppm , δH 1.02 ppm  and δH 1.71 ppm . A multiplet of one proton at δH 2.23 ppm  has been assigned to 19-H while 3-H proton displayed a multiplet at δH 3.02 ppm . A pair of broad singlets at δH 4.72 ppm  and δH 4.61 ppm  was indicative of olefinic protons at (29a-H and 29b-H). This supported the double bond between methylene carbon (C-29) and quaternary carbon (C-20). Finally, data comparison with the literature confirmed the isolation of lupeol, a pentacyclic triterpenoid.The d-Galactopyranose (3), a monosaccharide sugar showed a [M-H]− ion peak at m/z 179.0557 matched with the molecular formula of C6H12O6. The 1H-NMR spectrum showed the anomers proton at δH 5.16 ppm (H-1α), δH 4.52 ppm (H-1β) and δH 3.32 ppm (H-3β) give rise to peaks positioned outside this envelope of ring protons atom signals between δH 4.52 ppm (H-1β) and δH 5.16 ppm (H-1α). According to a literature report, the α anomeric proton atom signals of d-sugars are mainly found between δH 4.9 ppm and δH 5.5 ppm, whilst the β proton atom signals are mostly found between δH 4.3 ppm and δH 4.7 ppm [ 4.7 ppm . A spect4) showed a [M-H]− ion peak at m/z 487.3448 [M-Glc-Rha-Xyl-H]− matched with the molecular formula of C47H76O18. The 1H-NMR spectrum displayed six methyl signals at δH 1.14 ppm , δH 1.19 ppm , δH 1.04 ppm , δH 0.95 ppm , δH 0.82 ppm  and δH 0.82 ppm . Moreover, the JH1, H2 coupling constants of two anomeric proton signals at δH 5.12 ppm , proposed that the β anomeric configuration for the xylopyranosyl. Furthermore, the HSQC spectrum showed correlation with carbons at δC 17.4 ppm (C-26), δC 17.6 ppm (C-25), δC 13.0 ppm (C-24), and δC 23.0 ppm (C-30) with protons at assigned to δH 1.14 ppm (26-H), δH 1.04 ppm (25-H), δH 0.95 ppm (24-H), and δH 0.86 ppm (30-H), respectively. In addition, the HMBC spectrum showed the signal at δH 5.27 ppm , assigned to the carbon at δC 122.0 ppm (C-12) which coupled with δC 144.1 ppm (C-13), indicated the presence of a double bond. The HMBC spectrum also showed correlations from the anomeric carbon signal at position C-1′ (δC 107.8 ppm) with 2′-H (δH 3.80 ppm), and 5′-H (δH 4.62 ppm), suggested the presence of D-xylopyranose.Bodinioside Q (O-Caffeoylquinic acid (5) showed the presence of 16 carbons, including two carbonyl groups at δC 145.4 ppm and δC 166.5 ppm, corresponding to carbons at positions 7 and 9′, respectively, on the 13C-NMR spectrum. Furthermore, the 13C-NMR spectrum presented two aromatic carbons bonded to hydroxyl groups at δC 148.5 ppm and δC 145.8 ppm corresponding to carbons at positions 4′ and 3′, and two olefinic carbons at δC 180.2 ppm and δC 113.6 ppm corresponding to carbons at positions 7 and 8′. The 1H-NMR spectrum showed two ortho-coupled doublets at δH 6.79 ppm and δH 6.95 ppm corresponding to protons at positions 5′ and 6′. A broad singlet at δH 7.06 ppm has been assigned to a proton at position 2′, confirming the presence of a tri-substituted aromatic ring. Moreover, the 1H-NMR revealed two doublets at corresponding to protons at δH 6.21 ppm and δH 7.52 ppm positions 7′ and 8′, indicating the presence of trans-di-substituted ethylene moiety in the compound. These assignments are in good agreement with the structure of 5-O-caffeoylquinic acid.5-6) displayed a molecular peak at m/z 341.1073, which is in agreement with the founded molecular formula of C12H22O11. The COSY spectrum showed coupling between protons H-3 (δH 3.72 ppm) with H-2 (δH 3.50 ppm) and H-4 (δH 3.39 ppm), H-2 (δH 3.50 ppm) and H-1 (δH 5.41 ppm), respectively. The only coupling constant value released for the protons of the fructoside ring was between H-4′ (δH 4.02 ppm) with H-5′ (δH 3.86 ppm) and H-6′ (δH 3.80 ppm). Furthermore, the HMBC spectrum revealed correlations between H-1′ (δH 3.63 ppm) with C-5′ (δC 81.6 ppm), H-4′ (δH 4.02 ppm) with C-6′ (δC 62.5 ppm), H-1 (δH 5.41 ppm) with C-3 (δC 72.2 ppm), H-5′ (δH 3.86 ppm) with C-2′ (δC 103.8 ppm), respectively. Moreover, the glucopyranosyl H-1′ (δH 3.63 ppm) gave a strong correlation to fructosyl C-2 (δC 70.2 ppm) establishing the linkage between the anomeric carbon of the glucopyranosyl residue and that of the fructoside one. Total assignment was done by a close examination of the 1D-NMR (1H-NMR and 13C-NMR), 2D- NMR (COSY and HMBC), HRMS and literature data.Sucrose (7) showed a triplet at δH 0.89 ppm  corresponding to protons at position 1 and 16 on the 1H-NMR spectrum. The 1H-NMR spectrum exhibited a long peak of multiplet at δH 1.28 ppm corresponding to protons assigned to 2-H, 3-H, 4-H, 5-H, 6-H, 7-H, 8-H, 9-H, 10-H, 11-H, 12-H, 13-H, 14-H and 15-H of the long chain, was evident as a multiplet that integrated for one proton. A single long peak multiplet at δC 29.3–29.7 ppm appeared from C-4, C-5, C-6, C-7, C-8, C-9, C-10, C-11, C-12 and C-13 on the 13C-NMR spectra. Furthermore, hexadecane (7) was isolated as white crystals with a melting point of 16–18 °C. Therefore, the spectroscopic data and physical property comparison with the literature confirmed the isolation of hexadecane.Hexadecane (8) revealed absorptions at 3436.00 cm−1 which is characteristic of O-H stretching, 2849.51 cm−1 which is due to aliphatics (CH3) stretching, 1703.71 cm−1 due to carbonyl (C=O) stretching and 2917.27 cm−1 due to overtone of the long chain (CH2)n bending frequency. The 1H-NMR spectrum showed a long peak of multiplet at δH 1.23 ppm assigned to protons at position 4-H, 5-H, 6-H, 7-H, 8-H, 9-H, 10-H, 11-H, 12-H and 13-H of the long chain, was evident as a multiplet that integrated for one proton. Furthermore, the 1H-NMR spectrum displayed a triplet at δH 2.27 ppm  and δH 0.83 ppm  corresponding to protons at positions 2 and 16, respectively. The 13C-NMR has shown double bonds of the acidic group (–COOH) at δC 178.4 ppm assigned to C-1 as singlet, the alkane carbon C-16 appeared at δC 14.1 ppm as a triplet. A single long peak multiplet at δC 29.0 ppm appeared from C-4 to C-13, whereas an alpha and beta carbon to the acidic group (-COOH) appeared at δC 31.9 ppm and δC 34.0 ppm corresponding to carbons at position 3 and 2 as doublets respectively. Furthermore, an alpha and beta carbon to the alkane carbon appeared as doublets at δC 22.6 ppm and δC 24.7 ppm corresponding to carbons at position 15 and 14.The infrared (IR) spectrum of palmitic acid  had the highest reducing power activity with an IC0.5 value of 0.1481 ± 0.1441 μg/mL, as shown in 50 values of 3.965 ± 0.067 mol TE/mol, 2.939 ± 0.037 mol TE/mol and 0.965 ± 0.015 mol TE/mol, respectively, and IC0.5 values of 0.793 ± 0.004 mol TE/mol, 1.032 ± 0.007 mol TE/mol and 1.018 ± 0.004 mol TE/mol, respectively ,,1 -glucuronide (1): Yellow powder, m.p. 215–217 °C . 1H-NMR : δH ppm: 8.25 , 7.00 , 6.70 , 6.39 , 6.25 , 3.21 , 3.19 . 13C-NMR : δC ppm: 168.0 (C-7″), 163.8 (C-7), 156.8 (C-5), 148.4 (C-2), 146.0 , 140.9 (C-4″), 132.8 (C-3), 130.2 , 121.0 , 115.4 , 109.1 , 106.3 (C-1‴), 99.4 (C-6), 94.2 (C-8), 79.4 (C-3‴), 79.2 (C-5‴), 74.0 (C-2‴), 71.25 (C-4‴). HRMS [M-H]−. m/z 613.08; calcd. for C28H22O16: 613.08129 (Kaempferol 3-curonide : Yellow 2): White powder, m.p. 217–218 °C : 3400 (O-H) cm−1, 2920.18 cm−1 (CH2), 2850.91 cm−1 (CH3) and 1686.74 cm−1 (C=C). 1H-NMR : δH (ppm): 4.72 , 4.61 , 3.02 , 2.23 , 1.91 , 1.71 , 1.52 , 1.43 , 1.30 , 1.02 , 0.98 , 0.96 , 0.87 , 0.77 , 0.74 . 13C-NMR : δH (ppm): 150.5 (C-20), 108.7 (C-29), 78.2 (C-3), 56.1 (C-5), 50.5 (C-9), 48.2 (C-18), 42.1 (C-14), 40.5 (C-8), 38.5 (C-4), 38.2 (C-1), 37.3 (C-13), 36.2 (C-10), 36.0 (C-16), 34.1 (C-7), 31.6 (C-21), 30.2 (C-23), 29.3 (C-2), 27.9 (C-15), 25.0 (C-12), 22.3 (C-11), 20.1 (C-30), 18.0 (C-6), 17.9 (C-28), 14.6 (C-26), 13.7 (C-24), 13.0 (C-27). HRMS [M-H]−: m/z 455.72; calcd. for C28H22O16: 455.3618 (3β-Lup-20(29)-en-3-ol . IR: νmd-Galactopyranose (3): Brown sticky oil. 1H-NMR : δH ppm: 5.16 , 4.52 , 4.41 , 4.10 , 3.91 , 3.89 , 3.71 , 3.32 . 13C-NMR : δC ppm: 97.8 (C-1β), 92.4 (C-1α), 76.4 (C-5β), 74.1 (C-3β), 73.4 (C-2β), 71.7 (C-2α), 70.3 (C-4β), 70.3 (C-3α), 70.3 (C-4α), 69.7 (C-5α), 63.1 (C-6β), 62.7 (C-6α). HRMS [M-H]−: m/z 179.156; calcd. for C6H12O6: 179.0557 -β-d-glucopyranoside : Brown amorphous solid, m.p. 203–205 °C . 1H-NMR : δH (ppm): 5.27 , 4.48 , 3.12 , 2.85 , 2.29 , 2.20 , 1.95 , 1.70 , 1.62 , 1.46 , 1.30 , 1.27 , 1.19 , 1.14 , 1.04 , 0.95 , 0.92 , 0.86 , 0.82 ; 3-O-sugar: Xyl: 5.12 , 4.62 , 4.10 , 3.80 , 28-O-sugar: Glc: 4.14 , 4.12 , 4.05 ; Rha: 1.67 . 13C-NMR : δC (ppm): 145.1 (C-13), 122.0 (C-12), 108.5 (C-28), 92.5 (C-3), 68.2 (C-2), 63.7 (C-23), 50.7 , 46.7 (C-1), 46.2 (C-5), 45.8 (C-19), 44.5 (C-14), 42.0 (C-18), 41.6 (C-4), 41.3 (C-8), 37.6 (C-10), 34.5 (C-21), 33.5 , 30.4 (C-27), 30.2 (C-20), 29.1 (C-15), 23.9 (C-11), 23.0 (C-30), 22.7 (C-16), 20.6 (C-6), 17.6 (C-25), 17.4 (C-26), 13.0 (C-24); 3-O-sugar: Xyl 107.8 (C-1′), 81.8 (C-3′), 76.0 (C-2′), 71.5 (C-4′), 68.0 (C-5′); 28-O-sugar: Glc 96.7 (C-1″), 82.8 (C-3″), 81.8 (C-5″), 75.6 (C-2″), 70.4 (C-4″), 61.4 (C-6″); Rha 101.7 (C-1‴), 74.8 (C-4‴), 73.5 (C-3‴), 72.4 (C-2‴), 69.8 (C-5‴), 19.4 (C-6‴). HRMS [M-H]−: m/z 487 [M–Glc–Rha–Xyl–H]−; calcd. for C47H76O18: m/z: 487.3448 [M–Glc–Rha–Xyl–H]− (3-O-Caffeolquinic acid (5): Green powder, m.p. 207–209 °C : quinic moiety δH ppm: 5.24 , 3.66 , 3.32 , 2.29–1.93 , 1.70–1.51 ; caffeoyl moiety δH ppm: 7.52 , 7.06 , 6.95 , 6.79 , 6.21 . 13C-NMR : quinic moiety δC ppm: 76.4 (C-1), 74.3 (C-5), 70.7 (C-4), 68.8 (C-3), 41.8 (C-6), 36.6 (C-2); caffeoyl moiety δC: 180.2 (COO-), 166.5 (C-9′), 148.5 (C-4′), 145.8 (C-3′), 145.4 (C-7′), 126.2 (C-1′), 121.6 (C-6′), 115.1 (C-5′), 113.6 (C-2′), 113.6 (C-8′) ppm. HRMS [M-H]−: m/z 367.311; calcd. for C16H18O9: 367.1107 . 1H-NMRO-α-d-Glucopyranosyl-(1→2)-β-d-fructofuranoside : Brown sticky oil. 1H-NMR : δH ppm: 5.41 , 4.14 , 4.02 , 3.86 , 3.83 , 3.81 , 3.80 , 3.72 , 3.63 , 3.50 , 3.39 . 13C-NMR : δC ppm: 103.8 (C-2′), 92.2 (C-1), 81.6 (C-5′), 77.8 (C-3′), 72.9 (C-4′), 72.2 (C-3), 72.0 (5), 70.2 (C-2), 67.9 (C-4), 62.5 (C-6′), 61.2 (C-1), 60.7 (C-6). HRMS [M-H]−: m/z 341.30; calcd. for C12H22O11: 341.1073 : White crystals, m.p. 16–18 °C : δH (ppm): 1.28 , 1.28 , 1.28 , 1.28 , 1.28 , 1.28 , 1.28 , 1.28 , 1.28 , 1.28 , 1.28 , 1.28 , 1.28 , 1.28 , 0.89 . 13C-NMR : δC (ppm): 31.95 , 29.3–29.7 , 22.7 , 14.1 , . 1H-NMR8): Green amorphous solid, m.p. 61–62 °C : 3436.00 cm−1 (O-H), 2917.27 cm−1 (CH2), 2849.51 (CH3) and 1703.71 cm−1 (C=O).1H-NMR : δH (ppm): 2.27 , 1.98 , 1.57 , 1.23 , 0.83 . 13C-NMR : δC (ppm): 178.4 (C-1), 34.0 (C-2), 31.9 (C-3), 29.0–29.6 , 24.7 (C-14), 22.6 (C-15), 14.1 (C-16). HRMS [M-H]−: m/z 255.40; calcd. for C16H32O2: 255.23295 . IR: νmTM tuneable microplate reader at 517 nm.The DPPH free radical scavenging activity of the crude extracts, fractions and pure compounds were determined according to the modified spectrophotometric method of Motamed and Naghibi (2010) . A solutThe percentage radical scavenging was determined by using the following formula:3Fe (CN)6] solution was added in each well. The mixture was incubated for 20 minutes at 45 °C. After incubation, a volume of 50 μL of 10% trichloroacetic acid solution was added to each well. An 80 μL volume of each mixture was transferred to another 96-well plate containing a volume of 80 μL of distilled water and 16 μL ferric chloride (0.1% w/v). Absorbance was determined using a VersaMax™ tuneable microplate reader at 700 nm.The reducing power was determined according to the modified method of Pereira et al. (2013) . A volump < 0.05 was considered statistically significant.Statistical analysis was undertaken using the SPSS package . The data was shown as mean ± standard deviation (SD). Mean differences of the crude extracts, fractions and pure compounds were assessed by one-way analysis of variance  in the antioxidant tests; Breonadia salicina. Eight compounds -glucuronide, lupeol, d-galactopyranose, bodinioside Q, 5-O-caffeoylquinic acid, sucrose, hexadecane and palmitic acid) were isolated from the stem bark, root and leaf extracts of B. salicina. This is the first study to report the isolation of these compounds from the genus Breonadia Ridsdale and B. salicina species. Consequently, a total of 25 metabolites were tentatively identified from different parts of B. salicina using UPLC-QTOF-MS. This is the first study to identify and report these metabolites from the genus Breonadia Ridsdale and B. salicina species. Furthermore, the study showed that the stem bark crude extract contained a significantly higher antioxidant capacity compared to the root and leaf samples. The findings in this work comprehensively indicate that polyphenols, hydroxycinnamic acids and flavonoids contribute to the biological activities evaluated.The main objective of this study was to determine the phytochemistry, phytochemical compositions and antioxidant activity of