Morus alba has led to the isolation and identification of three new isoprenylated flavonoids, namely sanggenon U (1), sanggenon V (2), and sanggenon W (3), along with four known isoprenylated flavonoids: euchrenone a7 (4), sanggenon J (5), kuwanon E (6), and kuwanon S (7). All compounds were isolated by repeated silica gel (SiO2), octadecyl SiO2 (ODS), and Sephadex LH-20 open column chromatography. The structure of the compounds were determined based on spectroscopic analyses, including nuclear magnetic resonance (NMR), mass spectrometry (MS), circular dichroism (CD), and infrared (IR). In addition, compounds 1–4 were isolated for the first time from the root bark of M. alba in this study.Phytochemical investigation of the root bark of  Morus alba L. in the family of Moraceae + in the EI/MS spectrum, and a molecular formula of C25H24O6 according to the high-resolved molecular ion peak m/z 420.1572 [M]+  in the HR/EI/MS. IR absorbance bands of hydroxyl (3382 cm−1), conjugated ketone (1666 cm−1), and aromatic  groups were observed. The 1H-NMR spectrum exhibited two olefinic methine signals of a 1,2,3,4-tetrasubstituted benzene ring B at δH 7.62  and 6.49  and two olefinic methine signals of a typical meta-coupled pattern due to a 1,2,3,5-tetrasubstituted benzene ring A at δH 6.38  and 6.17 . The olefinic methine signal at δH 7.04  indicated the characteristic of flavone ring C structure. In addition, the olefinic methine signals at δH 6.72  and 5.63  of a prenyl moiety indicated the formation of a pyran ring through cyclization between the oxygenated olefinic quaternary carbon at δC 110.84 (C-3′) and the oxygenated quaternary carbon at δC 81.37 (C-3′′). This cyclization, a type of isoprene side chain with an ortho-phenolic hydroxyl, leads to pyrano or furano derivatives + in the EI/MS spectrum, and the molecular formula of C25H26O6 according to the high-resolved molecular ion peak m/z 422.1726 [M]+  in the HR/EI/MS. The IR absorbance bands of hydroxyl (3376 cm−1), conjugated ketone (1659 cm−1), and aromatic  groups were observed. The 1H-NMR and 13C-NMR data of compound 3 were quite similar to those of 1, except for the respective replacements of the oxygenated methine signal  and the methylene signal  in the ring C of 1 by the oxygenated olefinic quaternary signal (δC 165.74) and the olefinic methine signal  in 3, and disappearance of the prenyl moiety signals at C-5′ position. The carbon chemical shift of ketone signal at C-4 (δC 184.16) was shifted upfield by 14.09 ppm, comparing with the carbon chemical shift of 1 (δC 198.25) due to the conjugation effects, indicating a double bond to be between C-2 and C-3. Those findings indicated compound 3 was a tetrahydroxyflavone compound with a geranyl moiety. The tetrahydroxyflavone structure and the location of the geranyl moiety in 3 was confirmed based on the COSY and HMBC experiments -flavone, a new isoprenylated flavonoid, and named sanggenon W.Compound eriments . Thus, tMorus alba L. (Moraceae) were supplied by the Korea Food Research Institute  in January 2012, and was identified by Professor Dae-Keun Kim, College of Pharmacy, Woosuk University, Jeonju, Korea. A voucher specimen (KHU-NPCL-201204) has been deposited at the Laboratory of Natural Products Chemistry, Kyung Hee University, Yongin, Korea.The dried root bark of 254 and RP-18 F254S (Merck) plates. The spots on TLC were detected using a UV lamp  and a 10% H2SO4 solution by spraying and heating. 1H- (400 MHz) and 13C-NMR  spectra were recorded on a Varian Unity Inova AS-400 FT-NMR spectrometer . Infrared (IR) spectra were obtained from a Perkin-Elmer model 599B spectrophotometer . Optical rotations were measured on a polarimeter . Electronic ionization mass spectrometry (EI/MS) and fast atom bombardment mass spectrometry (FAB/MS) spectra were obtained using a JMSAX 700 . Melting points were determined using Fisher-Johns melting point apparatus  and not corrected. Circular dichroism (CD) spectra were obtained from a Chirascan Plus instrument .Open column chromatography (CC) was carried out with a Kiesel gel 60 , LiChroprep RP-18 , and Sephadex LH-20 . The thin layer chromatography (TLC) analysis was performed using Kieselgel 60 FM. alba (10 kg) was extracted with 80% methanol (170 L) at room temperature for 24 h. The concentrated MeOH extract (1.7 kg) was suspended in 2 L of water and successively partitioned by increasing polarity gradients of ethyl acetate  and n-butyl alcohol . The organic and aqueous layers were concentrated to produce the EtOAc fraction , the n-BuOH fraction , and the H2O fraction  residues, respectively. The MRE fraction (122 g) was subjected to a SiO2 CC (ϕ 12.5 × 15 cm) and eluted with n-hexane–EtOAc  → CHCl3–MeOH  with monitoring by TLC to obtain 41 fractions (MRE-1 to MRE-41). Fraction MRE-8  was subjected to an ODS CC (ϕ 6.5 × 12 cm) and eluted with MeOH–H2O , yielding 12 fractions (MRE-8-1 to MRE-8-12). Subfraction MRE-8-4  was subjected to the ODS CC (ϕ 3.5 × 12 cm) and eluted with acetone–H2O , yielding 10 fractions (MRE-8-4-1 to MRE-8-4-10). Subfraction MRE-8-4-8  was subjected to a Sephadex LH-20 CC (ϕ 1.5 × 60 cm) and eluted with MeOH–H2O , yielding six fractions (MRE-8-4-8-1 to MRE-8-4-8-6) including a purified compound 5 at MRE-8-4-8-3 . Fraction MRE-23  was applied to the ODS CC (ϕ 4.5 × 6 cm) and eluted with MeOH–H2O , yielding 12 fractions (MRE-23-1 to MRE-23-12). Subfraction MRE-23-6  was subjected to the Sephadex LH-20 CC (ϕ 1.5 × 57 cm) and eluted with MeOH–H2O , yielding seven fractions (MRE-23-6-1 to MRE-23-6-7) including a purified compound 1 at MRE-23-6-6 . Subfraction MRE-23-7  was subjected to the Sephadex LH-20 CC (ϕ 1.5 × 60 cm) and eluted with MeOH–H2O , yielding 10 fractions (MRE-23-7-1 to MRE-23-7-10) including a purified compound 6 at MRE-23-7-8 . Subfraction MRE-23-8  was subjected to the Sephadex LH-20 CC (ϕ 2 × 67 cm) and eluted with MeOH–H2O , yielding 10 fractions (MRE-23-8-1 to MRE-23-8-10) including a purified compound 2 at MRE-23-8-5  and a compound 3 at MRE-23-8-8 . Subfraction MRE-23-9  was subjected to the Sephadex LH-20 CC (ϕ 1.5 × 60 cm) and eluted with MeOH–H2O , yielding five fractions (MRE-23-9-1 to MRE-23-9-5). Subfraction MRE-23-9-4  was subjected to the SiO2 CC (ϕ 2 × 15 cm) and eluted with CHCl3–MeOH , yielding nine fractions (MRE-23-9-4-1 to MRE-23-9-4-9) including a purified compound 7 at MRE-23-9-4-4 . Fraction MRE-28  was applied to the ODS CC (ϕ 7 × 4 cm) and eluted with MeOH–H2O , yielding 16 fractions (MRE-28-1 to MRE-28-16). Subfraction MRE-28-3  was subjected to the Sephadex LH-20 CC (ϕ 1 × 60 cm) and eluted with MeOH–H2O , yielding 10 fractions (MRE-28-3-1 to MRE-28-3-10) along with a purified compound 4 at MRE-28-3-4  Rf 0.55, MeOH–H2O = 2:1).The fractionation and isolation procedure is described in Sanggenon U (1). Yellow amorphous powder (MeOH). m.p. 155–160 °C. c 0.62, MeOH). IRν (CaF2 plate) 3373, 2924, 1662, 1608, 1577 cm−1. HR/EI/MS m/z 510.2616 [M]+ . 1H-NMR (CD3OD) and 13C-NMR (CD3OD) data: see Sanggenon V (2). Yellow amorphous powder (MeOH). m.p. 145–150 °C. c 0.85, MeOH). IRν (CaF2 plate) 3382, 2944, 2863, 1666, 1598, 1545 cm−1. HR/EI/MS m/z 420.1572 [M]+ . 1H-NMR (CD3OD) and 13C-NMR (CD3OD) data: see Sanggenon W (3). Yellow amorphous powder (MeOH). m.p. 100–105 °C. IRν (CaF2 plate) 3376, 2935, 2869, 1659, 1588, 1541 cm−1. HR/EI/MS m/z 422.1726 [M]+ . 1H-NMR (CD3OD) and 13C-NMR (CD3OD) data: see 7Euchrenone a (4). Yellow amorphous powder (MeOH). m.p. 110–115 °C. c 0.45, MeOH). IRν (CaF2 plate) 3310, 2922, 2887, 1667, 1601, 1518 cm−1. EI/MS m/z 340 [M]+. 1H-NMR  7.58 , 7.27 , 6.50 , 6.33 , 6.32 , 5.60 , 5.19 , 3.28 , 2.92 , 2.72 , 1.75 , 1.75 ; 13C-NMR  195.03 (C-4), 163.84 (C-8a), 163.57 (C-7), 159.51 (C-4′), 156.65 (C-2′), 132.11 (C-3′′), 128.65 (C-6′), 126.74 (C-5), 123.38 (C-2′′), 118.57 (C-1′), 117.13 (C-8), 115.02 (C-4a), 110.69 (C-6), 107.64 (C-3′), 103.36 (C-5′), 76.29 (C-2), 44.03 (C-3), 25.98 (C-4′′), 22.99 (C-1′′), 17.95 (C-5′′).Sanggenon J (5). Yellow amorphous powder (MeOH). m.p. 140–145 °C. c 0.03, CHCl3). EI/MS m/z 488 [M]+. IR  3374, 2928, 2892, 1661, 1605, 1514 cm−1. 1H-NMR  6.97 , 6.75 , 6.39 , 6.26 , 6.17 , 5.63 , 5.09 , 5.07 , 3.07 , 2.11 , 1.71 , 1.65 , 1.58 , 1.56 , 1.39 , 1.33 ; 13C-NMR  183.66 (C-4), 165.61 (C-7), 163.27 (C-5), 162.81 (C-2), 159.94 (C-8a), 157.36 (C-4′), 151.92 (C-2′), 132.83 (C-3′′′′), 132.49 (C-3′′′), 131.24 (C-6′), 129.35 (C-2′′), 125.24 (C-2′′′), 122.52 (C-2′′′′), 122.23 (C-3), 118.14 (C-1′′), 114.97 (C-1′), 111.72 (C-3′), 109.33 (C-5′), 105.50 (C-4a), 99.56 (C-6), 94.61 (C-8), 79.67 (C-3′′), 42.19 (C-5′′), 26.76 (C-4′′′′), 25.85 (C-5′′′), 25.85 (C-5′′′′), 24.68 (C-1′′′′), 23.75 (C-1′′), 17.69 (C-4′′′), 17.63 (C-4′′′′).Kuwanon E (6). Yellow amorphous powder (MeOH). m.p. 120–125 °C. c 0.29, CH3OH). EI/MS m/z 424 [M]+. IR  3379, 2922, 2889, 1666, 1612, 1588 cm−1. 1H-NMR  7.06 , 6.33 , 5.89 , 5.86 , 5.58 , 5.28 , 5.08 , 3.19 , 3.03 , 2.68 , 2.07 , 2.00 , 1.66 , 1.59 , 1.55 ; 13C-NMR  198.45 (C-4), 168.20 (C-8a), 165.43 (C-7), 165.35 (C-5), 156.95 (C-4′), 154.42 (C-2′), 136.44 (C-3′′), 132.11 (C-3′′′), 128.65 (C-6′), 125.41 (C-2′′′), 124.39 (C-2′′), 120.55 (C-5′), 117.29 (C-1′), 103.35 (C-3′), 103.34 (C-4a), 96.88 (C-6), 96.17 (C-8), 76.07 (C-2), 43.13 (C-3), 40.85 (C-5′′), 28.44 (C-1′′), 27.77 (C-1′′′), 25.86 (C-4′′′), 17.76 (C-5′′′), 16.18 (C-4′′).Kuwanon S (7). Yellow amorphous powder (MeOH). m.p. 78–80 °C. EI/MS m/z 406 [M]+. IR  3359, 2913, 2884, 1671, 1601, 1584 cm−1. 1H-NMR  7.59 , 7.58 , 6.84 , 6.44 , 6.36 , 6.15 , 5.32 , 5.09 , 3.31 , 2.11 , 2.05 , 1.72 , 1.59 , 1.56 ; 13C-NMR  183.72 (C-4), 166.44 (C-2), 166.03 (C-7), 163.11 (C-5), 160.51 (C-4′), 159.30 (C-8a), 137.64 (C-3′′), 132.11 (C-3′′′), 130.28 (C-3′), 128.79 (C-6′), 126.81 (C-2′′′), 125.26 (C-2′′′), 123.26 (C-2′′), 116.17 (C-5′), 105.21 (C-4a), 103.53 (C-3), 100.11 (C-6), 95.05 (C-8), 40.86 (C-5′′), 28.99 (C-1′′), 27.70 (C-1′′′), 25.85 (C-4′′′), 17.79 (C-5′′′), 16.27 (C-4′′).1–3 and four known ones 4–7 were isolated from the root bark of M. alba. Chemical structures of the isolated compounds were identified on the basis of NMR, MS, CD, and IR spectroscopic data. Compounds 1–4 were isolated for the first time from the root bark of M. alba in this study.Three new isoprenylated flavonoids


The structures were elucidated by a study of their physical and spectral data. Compounds 1 and 2 displayed inhibitory effects towards chronic myelogenous leukemia K562 and human gastric cancer SGC-7901 cell lines.A new pregnanone, named calotropone ( Calotropis (Asclepiadaceae) is comprised of about six species of shrubs distributed throughout tropical and subtropical Africa and Asia. Two of them, Calotropis gigantea L. and Calotropis procera L. occur in China, and are two sister species. C. gigantea is a high biomass, fast growing perennial shrub growing as a weed in the Hainan province of China + at m/z 491.2409  in the high-resolution ESI-Mass spectrum corresponded to the molecular formula C28H36O6. This formula can also be validated through 1H-NMR, 13C-NMR and DEPT spectra. The IR spectrum of 1 showed absorption bands due to a hydroxyl  and a carbonyl  group, whereas the UV spectrum of 1 suggested the presence of a benzoyl group . The 1H-NMR spectrum of 1 suggested the presence of a benzoyl group . In addition, one olefinic proton  and three high field methyl singlet at δ 2.06, 1.41, and 0.98 were also observed. The 13C NMR (DEPT) spectra of 1 showed the coexistence of three methyl groups, seven methylene groups, two aliphatic sp3 methine carbons, two oxygenated sp3 methine carbons, four sp3 quaternary carbon atoms, one tri-substituted double bond, one benzoyl group, and one ketone. This observation suggested that 1 was likely to be a lineolon-type compound. Comparing the 13C-NMR spectral data with those of the 12-O-benzoyllineolon showed that 1 had one tertiary carbon more and one quarternary carbon less than 12-O-benzoyllineolon −89.7° ; HR-ESI-MS: m/z [M+Na]+ 491.2409; IRν (cm-1): 3431, 2918, 2849, 1712, 1629, 1463, 1275, 1110; UVλmax nm (CHCl3): 241, 267, 284; 1H-NMR , 13C-NMR : Gofruside (2) −; 1H-NMR : δ 5.88 , 5.02, 4.89 , 4.70 , 3.69 , 3.65 , 3.29 , 3.23 , 3.14 , 1.20 , 0.91 ; 13C-NMR : δ 32.1 (C-1), 30.2 (C-2), 78.7 (C-3), 37.0 (C-4), 45.1 (C-5), 30.0 (C-6), 28.6 (C-7), 43.8 (C-8), 51.9 (C-9), 52.8 (C-10), 22.9 (C-11), 40.6 (C-12), 50.9 (C-13), 86.1 (C-14), 32.8 (C-15), 27.9 (C-16), 52.0 (C-17), 16.3 (C-18), 210.4 (C-19), 178.3 (C-20), 75.3 (C-21), 117.9 (C-22), 177.2 (C-23), 99.9 (C-1'), 72.4 (C-2'), 72.9 (C-3'), 74.3 (C-4'), 70.5 (C-5'), 18.2 (C-6').side (2) ,16: C29H1 and 2 were examined for their cytotoxic activity against chronic myelogenous leukemia K562 and human gastric cancer SGC-7901 cell lines. Cancer cells were incubated for 3 days at 37 °C in the presence of various concentrations of compounds from DMSO-diluted stock solutions. The growth inhibitory property was determined using 3--2,5-diphenyltetra-zolium bromide (MTT) assay as described by Mosmann [Compounds  Mosmann .


UJNMF0008. Their structures were assigned by detailed analyses of spectroscopic data, while the absolute configurations of 1 and 5 were established by electronic circular dichroism (ECD) calculations and that of 2 was determined by modified Mosher ester method. Compounds 3 and 8 exhibited potent antioxidant property with DPPH and ABTS radical scavenging activities, with IC50 values ranging from 16.9 to 18.7 μM. Meanwhile, no compounds indicated obvious bioactivity in our antimicrobial and anti-inflammatory assays at 50.0 μM.Five new chromone derivatives, arthones A–E ( Arthrinium has wide geographic distribution and host range as plant pathogens, endophytes, saprobes, etc., while Poaceae and Cyperaceae are the major host plant families −) and NMR data. Detailed analysis of the 1H- and 13C-NMR data (1 possessed the same A and B rings (5-hydroxy-7-(hydroxymethyl)-4H-chromen-4-one, 9–12 + , indicating eight degrees of unsaturation. 1D-NMR data analysis -4H-chromen-4-one fragment but with larger chemical shifts for C-5a (δC 165.3) and C-8a (δC 114.1) compared with those of 1 . Correlated spectroscopy (COSY) correlations from H-7 (δH 3.99) to H2-6 (δH 1.80/1.87) and H2-8 (δH 2.32/2.61), and H-5 (δH 2.71/2.79) to H-6 (δH 1.80/1.87), revealed the spin-coupling system from H2-5–H2-8 - and (S)-MTPA esters (Δδ = δS − δR) led to the assignment of 7R-configuration for 2  was yielded as a yellow powder and displayed an HR-ESIMS peak at m/z 333.0607 [M + H]+  corresponding to the molecular formula C16H12O8, with 16 amu more than that of its cometabolite sydowinin B (10) +. Its NMR data (H-chromene-5-carboxylate moiety as arthone D (4). COSY correlations from H-11 (δH 1.93/2.08) to H-10 (δH 2.84) and H-12 (δH 4.14), and 12-OH (δH 5.63) to H-12 (δH 4.14), and HMBC correlations from OCH3 to C-13, and H-11/H-12 to C-13, revealed the moiety of C-10–C-13–OCH3 . Only compounds 3 and 8 exhibited significant antioxidant activities, with IC50 values of 16.9 and 22.1 μM for DPPH assay, and 18.7 and 18.0 μM for ABTS assay, respectively, while others showed no significant effect at 50.0 μM. Meanwhile, the antimicrobial activity against Gram-positive bacterial strains Mycobacterium smegmatis ATCC 607 and Staphylococcus aureus ATCC 25923, Gram-negative Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027, and fungus Candida albicans ATCC10231, as well as anti-inflammatory activity based on the inhibition effect of NO production in lipopolysaccharide (LPS)-induced mouse macrophages RAW 264.7 cells, were evaluated for 1–13. However, no compounds displayed obvious bioactivity in the two assays up to 50.0 μM.In order to evaluate the biological properties of d6: δH 2.50, δC 39.52). ESIMS analyses were carried out on an Agilent 1260-6460 Triple Quad LC-MS instrument . HR-ESIMS data were acquired on an Agilent 6545 Q-TOF mass spectrometer . UV spectra were obtained on a Shimadzu UV-2600 spectrophotometer  with 1 cm pathway cell. Optical rotations were measured on a Rudolph VI polarimeter  with a 10 cm length cell. ECD spectra were acquired on a Chirascan circular-dichroism spectrometer . IR spectra were recorded on an FT-IR VERTEX 70 . All HPLC analyses and separations were carried out on Agilent 1260 series LC instruments  and a YMC-Pack ODS-A column  was used for HPLC separations. Column chromatography (CC) was performed on Silica gel  and Sephadex LH-20 gel . All solvents used for CC were of analytical grade  and solvents used for HPLC were of HPLC grade .NMR spectra were recorded on a Bruker Avance DRX600 NMR spectrometer , with residual solvent peaks as references ). The BLAST sequenced data were deposited at GenBank (No. MG010382). The strain was deposited at the CGMCC center, Institute of Microbiology, Chinese Academy of Sciences .The fungus strain UJNMF0008 was isolated from a marine-sediment sample collected in the South China Sea . This strain was identified as an Arthrinium sp. UJNMF0008 from a PDA culture plate was inoculated in 500 mL Erlenmeyer flasks containing 150 mL soluble-starch medium  at 28 °C on a rotary shaker at 180 rpm for 3 days as seed cultures. Then, each of the seed cultures (20 mL) was transferred into autoclaved 1 L Erlenmeyer flasks with solid-rice medium . After that, the strain was incubated statically for 30 days at 28 °C.After fermentation, the total 4.8 kg rice culture was crushed and extracted with 15.0 L 95% EtOH three times. The EtOH extract was evaporated under reduced pressure to afford an aqueous solution and then extracted with 2.0 L ethyl acetate three times to give 80 g crude gum.2Cl2-MeOH  to give 10 fractions (Fr.1–Fr.10) based on TLC and HPLC analysis. Fr.6 (30.2 g) was applied to CC over D101-macroporous absorption resin eluted with EtOH-H2O  to afford four subfractions (Fr.6-1–Fr.6-4). Fr.6-3 (11.6 g) was fractionated by the silica gel column with step gradient CH2Cl2-(CH3)2CO (v/v 100:0–0:100) and divided into nine subfractions (Fr.6-3-1–Fr.6-3-9) and a portion of (32.5 mg) Fr.6-3-3 was further purified by HPLC eluting with MeOH-H2O  to give 13 . Fr.6-3-2 (0.67 g), Fr.6-3-4 (510 mg), Fr.6-3-5 (1.04 g), Fr.6-3-6 (3.9 g), and Fr.6-3-7 (2.2 g) were separated by MPLC with an ODS column eluting with gradient MeOH-H2O (v/v 20:80–100:0) to obtain five, five, six, six and five subfractions, respectively. Fr.6-3-2-4 (37.0 mg) was further purified by HPLC eluting with MeOH-H2O  to give 9 . Fr.6-3-4-3 (37.0 mg) was further purified by HPLC eluting with MeOH-H2O-CH3CO2H  to afford 4 , while Fr.6-3-4-5 (50.2 mg) was further purified by HPLC eluting with MeOH-H2O  to give 7 . Fr.6-3-5-2 (25.0 mg) was further purified by HPLC eluting with MeOH-H2O  to give 1 , while Fr.6-3-5-5 (35.2 mg) was further purified by HPLC eluting with MeOH-H2O  to give 10  and 11 , and Fr.6-3-5-6 (25.1 mg) was further purified by HPLC eluting with MeOH-H2O  to give 12 . Fr.6-3-6-2 (55.2 mg) was further purified by HPLC eluting with MeOH-H2O  to give 6 . Fr.6-3-7-2 (80.1 mg) was further purified by HPLC eluting with MeOH-H2O  to give 2 . Fr.7 (15.2 g) was chromatographed on a silica gel column with step gradient CH2Cl2-(CH3)2CO (v/v 100:0–0:100) and divided into three subfractions (Fr.7-1–Fr.7-3). Fr.7-1 (3.5 g) was divided into three subfractions (Fr.7-1-1–Fr.7-1-3) by Sephadex LH-20 CC eluting with MeOH-CH2Cl2 (v/v 1:1), and Fr.7-1-2 (1.2 g) was fractionated by MPLC with an ODS column eluting with step gradient MeOH-H2O (v/v 20:80 to 0:100) and further purified by HPLC eluting with MeOH-H2O  to provide 5 . Fr.9 (7.6 g) was subject to silica gel column with step gradient CH2Cl2-(CH3)2CO (v/v 100:0–0:100) and divided into five subfractions (Fr.9-1–Fr.9-5). Fr.9-2 (1.6 g) was separated by Sephadex LH-20 CC eluting with MeOH-CH2Cl2 (v/v 1:1) to obtain four subfractions (Fr.9-2-1–Fr.9-2-4), and Fr.9-2-2 (42.6 mg) was further purified by HPLC eluting with MeOH-H2O-CH3CO2H  to give 3 , while Fr.9-2-3 (17.0 mg) was further purified by HPLC eluting with MeOH-H2O  to give 8 .The ethyl acetate extract (80 g) was fractionated by a silica gel column eluting with step gradient CHc 0.56, MeOH); ECD  λ (Δε) 322 (2.76), 291 (0.56), 258 (−15.78), 224 (2.50), 212 (−5.55), 204 (1.71) nm; UV (MeOH) λmax (log ε) 238 (4.35), 259 (4.23), 331 (3.64) nm; IR (KBr) νmax 3404, 2957, 1742, 1658, 1594, 1493, 1451, 1299, 1195, 1051, 1022, 821 cm−1; 1H- and 13C-NMR data, m/z 332.9 [M − H]−; (−)-HR-ESIMS m/z 333.0623 [M − H]− .Pale yellow powder; c 0.16, MeOH); ECD  λ (Δε) 328 (0.42), 286 (0.01), 276 (0.07), 258 (−0.29), 245 (−0.01), 206 (−2.21) nm; UV (MeOH) λmax (log ε) 239 (5.18), 328 (4.35) nm; IR (KBr) νmax 3431, 2935, 1659, 1625, 1597, 1499, 1459, 1292, 1117, 1041 cm−1; 1H- and 13C-NMR data, m/z [M − H]− 260.9; (+)-HR-ESIMS m/z 263.0916 [M + H]+ .Pale yellow powder; λmax (log ε) 233 (4.20), 246 (4.14), 254 (4.13), 296 (3.69), 377 (4.01) nm; IR (KBr) νmax 3307, 1702, 1651, 1607, 1586, 1499, 1436, 1381, 1366, 1286, 1250, 1011, 830 cm−1; 1H- and 13C-NMR data, m/z 330.9 [M − H]−; (+)-HR-ESIMS m/z 333.0607 [M + H]+ .Yellow powder; UV (MeOH) λmax (log ε) 234 (4.27), 267 (4.26), 391 (3.33) nm; IR (KBr) νmax 3427, 1725, 1613, 1501, 1459, 1435, 1297, 1235, 1041 cm−1; 1H- and 13C-NMR data, m/z 314.9 [M − H]−; (+)-HR-ESIMS m/z 317.0664 [M + H]+ .Yellow powder; UV (MeOH) c 0.73, MeOH); UV (MeOH) λmax (log ε) 209 (4.30), 314 (3.64) nm; IR (KBr) νmax 3413, 2957, 1736, 1611, 1438, 1309, 1227, 1176, 1040, 1037, 861 cm−1; 1H- and 13C-NMR data, m/z 351.0 [M + H]+; (+)-HR-ESIMS m/z 351.1073 [M + H]+ .Pale yellow powder; 1–13 were determined by DPPH and ABTS methods. DPPH radical scavenging method was conducted as described formerly [50 values were defined as the concentrations of tested compounds resulting in 50% loss of the ABTS radical. All determinations were carried out in triplicate, and curcumin was applied as positive control.The antioxidant activities for compounds formerly , while Aformerly  with som1–13 was assayed against the Gram-positive bacterial strains Mycobacterium smegmatis ATCC 607 and Staphylococcus aureus ATCC 25923, Gram-negative Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027, and fungus Candida albicans ATCC10231 by the two-fold serial dilution method in 96-well microplates as described previously [The antimicrobial activity of compounds eviously . Penicil−1 LPS. After 24 h incubation, detection of accumulated nitric oxide in the cell supernatants was assayed by Griess reagent kit  according to the manufacturer’s instructions. Equal volumes of culture supernatant and Griess reagent were mixed, and the absorbance at 540 nm was measured using a Microplate Reader .Determination of nitric oxide production. Briefly, RAW 264.7 cells were plated into 96-well plates and pretreated with a series of concentrations of compounds  for 1 h before treatment with 1 μg mL4 cells well−1 and allowed to attach for 24 h. The medium was replaced with a 100 μL medium containing the indicated concentrations of compounds and further incubated for 24 h. 10 μL of MTT (5 mg mL−1 in PBS) was added into each well and the plates were incubated for 4 h at 37 °C. Supernatants were aspirated and formed formazan was dissolved in 100 μL of dimethyl sulfoxide (DMSO). The optical density (OD) was measured at an absorbance wavelength of 490 nm using a Microplate Reader .Cell viability assay. RAW 264.7 cells were seeded into 96-well plates at 1 × 10−1 was performed using the OPLS3 molecular mechanics force field via the MacroModel [p) level for 1 and B3LYP/6-311G level for 5, respectively, and the harmonic vibrational frequencies were also calculated to confirm their stability. Then, the 30 lowest electronic transitions for the obtained conformers in vacuum were calculated using time-dependent density functional theory (TD-DFT) method at the CAM-B3LYP/6-311G level for 1 and B3LYP/6-311G level for 5, respectively. ECD spectra of the conformers were simulated using a Gaussian function with a half-bandwidth of 0.25 eV for 1 and 0.35 eV for 5. The overall theoretical ECD spectra were obtained according to the Boltzmann weighting of each conformer.Conformational analysis within an energy window of 3.0 kcal molcroModel  panel ofcroModel  at the C


O-β-d-glucopyranosyl-3-O-(4-O-α-d-glucopyranosyl)-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid-[(4-O-α-d-glucopyranosyl-β-d-glucopyranosyl) ester] (1), 13-[(2-O-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid-[(4-O-(4-O-(4-O-α-d-glucopyranosyl)-α-d-glucopyranosyl)-α-d-glucopyranosyl)-β-d-glucopyranosyl ester] (2), 13-[(2-O-β-d-glucopyranosyl-3-O-(4-O-(4-O-(4-O-α-d-glucopyranosyl)-α-d-glucopyranosyl)-α-d-glucopyranosyl)-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid β-d-glucopyranosyl ester (3), and 13-[(2-O-β-d-glucopyranosyl-3-O-(4-O-(4-O-(4-O-α-d-glucopyranosyl)-α-d-glucopyranosyl)-α-d-glucopyranosyl)-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid-[(4-O-α-d-glucopyranosyl-β-d-glucopyranosyl) ester] (4) on the basis of extensive NMR and mass spectral (MS) data as well as hydrolysis studies. Four new minor diterpene glycosides with a rare α-glucosyl linkage were isolated from a cyclodextrin glycosyltransferase glucosylated stevia extract containing more than 98% steviol glycosides. The new compounds were identified as 13-[(2- Stevia rebaudiana Bertoni (Bertoni) is a perennial shrub of the Asteraceae (Compositae) family native to certain regions of South America (Paraguay and Brazil) + and [M+Na]+ ions at m/z 1291.5458 and 1313.5277, respectively; this was supported by 13C-NMR spectral data. The 1H-NMR spectrum of 1 , 5.87 (δC 102.7), 5.81 (δC 102.9), and 5.32 (δC 104.2) in the 1H-NMR spectrum. One of the other two anomeric protons was observed at δH 5.07 (δC 97.8) and was partially overlapped with one of the H-17 protons, whereas the remaining anomeric proton was observed at δH 5.58 (δC 104.2) in the HSQC data but was co-suppressed with the residual H2O peak in the 1H spectrum. Two of the anomeric protons (δH 5.81 and 5.87) had small coupling constants (J < 4 Hz) indicating that they have an α-configuration similar to the steviol glycosides reported earlier from S. rebaudiana  ent-kaur-16-en-19-oic acid-[(4-O-α-d-glucopyranosyl-β-d-glucopyranosyl) ester].Identification of sugars present in yl units . A close2 was also determined to be C56H90O33 by high resolution mass spectral data. It’s 1H-NMR spectrum of 2 showed the presence of two methyl singlets, two olefinic protons as singlets of an exocyclic double bond, nine methylene and two methine protons , 5.13 , and 5.32  suggested their β-orientation as reported for steviol glycosides, whereas the coupling constants for the other three anomeric protons of the sugars IV, V and VI appeared at δ 5.80, 5.75, and 5.89 were obtained as 3.7, 3.8 and 3.7 Hz respectively, suggested their α-orientation, similar to 1.The molecular formula of compound  protons ; similar2 and their configurations were achieved by preparing their thiocarbamoyl-thiazolidine carboxylate derivatives as described for 1 suggesting the sugar moieties as d-glucopyranosylunits. From the above spectral data and hydrolysis results, it was clear that both compounds 1 and 2 are identical except for the connectivity of the glucosyl units to the basic aglycone (steviol) skeleton and the orientation. A close comparison of the 1H and 13C-NMR chemical shift values of 2 with 1, and stevioside suggested the presence of a β-d-glucosyl substituent at C-19 and 2-β-d-glucobiosyl substituent at C-13 leaving the assignment of the additional three α-d-glucosyl units. A series of COSY, HSQC-TOCSY, HSQC and HMBC experiments suggested the placement of the other three d-glucosyl units as α-d-glucopyranosyl-(1→4)-O-[α-d-glucopyranosyl-(1→4)]-O-α-d-glucupyranosyl at the C-4 position of the β-d-glucosyl unit at C-19, as shown in 2 was established as 13-[(2-O-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid –[(4-O-(4-O-(4-O-α-d-glucopyranosyl)-α-d-glucopyranosyl)-α-d-glucopyranosyl)-β-d-glucopyranosyl ester].Further, the identification of sugars present in 3 was determined to be C62H100O38 by high resolution mass spectral data which showed [M+H]+ and [M+Na]+ ions at m/z 1453.6035 and 1475.5829, respectively. It’s 1H-NMR spectrum showed the presence of two methyl singlets, two olefinic protons as singlets of an exocyclic double bond, nine methylene and two methine protons , 5.06 , 5.60 , and 5.45  suggested their β-orientation as reported for steviol glycosides, whereas the coupling constants for the other three anomeric protons of the sugars V, VI and VII appeared at δ 5.74, 5.78, and 5.90 were obtained as 3.1, 3.1 and 3.2 Hz respectively, suggesting their α-orientation. Further, the identification of sugars present in 3 and their configurations were achieved by preparing their thiocarbamoyl-thiazolidine carboxylate derivatives as described for 1 suggesting the sugar moieties as d-glucopyranosyl units.The molecular formula of compound  protons ; similar1H and 13C-NMR chemical shift values of 3 with 1, and rebaudioside A suggested the presence of a β-d-glucosyl substituent at C-19 and a 2,3-branched β-d-glucotriosyl substituent at C-13, leaving the assignment of the additional three α-d-glucosyl units. From the key COSY, HSQC-TOCSY, HSQC and HMBC experiments, the placement of the other three α-d-glucosyl units were assigned as α-d-glucopyranosyl-(1→4)-O-[α-d-glucopyranosyl-(1→4)]-O-α-d-glucupyranosyl to the C-4 position of the sugar-IV as shown in A close comparison of the 4 was determined to be C68H110O43 by high resolution mass spectral data which showed [M+H]+ and [M+Na]+ ions at m/z 1615.6498 and 1637.6302, respectively; this was supported by 13C-NMR spectral data. It’s 1H-NMR spectrum showed the presence of two methyl singlets, two olefinic protons as singlets of an exocyclic double bond, nine methylene and two methine protons , 5.05 , 5.60 , and 5.47  suggested their β-orientation as reported for steviol glycosides,whereas the coupling constants for the other four anomeric protons of the sugars V, VI, VII and VIII appeared at δ 5.87, 5.73, 5.78, and 5.90 were obtained as 3.4, 3.2, 3.2 and 3.2 Hz respectively, suggested their α-orientation.The molecular formula of compound  protons ; similar4 and their configurations were achieved by preparing their thiocarbamoyl-thiazolidine carboxylate derivatives as described for 1 suggesting the sugar moieties as d-glucopyranosyl units. A close comparison of the 1H and 13C-NMR chemical shift values of 4 with 1–3, and rebaudioside A suggested the presence of a β-d-glucosyl substituent at C-19 and a 2,3-branched β-d-glucotriosyl substituent at C-13, leaving the assignment of the additional four α-d-glucosyl units. From the key COSY, HSQC-TOCSY, HSQC and HMBC experiments, the placement of the other four α-d-glucosyl units were assigned as α-d-glucopyranosyl-(1→4)-O-[α-d-glucopyranosyl-(1→4)]-O-α-d-glucupyranosyl to the C-4 position of the sugar-IV with an additional α-d-glucupyranosyl attached to the C-6 position of the sugar-I as shown in Further, the identification of sugars present in H 8.71, δC 149.9 for pyridine-d5), chemical shifts are given in δ (ppm), and coupling constants are reported in Hz. MS and MS/MS data were generated with a Waters Premier Quadrupole Time-of-Flight (Q-Tof) mass spectrometer equipped with an electrospray ionization source operated in the positive-ion mode and ThermoFisher Discovery OrbiTrap in the positive mode electrospray. Samples were diluted with water: acetonitrile (1:1) containing 0.1% formic acid and introduced via infusion using the onboard syringe pump. Analytical HPLC was carried out with a Waters 600E multisolvent delivery system using Luna C18 column  and Phenomenex Luna C18  column. The details of various columns and other parameters used for HPLC purification for methods 1–3 .Column: Phenomenex Prodigy ODS(3) with a Phenomenex guard column, 250 × 21.2 mm, 5 μm (p/n 00G-4097-P0); UV Detection: 210 nm; Mobile Phase A: H2O; Mobile Phase B: Acetonitrile; Flow Rate: 5.0 mL/min; Injection volume: 150 µL prepared in H2O; Detection was by UV (210 nm).Column: Phenomenex spherex diol, 250 × 10 mm, 5 µm (p/n 00G-0021-NO); Column Temp: 25 °C; Mobile Phase A: H18 with guard column, 250 × 10 mm, 5 µm (p/n 186003694); Column Temp: 25 °C; Mobile Phase A: H2O; Mobile Phase B: Acetonitrile; Flow Rate: 5.0 mL/min; Injection volume: 150 µL prepared in H2O; Detection was by UV (210 nm).Column: Atlantis CBacillus stearothermophilus) of the stevia extract with content of stevioside, rebaudioside A, rebaudioside C, and dulcoside more than 98% 13-[(2-O-β-d-glucopyranosyl-β-d-glucopyranosyl) ester]ent-kaur-16-en-19-oic acid- ent-kaur-16-en-19-oic acid-[(4-O-(4-O-(4-O-α-d-glucopyranosyl)-α-d-glucopyranosyl)-α-d-glucopyranosyl)-β-d-glucopyranosyl ester]13- ent-kaur-16-en-19-oic acid β-d-glucopyranosyl esterglucopyranosyl)-β- (3). White powder; 1H-NMR  and 13C-NMR  spectroscopic data see +m/z 1453.6035 ; (M+Na)+m/z 1475.5829 .d-glucopyranosyl-3-O-(4-O-(4-O-(4-O-α-d-glucopyranosyl)-α-d-glucopyranosyl)-α-d-13-[(2-O-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid-[(4-O-α-d-glucopyranosyl-β-d-glucopyranosyl) ester]glucopyranosyl)-β- (4). White powder; 1H-NMR  and 13C-NMR  spectroscopic data see +m/z 1615.6498 ; (M+Na)+m/z 1637.6302 .Acid Hydrolysis of Compounds1–4. To a solution of each compound 1–4 (250 μg) in MeOH (3 mL) was added 3 mL of 5% H2SO4 and the mixture was refluxed for 8 hours. The reaction mixture was then neutralized with saturated sodium carbonate and extracted with ethyl acetate (EtOAc) (2 × 15 mL) to give an aqueous fraction containing sugars and an EtOAc fraction containing the aglycone part. The aqueous phase was concentrated and compared with standard sugars using the TLC systems EtOAc/n-butanol/water (2:7:1) and CH2Cl2/MeOH/water (10:6:1); the sugar was identified as d-glucose in all four experiments  [Each compound 08 min)] .O-β-d-glucopyranosyl-3-O-(4-O-α-d-glucopyranosyl)-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid-[(4-O-α-d-glucopyranosyl-β-d-glucopyranosyl) ester] (1), 13-[(2-O-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid-[(4-O-(4-O-(4-O-α-d-glucopyranosyl)-α-d-glucopyranosyl)-α-d-glucopyranosyl)-β-d-glucopyranosyl ester] (2), 13-[(2-O-β-d-glucopyranosyl-3-O-(4-O-(4-O-(4-O-α-d-glucopyranosyl)-α-d-glucopyranosyl)-α-d-glucopyranosyl)-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid β-d-glucopyranosyl ester (3), and 13-[(2-O-β-d-glucopyranosyl-3-O-(4-O-(4-O-(4-O-α-d-glucopyranosyl)-α-d-glucopyranosyl)-α-d-glucopyranosyl)-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid-[(4-O-α-d-glucopyranosyl-β-d-glucopyranosyl) ester] (4), respectively, on the basis of extensive NMR and mass spectroscopic data and chemical studies. To the best of our knowledge this is the first report of the isolation of these four new steviol glycosides 1–4.Four new minor diterpenoid steviol glycosides were isolated from a glucosylated steviol glycoside mixture obtained by cyclodextrin glycosyltransferase treatment of a stevia extract obtained from PureCircle. The new compounds were identified as 13-[(2-


The structures of compounds were assigned based on 1D and 2D NMR spectroscopic analyses, including HMQC, HMBC, and NOESY. Compounds 1 and 2 showed anti-inflammatory activity by superoxide generation and elastase release by human neutrophils in response to fMLP/CB.Two new steroidal saponins, named drangustosides A–B ( Dracaena (Agavaceae) includes more than 50 species found in tropical and subtropical regions of the eastern hemisphere. Dracaena angustifolia Roxb. is a native shrub or small tree in southern Taiwan, widely planted in Australia, India, Malaysia, and Philippines , one olefinic proton [δH 5.53 ], and three monosaccharide anomeric protons , two of which were considered to be L-rhamnosides. Based on the chemical shifts and coupling constants, the three monosaccharides were considered as a combination of one D-glucoside and two L-rhamnosides. The 13C-NMR spectrum  and the signal at δC 139.3 was assigned as a diakyl substituted olefinic carbon (C-5). The aforementioned data suggested that 1 is possibly a spirostanol glycoside with the aglycone being a 27-carbons skeletonal aglycone along with three sugar moieties . The NMR peaks of C-1 were at δC 78.1 [δH-1 3.68 ] and C-3 at δC 75.0 [δH-3 4.02 (m)] were in compound 1, while the anomeric proton H-1′ [δH 4.91 ] exhibited an HMBC correlation with δC 75.0 (C-3). These results lead to the conclusion that the -O-glycoside is unambiguously located at C-3 in compound 1. Comparing the NMR data of C-1 and C-3 between 1 and 3, the -O-glycosidation at C-3 in compound 1 and at C-3 in compound 3 agrees with the general knowledge. The coupling constant of H-1  can be assigned to an α-axial orientation. H-1 and H-3 have a NOESY correlation, therefore revealing that H-3 was located with the same α-axial orientation. In general, the difference in chemical shifts between axial and equatorial protons among on H2-23, H2-24, and H2-26 can be used to resolve the absolute configuration of C-25  and the spirostanol had a normal type F ring (δH 3.34) to H-25 (δH 1.58) and from H-23ax (δH 1.43) to H-20 (δH 1.87) and H3-27 (δH 1.06) in the NOESY spectrum  , indicat of C-25 ,18. The e F ring  [18]. Thspectrum  were conD-Glucose and L-rhamnose in a 1:2 ratio were obtained upon acidic hydrolysis of 1 with HCl in 1,4-dioxane  which correlated to carbon signals at δC 100.2, 102.8, and 104.1, respectively  showed connectivity with four consecutive methines as follows: δH 3.99 (H-2'), 4.14 (H-3'), 4.03 (H-4'), and 3.78 (H-5'), and then two methylene protons . The TOCSY spectrum gave a result that showed two rhamnoside units connected on the glucoside unit. In the HMBC spectrum, a 3J correlation between H-1'' (δH 5.82) of rhamnoside and C-2' (δC 78.3) of glucoside, and between H-1''' (δH 5.73) of rhamnoside and C-3' (δC 87.6) of glucoside confirmed that two rhamnoside units located at C-2' and C-3' of glucoside units, respectively (1 was identified as 3-O-{α-L-rhamnopyranoside(1→2)-O-[α-L-rhamnopyranoside(1→3)]-β-D-glucopyranosyl}. Therefore, the structure 1 was ultimately identified as 3-O-{α-L-rhamnopyranoside(1→2)-O-[α-L-rhamno-pyranoside(1→3)]-β-D-glucopyranosyl} -spirost-5-ene-1β,3β-diol, to which the trivial name drangustoside A was given.-dioxane , as indi-dioxane . Analysiectively . In the ectively . Consequ2 was obtained as an amorphous solid with a molecular formula of C39H62O13 determined by the HRESIMS data (m/z 761.4085 [M+Na]+). The IR spectrum of 2 indicated the existence of hydroxyl groups  and the characteristic absorption bands of (25S)-spiroketal at 987, 920, 897, and 840 (intensity 920 > 897) cm−1  and the β-anomeric configurations of the D-glucoside units [δH 5.06 ] were confirmed by their coupling constants and chemical shifts  and named drangustoside B. Compound 97) cm−1 . The 1H-ety of 2  were verl shifts . In the aglycone . Consequ3)  25D −54.74 ; IR (KBr) νmax 3,389 (OH), 2,928, 1,681 (C=C), 1,367, 1,043 (C-O-C), 987, 918, 898 and 843  cm-1; for 1H- and 13C-NMR spectroscopic data, see m/z 907.4667 [M+Na]+ . Drangustoside B (2). white amorphous powder; [α]25D −38.25 ; IR (KBr) νmax 3,389 (OH), 2,929, 1,681 (C=C), 1,368, 1,044 (C-O-C), 987, 920, 897 and 840  cm−1; for 1H- and 13C-NMR spectroscopic data, see m/z 761.4085 [M+Na]+ .1 or 2 (2 mg each) were heated with 1N HCl  at 90 °C for 4 h, and then evaporated under reduced pressure to give a residue. The residue was partitioned with CH2Cl2and H2O three times. The aqueous (pH = 2.0) was neutralized with aqueous 1N NaOH and then evaporated under reduced pressure. The aqueous layer  was dissolved in MeOH (0.5 mL) and then analyzed by stereoselective HPLC under the following conditions: column: Chiralpak AD-H; solvent system: n-hexane-ethanol-TFA ; flow rate: 0.5 mL/min; inject volume: 20 μL; detector: refractive index. D-Glucose and L-rhamnose for compounds 1  and 2  were determined by comparison of the respective retention times of D-glucose (tR= 14.33 min) and L-rhmanose (tR =15.85 min) standards. Compound Human neutrophils were obtained using dextran sedimentation and Ficoll centrifugation. Blood was drawn from healthy human donors (20–32 years old) by venipuncture using a protocol approved by the institutional review board at Chang Gung Memorial Hospital. Neutrophils were isolated using a standard method as previously described ,30. Meas1–2, one known steroidal saponin 3, and seven known benzenoids 4–10 were isolated from the MeOH extract of D. angustifolia Roxb. Their structures were determined based on extensive spectroscopic analyses, and the anti-inflammatory activity of 1 and 2 were evaluated by superoxide anion generation and elastase assays, which revealed that the steroidal saponin -spirost-5-ene-1,3-diol with a disaccharide, 2, had better anti-inflammatory activity against superoxide generation than 1 with a trisaccharide. Another reported steroidal saponin with a disaccharide, 3-O-β-chacotriosyl-25(S)-spirost-5-en-3β-ol [2, but missing an hydroxyl group at C-1, also showed very potent anti-inflammatory activity against superoxide generation. These results implied that the number of sugars in this type of steroidal saponins could be important for the anti-inflammatory activity. Moreover, 3-O-β-chacotriosyl-25(S)-spirost-5-en-3-ol  had more potent anti-inflammatory activity against superoxide generation than compound 2 (IC50 = 18.55 ± 0.23 μM); whereas, 2 (IC50 = 1.74 ± 0.25 μM) was more active than 3-O-β-chacotriosyl-25(S)-spirost-5-en-3β-ol  in the human neutrophil elastase release assay [Two new steroidal saponins en-3β-ol , which wse assay . Thus, t


The steroidal thiosemicarbazones were prepared by the reaction of thiosemicarbazides with a steroidal ketone. The structures of these compounds were elucidated by IR, 1H-NMR, 13C-NMR, FAB mass spectroscopic methods, elemental analyses and TGA analysis. The antibacterial activity of these compounds were tested in vitro by the disk diffusion assay against two Gram-positive and two Gram-negative bacteria. The results showed that steroidal complexes are better inhibitors of both types of the bacteria (Gram-positive and Gram-negative) as compared to steroidal thiosemicarbazones. Compound Ia displays remarkable antibacterial activity as compared to amoxicillin.We have investigated the antibacterial activity of some new steroidal thiosemicarbazones and their Pd(II) metal complexes were prepared by the reaction of the thiosemicarbazones with [Pd(DMSO) All the compounds were isolated in good yields and were stable both in the solid and solution state. The structures of the ligands and complexes presented in 1H-NMR, 13C-NMR and FAB mass spectra) with the free ligand, along with their thermogravimetric analysis.Reaction of steroidal thiosemicarbazones with  in refluxing methanol. The solution was kept at 0 ºC overnight, the product was separated by filtration and finally washed with methanol. Recrystallization was effected in methanol/ DMF (6:4) [The metal complexes were prepared by mixing an equimolar ratio of ligand and [Pd (DMSO)MF (6:4) .Dichloro(cholest-5-en-3-one cyclopentyl thiosemicarbazone)palladium (II) (1a). Pd (C33H55N3S) Cl2); Yield: 76%; m.p. 256–258 ºC; IR (KBr) waxv  cm-1: 3442 (N-H), 1538 (C=N), 1528 (C=C), 1152 (C-N), 1024 (C=S), 516, 434 ; 1H-NMR (DMSO-d6) : 9.54 , 5.58 , 4.22 , 3.82 , 1.12, (C10-CH3), 0.82 (C13-CH3), 0.94, 1.06 (other methyl protons); 13C-NMR (DMSO-d6) : 184.5 (C=S), 153.8 (C=N), 133.8 (C-NH), 22.2 (C10-CH3), 20.4 (C13-CH3), 19.4, 19.8 (other methyl carbon); Mass spectra (M+.) at m/z 701, 666 (M- Cl), 731 (M-Cl2), 597 (M-Pd), 632 (M-C5H9), 617(M- C5H10N), 573 (M- C6H10NS), 558 (M- C6H11N2S). Anal. Calc. for Pd(C33H55N3S)Cl2: C, 56.38; H, 7.83; N, 5.98, Cl, 10.09, Pd, 15.12. Found: C, 56.34; H, 7.79; N, 5.95, Cl, 10.02, Pd, 15.09.Dichloro(cholest-5-en-3-one cyclohexyl thiosemicarbazone)palladium (II) (2a). Pd (C35H57N3S) Cl2; Yield: 72.00%; m.p. 264–265 ºC; IR (KBr) ν wax cm-1: 3456 (N-H), 1545 (C=N), 1538 (C=C), 1156 (C-N), 1022 (C=S), 508, 428 ; 1H-NMR (DMSO-d6) : 9.56 , 4.16 , 5.52 , 3.38 , 1.16 (C10-CH3), 0.86 (C13-CH3), 0.92, 1.06 (other methyl protons); 13C-NMR (DMSO-d6) : 183.5 (C=S), 152.2 (C=N), 131.5 (C-NH), 22.4(C10-CH3), 18.4 (C13-CH3); Mass spectra (M+.) at m/z 715, 680 (M- Cl), 645 (M-Cl2), 611 (M-Pd) 632 (M-C6H11), 617 (M-C6H12N), 573 (M- C7H12NS), 558 (M- C7H13N2S); Anal. Calc. for Pd (C34H57N3S) Cl2: C, 56.95; H, 7.98; N, 5.86, Cl, 9.89, Pd, 14.85. Found: C, 56.89; H, 7.95; N, 5.85, Cl, 9.85, Pd, 14.82.Dichloro(cholest-5-en-3-one cyclooctyl thiosemicarbazone)palladium (II) (3a). Pd(C36H61N3S)Cl2;Yield: 76.8%; m.p. 272–273 ºC; IR (KBr) ν wax cm-1: 3438 (N-H), 1532 (C=N), 1522(C=C), 1172 (C-N), 1018 (C=S), 521, 437 ; 1H-NMR (DMSO-d6) : 9.45 , 4.12 , 5.62 , 3.65 , 1.14 (C10-CH3), 0.84 (C13-CH3), 0.96, 1.04 (other methyl protons); 13C-NMR (DMSO-d6) : 182.2 (C=S), 156.7 (C=N), 128.4 (C-NH), 23.4 (C10-CH3), 19.5 (C13-CH3); Mass spectra (M+.) at m/z 742, 707 (M-Cl), 672 (M-Cl2), 637 (M-Pd), 633 (C8H13), 618 (M-C8H14N), 574 (M-C9H14NS), 559 (M-C9H15N2S); Anal. Calc. for Pd(C36H61N3S)Cl2: C, 58.03; H, 8.19; N, 5.64, Cl, 9.52, Pd, 14.29. Found: C, 58.01; H, 8.18: N, 5.62, Cl, 9.48, 14.26.S. aureus, S. pyogenes, S. typhimurium, and E. coli were subcultured in BHI medium and incubated for 18 h at 37 ºC, and then the bacterial cells were suspended, according to the McFarland protocol in saline solution to produce a suspension of about 10-5 CFU mL-1. Ten μL of this suspension was mixed with sterile antibiotic agar (10 mL) at 40 ºC and poured onto an agar plate in a laminar flow cabinet. Five paper disks (6.0 mm diameter) were fixed onto nutrient agar plate. Ten mg of each test compound was dissolved in DMSO (100 μL) to prepare stock solution and from stock solution different concentration of 10 (1 μL stock solution + 9 μL solvent), 20 (1 μL stock solution + 4 μL solvent), 25 (1 μL stock solution + 3 μL solvent), 50 (1 μL stock solution + 1 μL solvent), and 100 μg/μL of each test compound were prepared. These compounds of different concentration were poured over disk plate on to it. Amoxicillin (30 μg) was used as standard drug (positive control). A DMSO-wetted disk was used as negative control. The susceptibility of the bacteria to the test compounds was determined by the formation of an inhibitory zone after 18 h of incubation at 36 ºC. Antibacterial activity was assayed by the disk diffusion method with minor modifications. In vitro antibacterial activity of these compounds was tested by the disk diffusion assay against two gram-positive and gram-negative bacteria. The result showed that cyclopentyl steroidal thiosemicarbazone derivative was found the most active among all thiosemicarbazones tested. Compound Ia displays remarkable antibacterial activity as compared to amoxicillin.This research examined the synthesis, characterization and antibacterial activity of some new steroidal thiosemicarbazone derivatives and their Pd (II) complexes.


Context:Paramignya trimera (Oliv.) Burkill (Rutaceae) has been used to treat liver diseases and cancer. However, the anti-inflammatory effects of this medicinal plant and its components have not been elucidated.Objective: This study investigated chemical constituents of the P. trimera stems and evaluated anti-inflammatory effects of isolated compounds.Materials and methods: Cytotoxicity of isolated compounds (5–40 μM) toward BV2 cells was tested using 3--2,5-diphenyltetrazolium bromide (MTT) for 24 h. Inhibitory effects of isolated compounds (5-40 μM) on nitrite and PGE2 concentrations were determined using Griess reaction and PGE2 ELISA kit, respectively (pretreated with the compounds for 3 h and then stimulated for 18 h with LPS). Inhibitory effects of compounds (5-40 μM) on iNOS and COX-2 protein expression were evaluated by Western blot analysis (pretreated with the compounds for 3 h and then stimulated for 24 h with LPS).Results: Seven coumarins were isolated and identified as: ostruthin (1), ninhvanin (2), 8-geranyl-7-hydroxycoumarin (3), 6--7-hydroxycoumarin (4), 6--7-hydroxycoumarin (5), 6-(2-hydroxyethyl)-2,2-dimethyl-2H-1-benzopyran (6), and luvangetin (7). Compounds 1–4 and 7 inhibited NO and PGE2 production in LPS-stimulated BV2 cells, with IC50 values ranging from 9.8 to 46.8 and from 9.4 to 52.8 μM, respectively. Ostruthin (1) and ninhvanin (2) were shown to suppress LPS-induced iNOS and COX-2 protein expression.Discussion and conclusion: The present study provides a scientific rationale for the use of P. trimera in the prevention and treatment of neuroinflammatory diseases. Ostruthin and ninhvanin might have potential therapeutic effects and should be considered for further development as new anti-neuroinflammatory agents. Paramignya trimera (Oliv.) Burkill (Rutaceae) is a woody shrub, mostly distributed in the Southern regions of Vietnam. In the Vietnamese traditional medicine, P. trimera is well known as a medicinal plant used to treat liver diseases and cancer  from the methanol extract of the stems of P. trimera.2 (PGE2) and cytokines  that play an important role in response to neuronal damage and removal of the damaged cells. In response to extracellular stimuli, including LPS, microglia are activated and produce pro-inflammatory mediators, such as nitric oxide (NO) and prostaglandin E2, the inflammatory products of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), are considered as the major factors in the inflammatory response. Excessive production of these pro-inflammatory mediators was shown to be the cause of the neurodegenerative diseases  were recorded on a Bruker AM500 FTNMR spectrometer: 500 MHz (1H NMR), 125 MHz (13C NMR). The electrospray ionization (ESI) mass spectra were recorded on an AGILENT 1200 LC-MSD trap spectrometer. Column chromatography (CC) was performed using silica gel  and RP-18 resins . Thin layer chromatography (TLC) was performed on DC-Alufolien 60 F254  and RP18 F254s (Merck) plates.1D and 2D NMR experiments  was deposited at the Herbarium of the Institute of Marine Biochemistry, VAST.The stems of Paramignya trimera (3 kg) were ground and extracted with MeOH under sonication at room temperature. After concentration under reduced pressure, the MeOH extract (150 g) was suspended in water and then partitioned successively with CHCl3 and EtOAc to give CHCl3-, EtOAc-, and water-soluble fractions. The CHCl3-soluble fraction (60 g) was subjected to fractionation over silica gel, eluting with EtOAc in n-hexane  to give fractions TR1A-E. Fraction TR1A was separated by column chromatography (CC) over silica gel, using CH2Cl2-MeOH  as eluent to yield 5 (8 mg). Fraction TR1C was subjected to silica gel CC and eluted with n-hexane-EtOAc  to provide 1 (50 mg) and 2 (20 mg). Fraction TR1E was chromatographed over a silica gel column, eluting with n-hexane-acetone , and further purified by silica gel CC, using n-hexane-acetone  as eluent to give 6 (8 mg) and 7 (8 mg). The EtOAc-soluble fraction (40 g) was subjected to reversed phase (RP) C18 CC, eluting with a gradient of acetone in water  to give fractions TR2A-C. Fraction TR2A was separated by silica gel CC, using CH2Cl2-MeOH  as eluent to provide 4 (8 mg). Fraction TR2B was separated by silica gel CC, eluting with n-hexane-acetone  to give 3 (10 mg).The dried stems of 1H NMR : δ 6.23 , 7.65 , 7.20 , 7.07 , 3.39 , 5.34 , 2.10 , 2.12 , 5.10 , 1.68 , 1.60 , 1.73 ; 13C NMR : δ 162.6 (C-2), 111.9 (C-3), 144.5 (C-4), 128.1 (C-5), 126.1 (C-6), 158.9 (C-7), 103.0 (C-8), 154.1 (C-9), 112.1 (C-10), 28.1 (C-1′), 120.9 (C-2′), 138.3 (C-3′), 39.7 (C-4′), 26.5 (C-5′), 123.9 (C-6′), 131.7 (C-7′), 25.7 (C-8′), 17.7 (C-9′), 16.1 (C-10′).White, amorphous powder; 1H NMR : δ 6.22 , 7.60 , 6.95 , 3.37 , 5.32 , 2.09 , 2.13 , 5.11 , 1.68 , 1.60 , 1.71 , 4.10 ; 13C NMR : δ 160.7 (C-2), 112.2 (C-3), 144.4 (C-4), 122.3 (C-5), 125.2 (C-6), 150.2 (C-7), 133.1 (C-8), 145.3 (C-9), 112.2 (C-10), 27.5 (C-1′), 120.8 (C-2′), 137.4 (C-3′), 39.6 (C-4′), 26.5 (C-5′), 124.0 (C-6′), 131.4 (C-7′), 25.6 (C-8′), 17.6 (C-9′), 16.0 (C-10′), 61.6 (8-OCH3).White, amorphous powder; 1H NMR : δ 6.18 , 7.85 , 7.31 , 6.83 , 3.53 , 5.26 , 1.98 , 2.06 , 5.03 , 1.57 , 1.53  1.85 ; 13C NMR : δ 164.0 (C-2), 111.4 (C-3), 146.6 (C-4), 127.6 (C-5), 113.8 (C-6), 161.1 (C-7), 117.0 (C-8), 154.8 (C-9), 113.1 (C-10), 22.6 (C-1′), 122.8 (C-2′), 136.5 (C-3′), 40.8 (C-4′), 27.5 (C-5′), 125.3 (C-6′), 132.0 (C-7′), 25.7 (C-8′), 17.6 (C-9′), 16.4 (C-10′).White, amorphous powder; m/z 331.1571 [M-H]- ; 1H NMR : δ 6.17 , 7.83 , 7.30 , 6.72 , 3.34 , 5.42 , 2.12 , 2.34 , 1.43 , 1.81 , 3.29 , 1.18 , 1.15 , 1.75 ; 13C NMR : δ 164.0 (C-2), 112.0 (C-3), 146.3 (C-4), 129.4 (C-5), 127.9 (C-6), 160.8 (C-7), 102.6 (C-8), 155.4 (C-9), 112.9 (C-10), 28.6 (C-1′), 123.1 (C-2′), 137.8 (C-3′), 37.8 (C-4′), 30.6 (C-5′), 78.9 (C-6′), 73.7 (C-7′), 25.7 (C-8′), 24.9 (C-9′), 16.2 (C-10′).White, amorphous powder; HRESIMS: 1H NMR : δ 6.14 , 7.83 , 7.29 , 6.71 , 3.37 , 5.42 , 2.80 , 5.66 , 1.31 , 1.73 ; 13C NMR : δ 164.2 (C-2), 111.5 (C-3), 146.3 (C-4), 129.3 (C-5), 128.0 (C-6), 162.0 (C-7), 102.7 (C-8), 155.6 (C-9), 112.6 (C-10), 28.6 (C-1′), 123.9 (C-2′), 136.6 (C-3′), 43.6 (C-4′), 129.4 (C-5′), 137.0 (C-6′), 82.4 (C-7′), 24.9 (C-8′ and C-9′), 16.2 (C-10′).White, amorphous powder; 1H NMR : δ 5.60 , 6.28 , 6.82 , 6.94 , 6.71 , 2.75 , 3.79 , 1.42 2); 13C NMR : δ 76.0 (C-2), 130.9 (C-3), 122.1 (C-4), 126.7 (C-5), 130.4 (C-6), 129.4 (C-7), 116.3 (C-8), 151.5 (C-9), 121.2 (C-10), 38.3 (C-11), 63.7 (C-12), 27.9 (2-(CH3)2).White, amorphous powder; 1H NMR : δ 6.24 , 7.57 , 6.82 , 6.33 , 5.71 , 1.51 , 3.98 ; 13C NMR : δ 160.6 (C-2), 113.0 (C-3), 143.5 (C-4), 113.2 (C-4a), 119.0 (C-5 and C-6), 149.3 (C-7), 135.6 (C-8), 148.3 (C-8a), 121.0 (C-1′), 131.2 (C-2′), 77.7 (C-3′), 28.2 (C-4′ and C-5′), 61.4 (8-OCH3).White, amorphous powder; 5 cells/mL in 100-mm dishes in DMEM supplemented with 10% heat-inactivated FBS, penicillin G (100 units/mL), streptomycin (100 μg/mL), and l-glutamine (2 mM), and were incubated at 37 °C in a humidified atmosphere containing 5% CO2 and 95% air. The effects of the various experimental modulations on cell viability were evaluated by determining the mitochondrial reductase function based on reduction of the tetrazolium salt, 3--2,5-diphenyltetrazolium bromide (MTT) to a formazan crystal (Berridge & Tan 4 cells/200 μL in each well of the 96-well plates) were incubated with MTT at a final concentration of 0.5 mg/mL for 4 h. The formazan formed was dissolved in acidic 2-propanol, and the optical density was measured at 590 nm. The optical density of the formazan formed in the control (untreated) cells was considered as 100% viability. For determination of cytoprotective effects, HT22 cells (5 × 105 cells/mL in DMEM medium) were treated with compounds in the presence of 20 mM glutamate and incubated for indicated times and cell viability by MTT assay.BV2 microglia cells were obtained from Prof. Hyun Park at Wonkwang University  and mouse hippocampal HT22 cells were received from Dr. Inhee-Mook Seoul National University . The cells were maintained at 5 × 10The production of nitrite, the stable end product of NO oxidation, was used as a measure of iNOS activity. The nitrite present in the conditioned media was determined by a method based on the Griess reaction (Titheradge 2 concentration using an ELISA kit (R & D Systems). Butein was used as the positive control.BV2 microglial cells were cultured in 24-well plates, pre-incubated for 3 h with different concentrations of compounds, and then stimulated for 18 h with LPS (Sigma-Aldrich). Supernatant of the culture media (100 μL) was collected to determine the PGEBV2 cells were lysed in 20 mM Tris-HCl buffer (pH 7.4) containing a protease inhibitor mixture . The protein concentration was determined using the Lowry protein assay kit . An equal amount of protein from each sample was resolved using 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrophoretically transferred to the Hybond enhanced chemiluminescence (ECL) nitrocellulose membrane . The membrane was blocked using 5% skim milk and subsequently incubated with the primary antibody  and horseradish peroxidase-conjugated secondary antibody, followed by ECL detection .post hoc test. Statistical analysis was performed using GraphPad Prism software, version 3.03 .Data expressed as the mean ± standard deviation (SD) of at least three independent experiments. Three or more groups were compared using one-way analysis of variance followed by the Newman-Keuls P. trimera was suspended in H2O and successively partitioned with CHCl3 and EtOAc to give CHCl3-, EtOAc-, and H2O-soluble fractions. The CHCl3- and EtOAc-soluble fractions were subjected to multiple chromatographic steps over silica gel and reversed phase C18, yielding compounds 1–7. The structures of these compounds were identified as: ostruthin (1)      (Wattanapiromsakul & Waterman 7)  is cytotoxic toward two human pancreatic cancer cell lines, including PANC-1 and PSN-1 cells in nutrient-deprived medium . Thus, for all subsequent experiments, the concentrations of the tested compounds were used within the range of 5–40 μM.To exclude the possibility that our results could be misinterpreted by cytotoxic effects, we used the MTT assay to evaluate the viability of BV2 cells treated with various concentrations (5–40 μM) of isolated compounds. As the result, all of the tested compounds did not exhibit significant cytotoxicity in BV2 microglia cells at the tested concentrations (2 (PGE2) and pro-inflammatory cytokines (e.g. tumor necrosis factor-α), interleukins, and cytotoxic factors  of compounds 1–4, 6, and 7 followed by treatment with LPS (1 μg/mL) for 18 h. As shown in 1–4, and 7 for 3 h, the production of NO, as indicated by the nitrite concentration, was decreased in a dose-dependent manner, with IC50 values ranging from 9.8–46.8 μM , prostaglandin E–46.8 μM . The PGEwith LPS . However–52.8 μM . Compoun2 are pro-inflammatory mediators produced by their inducible enzymes iNOS and COX-2, respectively. These mediators play key roles in the activation of macrophages during the inflammation response  , ninhvanin (2), 8-geranyl-7-hydroxycoumarin (3), 6--7-hydroxycoumarin (4), 6--7-hydroxycoumarin (5), 6-(2-hydroxyethyl)-2,2-dimethyl-2H-1-benzopyran (6), and luvangetin (7). Although some pharmacological effects of several coumarins have been reported as aforementioned, their anti-inflammatory effects in LPS-stimulated BV2 microglia and protective activity against glutamate-induced toxicity in HT22 cells have yet to be elucidated. In addition to the first report of compounds 3–5 and 7 from the Paramignya genus and the NMR data of compound 4, the present study reported the anti-inflammatory effects of the isolated coumarins for the first time. The results demonstrated that compounds 1–4, and 7 inhibited the NO and PGE2 production in LPS-stimulated BV2 cells in a dose-dependent manner. Furthermore, the inhibitory effects of compounds 1 and 2 on NO and PGE2 production were confirmed by the inhibition of LPS-induced iNOS and COX-2 expression. These results provide a scientific rationale for the use of P. trimera in the prevention and treatment of neuroinflammatory diseases and suggest that ostruthin (1) and ninhvanin (2) should be considered as lead compounds for further development as new anti-neuroinflammatory agents.In summary, chemical investigation of the stems of


The biological activity of the compounds against the root rot fungus Heterobasidion occidentale, as well as against Fusarium oxysporum and Penicillium canescens, was also investigated, but only o-orsellinaldehyde was found to have any antifungal activity in the concentration range tested.Three cyclopentanoids (phlebiopsin A–C), one glycosylated  Heterobasidion are, by far, the most widespread and destructive pathogens + .Compound 2 was obtained as a yellowish oil; 1.03 mg; [α]c 0.09, CH3OH); UV (CH3OH) λmax(log ε) 353 (3.9), 273 (4.0), 246 (4.0), 203 (4.3) nm; 1H- and 13C-NMR in MeOH-d4, see m/z 281.0808 [M + H]+ .Compound 3 was obtained as a yellow-brownish oil; 1.29 mg; [α]c 0.12, CH3OH); UV (CH3OH) λmax(log ε) 288 (9.6), 254 (10.0), 205 (10.4) nm; 1H- and 13C-NMR in THF-d8, see m/z 295.0970 [M + H]+ .Compound 4 was obtained as a yellow-brownish oil; 3.18 mg; [α]c 0.16, CH3OH); 1H- and 13C-NMR in acetone-d6, see m/z 477.1521 [M + Na]+ .Compound


Replacement of the N-acetyl side chain of agomelatine by oxathiadiazole-2-oxide (compound 3), oxadiazole-5(4H)-one (compound 4), tetrazole (compound 5), oxazolidinone (compound 7a), pyrrolidinone (compound 7b), imidazolidinedione (compound 12), thiazole (compounds 13 and 14) and isoxazole moieties (compound 15) led to a decrease of the melatoninergic binding affinities, particularly at MT1. Compounds 7a and 7b exhibiting nanomolar affinity towards the MT2 receptors subtypes have shown the most interesting pharmacological results of this series with the appearance of a weak MT2-selectivity.Novel conformationally restricted analogues of agomelatine were synthesized and pharmacologically evaluated at MT N-acetyl-5-methoxytryptamine  ncertain . Nonethe the rat  and in t1, MT2 and 3MT biological functions, over the last years several ligands were synthesized Iodomelatonin (2200 Ci/mmol) was purchased from NEN . Other drugs and chemicals were purchased from Sigma–Aldrich .2-[125I]Iodomelatonin binding assay conditions were essentially as previously described [1 or MT2 diluted in binding buffer  to 2-[125I]iodomelatonin (20 pM for MT1 and MT2 receptors expressed in CHO cells) and the tested drug. Non-specific binding was defined in the presence of 1 µM melatonin. After 120 min incubation at 37 °C, reaction was stopped by rapid filtration through GF/B filters presoaked in 0.5% (v/v) polyethylenimine. Filters were washed three times with 1 mL of ice-cold 50 mM Tris–HCl buffer, pH 7.4. Data from the dose–response curves (seven concentrations in duplicate) were analysed using the program PRISM  to yield IC50 (inhibitory concentration 50). Results are expressed as pKi (pKi = −Log10 (Ki)) with Ki = IC50/1 + ([L]/KD), where [L] is the concentration of radioligand used in the assay and KD, the dissociation constant of the radioligand characterizing the membrane preparation.2-+.N-[(Ethoxycarbonyl)oxy]-2-(7-methoxynaphth-1-yl)acetamidoxime (2). Ethyl chloroformate  was added dropwise to a suspension of 1  and triethylamine  in 25 mL of THF. Stirring was maintained for 3 h at ambient temperature. The reaction mixture was then filtered and the filtrate was evaporated under reduced pressure. The crude was purified by column chromatography  to give 2.3 g (88% yield) of 2 as a pink solid, mp: 104–106 °C. 1H-NMR  δ: 1.22 , 3.82 , 3.91 , 4.14 , 6.13 , 7.16 , 7.28 , 7.51 , 7.73 , 7.64 , 7.81 . IR (cm−1) 1680 (C=N), 1730 (C=O), 3340, 3460 (NH). MS (EI): m/z = 303 [M+H]+.4-[(7-Methoxynaphth-1-yl)methyl]-3H-1,2,3,5-oxathiadiazole 2-oxide (3). Thionyl chloride  in methylene chloride (2 mL) was added dropwise to a mixture of 1  in methylene chloride (20 mL) and pyridine  at 0 °C. After stirring for 3 h at room temperature, the reaction mixture was poured into water and extracted with ethyl acetate. The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure. The residue was recrystallized from cyclohexane/toluene and ethyl ether/petroleum ether to afford 791 mg (47% yield) of 4 as a white solid, mp: 121–123 °C (decomp.). 1H-NMR  δ: 3.88 , 4.36 , 4.42 , 7.21 , 7.29–7.49 , 7.78–7.94 , 11.40 . IR (cm−1) 1620 (C=N), 3300 (NH). MS (EI): m/z = 277 [M+H]+. Anal. Calcd for C13H12N2O3S: C, 56.51%; H, 4.38%; N, 10.14%. Found: C, 56.55%; H, 4.43%, N, 10.15%.3-[(7-Methoxynaphth-1-yl)methyl]-1,2,4-oxadiazole-5(4H)-one (4). Potassium carbonate  was added to a solution of 2  in DMF (10 mL). After 5 h of stirring at 60 °C, the reaction mixture was poured into water and extracted twice with ethyl acetate. The aqueous phase was acidified with a 3 M HCl solution and extracted with ethyl acetate. The combined organic phases were washed with water and brine, dried (MgSO4), filtered, and concentrated under reduce pressure. The residue was recrystallized from toluene to give 712 mg of 3 (yield 70%) as a white solid, mp: 185–187 °C (decomp.). 1H-NMR  δ:3.89 , 4.33 , 7.23 , 7.34 , 7.39 , 7.44 , 7.83 , 7.89 , 11.40 . IR (cm−1) 1725 (C=N), 3440 (NH). MS (EI): m/z = 257 [M+H]+. Anal. Calcd for C14H12N2O3: C, 65.61%; H, 4.72%; N, 10.93%. Found: C, 65.64%; H, 4.76%; N, 10.90%.5-(7-Methoxynaphth-1-yl methyl)-1H-tetrazole (5). A mixture of 2-(7-methoxynapht-1-yl)acetonitrile , sodium azide  and tributyltin chloride  in dry DMF (20 mL) was heated to reflux and monitored by TLC until the reaction was complete (~7 h). After being cooled to room temperature, 1 M HCl (50 mL) was added to precipitate the crude product. The white solid product was collected, washed with water and ether, and dried with phosphorous pentoxide under vacuum and recrystallized from cyclohexane affording 1.21 g (50% yield) of tetrazole 5 as a beige solid, mp: 177–179 °C (decomposition). 1H-NMR  δ: 3.88 , 4.74 , 7.22 , 7.31–7.47 , 7.83 , 7.90 , 7.81 . MS (EI): m/z = 241 [M+H]+.N-(2-Chloroethyl)oxycarbonyl)-2-(7-methoxynaphth-1-yl)ethylamine (6a). 2-Chloroethyl chloroformate  and triethylamine  were added to a solution of 2-(7-methoxynaphth-1-yl)ethylamine hydrochloride  in THF (30 mL). The reaction mixture was stirred at room temperature for 36 h. After filtration, the filtrate was concentrated under vacuum and purified by column chromatography (SiO2) using cyclohexane/ethyl acetate; 8/2 as eluant to gave 1.7 g (65% yield) of carbamate 6a as a white solid, mp: 73–75 °C. 1H-NMR  δ: 3.04–3.34 , 3.80 , 3.94 , 4.24 , 7.18 , 7.22–7.42 , 7.53 , 7.60 , 7.72 , 7.84 . MS (EI): m/z = [M+H]+.N-[(2-(7-methoxynaphth-1-yl)]-1,3-oxazolidin-2-one (7a). A mixture of carbamate 6a  and 20% NaOH  in THF (20 mL) were refluxed for 39 h. The residue was evaporated to dryness, water was added, and the mixture was stirred and extracted with ethyl acetate. The combined organic phases were washed with water (50 mL), dried (MgSO4), filtered and concentrated under vacuum. The residue was purified by column chromatography  to afford 868 mg (82% yield) of 7a as a white solid, mp 78–80 °C. 1H-NMR  δ: 3.24 , 3.47 , 3.56 , 3.92 , 4.22 , 7.17 , 7.30 , 7.36 , 7.46 ; 7.72 , 7.83 . MS (EI): m/z = 272 [M+H]+.2) using ether as eluant.A solution of agomelatine  in DMSO (100 mL) was refluxed for 15 h. The reaction mixture was poured into ice and extracted twice with ether, organic phase was washed with water and brine and then concentrated under reduced pression. The crude was purified by column chromatography ethanone (9a). Recrystallized from isopropyl ether to furnish 1.59 g (38% yield) as a white solid, mp: 107–108 °C. 1H-NMR  δ: 1.88 , 3.44 , 3.81 and 3.87 , 3.93 and 3.97 , 5.11 and 5.20 , 7.21–7.30 , 7.34 and 7.41 , 7.69–7.73 , 7.77 and 7.83 . IR (cm−1) 1625 (C=O). MS (EI) m/z = 256 [M+H]+. Anal. Calcd for C16H17NO2: C, 75.27%; H, 6.71%; N, 5.49%. Found: C, 75.25%; H, 6.72%; N, 5.48%.1-ethanone (9b). Recrystallized from isopropyl ether, 9b was obtained with 2% yield as a white solid, mp: 141–143 °C. 1H-NMR  δ: 2.14 , 3.10 , 3.84 , 3.92 , 4.75 , 7.16 , 7.17 , 7.27 , 7.69 , 7.81 . IR (cm−1) 1620 (CO). MS (EI): m/z = 256 [M+H]+. Anal. Calcd for C16H17NO2: C, 75.27%; H, 6.71%; N, 5.49%. Found: C, 75.29%; H, 6.72%; N, 5.45%.2-(7-Methoxynaphth-1-yl)ethanol (10). Thionyl chloride  was added dropwise to a solution of (7-methoxynaphth-1-yl) acetic acid  in methanol (350 mL) at 0 °C. After stirring for 5 h, the reaction mixture was evaporated under reduced pressure. The residue was dissolved in ethyl acetate and washed with 10% aqueous potassium carbonate solution and water. The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure affording the intermediate ester. The residue was dissolved in ether (100 mL) and added dropwise to a suspension of lithium aluminium hydride  in ether (200 mL) at 0 °C. The stirring was maintained at room temperature for 4 h. Water (50 mL) and 20% NaOH aqueous solution (12 mL) were added and the mixture was stirred and filtered. After evaporation under reduced pressure, the residue was recrystallized from cyclohexane to furnish 13.7 g (73% yield) of alcohol 10 as a white solid, mp: 79–81 °C. 1H-NMR  δ: 3.30 , 3.94 , 3.99 , 7.17 , 7.24–7.38 , 7.68 , 7.77 . IR (cm−1) 3000–3330 (OH). MS (EI): m/z = 203 [M+H]+.2-(7-Methoxynaphth-1-yl)acetaldehyde (11). To a stirred solution of alcohol 10  in anhydrous CH2Cl2 (300 mL) under argon atmosphere was added Dess-Martin periodinane  and stirred at room temperature for 5 h. The reaction mixture was quenched by adding saturated Na2S2O4 (60 mL) and saturated NaHCO3 (8 mL). The heterogeneous mixture was extracted with CH2Cl2 and the organic layer was washed with saturated NaHCO3 and water. The combined organic layers were dried over MgSO4 and the solvent removed under reduced pressure. The residue was purified by column chromatography  to give 5.2 g (87% yield) of 11 as a yellow oil. 1H-NMR  δ: 3.91 , 4.03 , 7.08–7.87 , 9.73 . IR (cm−1) 1700 (C=O). MS (EI): m/z = 201 [M+H]+.5-[(7-Methoxynaphth-1-yl)methyl]-imidazolidin-2,4-diones (12). Ammonium carbonate  and potassium cyanide  were added to a stirred solution of aldehyde 14  in 20 mL (16 mL/4 mL) of ethanol/water, the mixture was refluxed overnight. After cooling, the reaction mixture was poured into cold water. The resulting precipitate was filtrated, washed with water and recrystallized from isopropyl ether affording 400 mg (42% yield) of 15 as a white solid, mp: 236–238 °C (decomp.). 1H-NMR  δ: 3.26 , 3.92 , 4.44 , 7.06–7.97 , 10.56 . IR (cm−1) 1680, 1750 (C=O), 3270 (NH). MS (EI): m/z = 271 [M+H]+. Anal. Calcd for C15H14N2O3: C, 66.65%; H, 5.22%; N, 10.36%. Found: C, 65.68%; H, 5.25%, N, 10.38%.11  and the appropriate heterocyclic amine (10 mmol) in methanol (20 mL) and 0.2 mL of DMF, were added a solution of sodium cyanoborohydride  and zinc iodide  in methanol (5 mL). The reaction was stirred at room temperature for 5 h and concentrated under reduced pressure to dryness, hydrolyzed and extracted with ethyl acetate. The organic layer was dried (MgSO4), filtered, and concentrated under reduced pressure.To a mixture of compound 2-[2-(7-Methoxynaphth-1-yl)]-ethylamino]-1,3-thiazole hydrochloride (13). The crude was purified by column chromatography (SiO2) using cyclohexane/ethyl acetate; 7/3 as eluant, treatment with gaseous HCl in ether provided 280 mg (35% yield) of hydrochloride salt 13 as a white solid, mp: 156–158 °C. 1H-NMR  δ: 3.38 , 3.81 , 3.93 , 6.92 , 7.19 , 7.25–7.49 , 7.75 , 10.22 . IR (cm−1) 2500–3160 (N+H). MS (EI): m/z = 285 [M+H]+. Anal. Calcd for C16H17ClN2OS: C, 59.90%; H, 5.34%; N, 8.73%. Found: C, 59.94%; H, 5.32%, N, 8.75%.2-[2-(7-Methoxynaphth-1-yl)ethylamino]-5-methyl-1,3-thiazole (14). Recrystallized from cyclohexane to furnish 364 mg (46% yield) of 14 as a white solid, mp: 150–152 °C (decomp.). 1H-NMR  δ: 2.22 , 3.28 , 3.47 , 3.95 , 6.71 , 7.11–7.89 . IR (cm−1) 3170 (NH). MS (EI): m/z = 299 [M+H]+. Anal. Calcd for C17H18N2OS: C, 68.43%; H, 6.08%; N, 9.39%. Found: C, 68.39%; H, 6.13%, N, 9.36%.3-[2-(7-Methoxynaphth-1-yl)ethylamino]-5-methyl-1,2-isoxazole hydrochloride (15). Recrystallized from cyclohexane to afford 333 mg (42% yield) of 15 as a white solid, mp 144–146 °C (decomp.). 1H-NMR  δ: 2.24 , 3.18–3.39 , 3.93 , 5.67 , 7.15 , 7.21–7.39 , 7.65 , 7.69 , 7.81 . IR (cm−1) 2200–3000 (N+H). MS (EI): m/z = 283 [M+H]+. Anal. Calcd for C17H19Cl N2O2: C, 64.05%; H, 6.01%; N, 8.79%. Found: C, 64.07%; H, 6.04%, N, 8.76%.1 and MT2 biological functions, we synthesized and pharmacologically evaluated a new series of constrained naphthalenic compounds. Indeed, replacement of the N-acetyl side chain of agomelatine by oxathiadiazole-2-oxide, oxadiazole-5(4H)-one, oxazolidinone, 2-oxopyrrolidine, imidazolidin-2,4-dione, thiazole, 5-methyl-1,3-thiazole, or 5-methyl-1,2-isoxazole hydrochloride resulted in a decrease of the melatoninergic binding affinities particularly towards the MT1 receptors leading to the appearance of a weak MT2-selectivity. Compound 7b conserves good affinity for both melatonin receptors subtypes and exhibited a selectivity of about 33-fold for the MT2 receptor subtype.In the search for pharmacological tools for the elucidation of MT


Viburnum mongolicum afforded six new nor-dammarane triterpenoids: 3β,12β-dihydroxy-25,26,27-trinordammara-22-en-24,20-olide (1), 3β,12β-dihydroxy-24α-methoxy-25,26,27-trinordammara-20,24-epoxy (2), 3β-O-acetyl-12β-hydroxy-23,24,25,26,27-hexanordammarane-20-one (3), 12β-O-acetyl-15α-hydroxy-17β-methoxy-3-oxo-20,21,22-23,24,25,26,27-octanordammanrane (4), 12β-O-acetyl-15α,17β-dihydroxy-3-oxo-20,21,22-23,24,25,26,27-octanordammanrane (5), and 12β,15α-dihydroxy-3-oxo-17-en-20,21,22-23,24,25,26,27-octanordammanrane (6), together with two known nor-dammarane triterpenoids, 12β-hydroxy-3-oxo-24α-methoxy-25,26,27-trinordammara-20,24-epoxy (7) and 3β,12β-dihydroxy-23,24,25,26,27-hexanordammarane-20-one (8). The structures of the isolated compounds were established based on 1D and 2D  NMR spectroscopy, in addition to high resolution mass spectrometry. The isolated compounds were tested in vitro for cytotoxic potential against seven tumor cell lines and radical scavenging activities. Compounds 4–6 exhibited significant cytotoxic activities against all tested tumor cell lines and radical scavenging activities against ABTS·+ radicals comparable with the standard drug Trolox.The ethanol extract of the whole plants of  Viburnum, which belongs to the family of Caprifliaceae, comprises more than 230 species +, consistent with the molecular formula C27H42O4 , accounting for seven degrees of unsaturation. The IR spectrum indicated the presence of OH (3450 cm−1) and C=O (1702 cm−1) groups, as well as a C=C bond (1640 cm−1). 1H-NMR signals for six methyl groups  and for two oxygenated CH groups [δH 3.21 (H-3) and 3.62 (H-12)] were observed , five quaternary carbons and one C=O group (δC 172.1). The distinct difference was that the ketone carbon signal at C-3 in cylindrictone B was replaced by an oxygenated methine  in 1, which was supported by the observation of a CH2-CH2-CH-O spin system in the 1H,1H-COSY spectrum and the HMBC correlations between C-3 (δC 78.5) with H-1 (δH 1.03 and 1.69), H-5 (δH 0.78), H-28 (δH 0.79) and H-29 (δH 0.99)  . Analysi2 was obtained as a white amorphous powder. Its positive HR-ESI-MS spectrum showed a quasimolecular ion peak at m/z 471.3454 [M+Na]+, consistent with the molecular formula C28H48O4 , accounting for five degrees of unsaturation. The IR spectrum absorptions at 3425 cm−1 indicated the presence of the OH group. The 1H-NMR spectrum , except for the replacement of the ketone carbonyl of C-3 by a hydroxymethylene moiety . This was supported by HMBC correlations from the H-3 to C-1 (δC 38.8), C-5 (δC 56.0), C-28 (δC 15.2), and C-29 (δC 27.9). These HMBC correlations of the oxygenated H-24 (δH 4.93) with the oxygenated quaternary C-atom C-20 (δC 80.4) and methoxy (δC 55.4) suggested that C-20 and H-24 were linked via O bridge to form a lactol ring, and the methoxy group was placed at C-24. The relative configuration of 2 was confirmed by a ROESY experiment. In the ROESY spectrum, the correlations of H-3/H-1ax and H-5 indicated that the OH-3 was β-oriented. The α-orientation of the methoxy group at C-24 was determined to be identical to that of 7 on the basis of ROESY correlation of H-24 with Hax-22 and of H-21 with Heq-22. Thus, the structure of 2 was established as 3β,12β-dihydroxy-24α-methoxy-25,26,27-trinordammara-20,24-epoxy.Compound spectrum  and 13C-NMR data , along w3, a white amorphous powder, exhibited a molecular formula of C26H42O4, based on the HRESIMS spectrum which showed a pseudomolecular ion at m/z 441.2978 [M+Na]+ . The IR absorptions at 3440 and 1705 cm−1 indicated the presence of OH and C=O groups, respectively. Its NMR data were similar to those of 2, suggesting that 3 possessed a dammarane-type triterpenoid skeleton with the exception of the side chain resonances. The presence of one isolated aceto-group on C-17 was deduced by the HMBC correlation of H-17 (δH 2.88) with Me-21 (δC 29.4), and of H-13 (δH 2.08) and H-16 (δH 1.72 and 2.04) with the ketone of C-20 (δC 215.4)  with C=O (δC 172.9) of acetyl group and of C-3 with H-1, H-5, H-28 and H-29. H-3 and H-12 were found to be α-oriented on the basis of ROESY cross-peaks of H-3/H-1ax and H-5, and of H-12/H-9 and H-30  . Compariand H-30 . Thus, c4 was obtained as a white amorphous solid. The HRESIMS displayed a pseudomolecular ion at m/z 443.2774 [M+Na]+  consistent with a molecular formula of C25H40O5, corresponding to six degrees of unsaturation. The IR of 4 showed the strong broad absorption of OH (3450 cm−1) and C=O (1735 cm−1). The 1H-NMR spectrum exhibited the signals of six singlet Me, one OMe, and three oxygenated CH . The 13C-NMR and DEPT spectra showed the signals of six quaternary carbons , six CH, six CH2, six Me, and one MeO). The unsaturation degrees (including two C=O) indicated that the skeleton of 4 was the derivative of a four-ring triterpenoid. Based on the NMR data, 4 had a 20,21,22,23,24,25,26,27-octanordammarane triterpenoid skeleton +). HR-ESI-MS (pos.) m/z: 453.2984 .3β,12β-Dihydroxy-24α-methoxy-25,26,27-trinordammara-20,24-epoxy (2). White amorphous powder.  = +27.1 . UV (CDCl3) λmax(log ε): 205 (0.56) nm. IR (KBr) νmax 3425, 2945, 2876, 1630, 1383, 1276, 1033 cm−1. 1H-NMR  data see 13C-NMR  data see m/z: 448 ([M]+). HR-ESI-MS (pos.) m/z: 471.3454 .3β-O-Acetyl-12β-hydroxy-23,24,25,26,27-hexanordammarane-20-one (3). White amorphous powder.  = +46.3 . UV (CDCl3) λmax(log ε): 200 (0.72) nm. IR (KBr) νmax 3440, 2968, 2945, 2870, 1705, 1463, 1384, 1365, 1177, 1042, 1007 cm−1. 1H-NMR  data see 13C-NMR  data see m/z: 418 ([M]+). HR-ESI-MS (pos.) m/z: 441.2978 .12β-O-Acetyl-15α-hydroxy-17β-methoxy-3-oxo-20,21,22-23,24,25,26,27-octanordammanrane (4). White amorphous powder.  = +15.4 . UV (CDCl3) λmax(log ε): 206 (0.66) nm. IR (KBr) νmax 3450, 1735, 1635, 1345 cm−1. 1H-NMR  data see 13C-NMR  data see m/z: 420 ([M]+). HR-ESI-MS (pos.) m/z: 443.2774 .12β-O-Acetyl-15α,17β-dihydroxy-3-oxo-20,21,22-23,24,25,26,27-octanordammanran (5). White amorphous powder.  = +11.4 . UV (CDCl3) λmax(log ε): 205 (0.69) nm. IR (KBr) νmax 3445, 1730, 1715, 1625, 1034 cm−1. 1H-NMR  data see 13C-NMR  data see m/z: 406 ([M]+). HR-ESI-MS (pos.) m/z: calcd. 429.2613 .12β,15α-Dihydroxy-3-oxo-17-en-20,21,22-23,24,25,26,27-octanordammanran (6). White amorphous powder.  = +10.7 . UV (CDCl3) λmax(log ε): 202 (0.54) nm. IR (KBr) νmax 3444, 1736, 1630, 1034 cm−1. 1H-NMR  data see 13C-NMR  data see m/z: 346 ([M]+). HR-ESI-MS (pos.) m/z: 369.2405 .1–8 were subjected to cytotoxic evaluation against A-549 cells (lung cancer), BGC-823 cells (human gastric carcinoma), HepG2 cells (human hepatocellular carcinoma), HL-60 (human myeloid leukemia), MCF-7 cells (human breast cancer), SMMC-7721 (hepatocellular carcinoma), and W480 (colon cancer) by employing the revised MTT method as described in the literature sample/[DPPH]blank). A calibrated Trolox  standard curve was also made. The percentage of remaining DPPH against the standard concentration was then plotted in an exponential regression, to obtain the amount of antioxidant necessary to decrease the initial DPPH concentration by 50% (IC50).Microplate DPPH assay was performed as described by . Briefly·+ radical scavenging activity was determined according to Re [·+ cation radical was produced by the reaction between 7 mM ABTS in H2O and 2.45 mM potassium persulfate, stored in the dark at room temperature for 16 h. Before usage, the ABTS·+ solution was diluted with phosphate buffer  to get an absorbance of 0.800 ± 0.035 at 734 nm. The solution is stable for 2 days. Different concentrations of extracts or pure compounds in methanol solution were added to 1 mL of ABTS·+ solution. The mixture was incubated at 37 °C in the dark. After 30 min of incubation, the percentage inhibition of absorbance at 734 nm was calculated for each concentration relative to a blank absorbance (methanol). All determinations were carried out at least three times, and in triplicate. The capability to scavenge the ABTS·+ radical was calculated using the following equation:Control is the initial concentration of the ABTS·+ and ASample is absorbance of the remaining concentration of ABTS·+ in the presence of different compounds. Trolox was used as reference. The stock concentrations of Trolox and of different compounds tested are the same as reported in DPPH assay.ABTSng to Re . The ABTV. mongolicum resulted in the isolation of six new nor-dammarane triterpenoids, 3β,12β-dihydroxy-25,26,27-trinordammara-22-en-24,20-olide (1), 3β,12β-dihydroxy-24α-methoxy-25,26,27-trinordammara-20,24-epoxy (2), 3β-O-acetyl-12β-hydroxy-23,24,25,26,27-hexanordammarane-20-one (3), 12β-O-acetyl-15α-hydroxy-17β-methoxy-3-oxo-20,21,22-23,24,25,26,27-octanordammanrane (4), 12β-O-acetyl-15α,17β-dihydroxy-3-oxo-20,21,22-23,24,25,26,27-octanordammanrane (5), and 12β,15α-dihydroxy- 3-oxo-17-en-20,21,22-23,24,25,26,27-octanordammanrane (6), together with two known compounds, 12β-hydroxy-3-oxo-24α-methoxy-25,26,27-trinordammara-20,24-epoxy (7) and 3β,12β-dihydroxy-23,24,25,26,27-hexanordammarane-20-one (8). Previous studies have indicated that significant antioxidant property of some natural products might be responsible for their antitumor property [in vitro for their cytotoxic activities against seven tumor cell lines and radical scavenging properties. Octanordammarane tertripenoids 4–6 showed particular cytotoxic activities against all tested tumor cell lines, with low IC50 values of less than 20 μM, and exhibited radical scavenging activities against ABTS·+ radicals with IC50 values comparable with that of the standard drug Trolox.A chemical investigation of the 80% EtOH extract of the dried whole plants of


Psoralea Fructus (PF) have demonstrated some anti-Alzheimer effects both in vitro and in vivo. To further reveal the active compounds of PF and their mechanisms regulating key targets of AD, in this study, we identified four prenylated compounds from the 70% ethanolic aqueous extract of PF, namely bavachin, bavachinin, bavachalcone, and isobavachalcone. Multi-target bioactivity analysis showed that these compounds could differentially inhibit neuroinflammation, oxidative damage, and key AD-related protein targets, such as amyloid β-peptide 42, β-secretase, glycogen synthase kinase 3β, and acetylcholinesterase. These compounds may generate beneficial effects in AD prevention and treatment.Alzheimer’s disease (AD) is an age-related neurodegenerative disease that is mediated by multiple signaling pathways. In recent years, the components of  Cullen corylifolium (L.) Medik. , is a medicinal plant widely distributed in China, India and Southeastern Asian countries. Psoralea Fructus (PF), the dried mature fruits  of this plant, has been used as a traditional Chinese medicine for the treatment of erectile dysfunction, pollakiuria, nephritis, osteoporosis, and cerebral and cardiac vascular diseases, etc. +. 1H-NMR  δ: 5.39 , 3.07 , 2.62 , 7.47 , 6.40 , 7.32 , 6.81 , 3.18 , 5.27 , 1.71 , 1.66 , 9.57 , 10.61 ; 13C-NMR  δ: 79.2 (C-2), 43.5 (C-3), 190.4 (C-4), 122.5 (C-5), 122.9 (C-6), 157.8 (C-7), 102.3 (C-8), 161.6 (C-9), 113.3 (C-10), 132.1 (C-1’), 128.4 , 115.4 , 162.7 (C-4’), 27.4 (C-1”), 127.0 (C-2”), 129.7 (C-3”), 25.8 (C-4”), 17.8 (C-5”).Bavachinin (2): Needles (MeOH). EI-MS m/z 338 [M]+. 1H-NMR  δ: 5.37 , 3.05 , 2.79 , 7.69 , 6.44 , 3.84 , 7.32 , 6.92 , 3.24 , 5.27 , 1.73 , 1.69 ; 13C-NMR  δ: 79.8 (C-2), 43.9 (C-3), 192.0 (C-4), 127.1 (C-5), 125.0 (C-6), 164.4 (C-7), 98.8 (C-8), 162.6 (C-9), 113.7 (C-10), 55.7 (7-OMe), 130.5 (C-1’), 127.9 , 115.7 , 156.5 (C-4’), 27.7 (C-1”), 121.6 (C-2”), 133.1 (C-3”), 25.8 (C-4”), 17.7 (C-5”).3): Square crystal (MeOH). EI-MS m/z 338 [M]+. 1H-NMR  δ: 7.43 , 7.82 , 7.54 , 6.90 , 6.44 , 7.59 , 3.26 , 5.28 , 1.77 , 1.74 , 3.87 , 13.58 ; 13C-NMR  δ: 191.9 (C=O), 118.0 (C-α), 144.1 (C-β), 127.7 (C-1), 130.6 , 116.0 , 158.1 (C-4), 113.3 (C-1’), 164.2 (C-2’), 99.3 (C-3’), 165.2 (C-4’), 121.8 (C-5’), 129.7 (C-6’), 28.0 (C-1”), 122.3 (C-2”), 133.0 (C-3”), 17.8 (C-4”), 25.8 (C-5”), 55.7 (4’-OMe).Bavachalcone : Yellowish amorphous powder (petroleum ether-EtOAc/4:1). EI-MS m/z 324 [M]+.1H-NMR  δ: 7.54 , 7.71 , 7.53 , 6.78 , 6.37 , 7.75 , 3.27 , 5.18 , 1.71 , 1.60 ; 13C-NMR  δ: 193.7 (C=O), 118.6 (C-α), 145.3(C-β), 127.9 (C-1), 131.7 , 116.9 , 161.4(C-4), 114.5 (C-1’), 163.6 (C-2’), 116.6 (C-3’), 165.1 ( C-4’), 108.2 (C-5’), 130.4 (C-6’), 22.5 (C-1”), 123.6 (C-2”), 131.8 (C-3”), 26.0 (C-4”), 17.9 (C-5”).2 and 95% air at 37 °C. For NO inhibition assay, BV-2 cells were seeded in 96-well culture plates at a density of 2 × 104 cells/well for 24 h, and then stimulated with LPS (0.5 μg/mL) and treated with various concentrations of assay compounds for 24 h. After that, the cell culture supernatant (100 μL) was collected to react with Griess reagent [2 was used to generate a standard curve, and NO production was determined by measuring the optical density at 540 nm by a Multiskan MK3 Automated Microplate Reader . The experiments were performed in parallel for three times, and curcumin was used as a positive control. Cell viability (>95%) was assessed using an MTT assay. The IC50 value of each compound is defined as the concentration (μM) that caused 50% inhibition of NO production. The IC50 values were calculated by the software SPSS 16.0 .BV-2 microglial cell line was purchased from the cell bank of Peking Union Medical College . Cells were maintained in DMEM containing 10% FBS, in a constant humidity atmosphere of 5% CO reagent  (100 μL)1–4 at the assayed concentrations (6.25–25.00 µM) for 1 h, and then stimulated with LPS for 24 h. The culture media were collected to determine the levels of IL-6 and TNF-α present in each sample using commercially available enzyme-linked immunosorbent assay (ELISA) kits . The assays were performed according to the manufacturer’s instructions.BV2 cells were treated with or without compounds 2O2 solution for 2 h. Curcumin was used as a positive control. The cell viability was measured using MTT assay to evaluate the oxidative damage of the cells and the protective effects of the compounds.PC-12 cells were maintained as BV-2 cells as described above. MTT assay was performed for cell toxicity analysis of the compounds. For anti-oxidative assay, PC-12 cells were seeded into 96-well culture plate for 24 h and incubated with different concentrations of compounds for 24 h followed by treatment with 450 µM of HBACE-1 and AChE activity assays were performed using assay kits from Sigma-Aldrich . GSK-3β activity assays were conducted using recombinant human GSK-3β and ADP-Glo™ kinase assay kit from Promega . All of the assays were carried out according to the manufacturer’s instructions. Epigallocatechin gallate (EGCG), tacrine, and tideglusib were used as positive controls, respectively.Synthetic Aβ42 peptide  was prepared and diluted in PBS (pH 7.4) to 50 μM, as described previously . The peppubchem.ncbi.nlm.nih.gov). The 3D NMR structure of Aβ42 monomer (PDB ID: 1Z0Q) were downloaded from the website of Protein Data Bank . Docking studies were conducted using Autodock Vina [All the 3D structures of the ligands were downloaded from the website of PubChem . Statistical significances were calculated by Student′s 2O2-induced neurodamage, and spontaneous Aβ42 aggregation. These new findings provided useful information for predicting anti-AD effective constituents of PF and their potential clinical values in the treatment of AD. The active ingredient bavachalcone has the potential to be developed into a drug and further investigation in vivo is needed.Taken together, the four structurally related PF compounds that were identified in this study demonstrated multi-target anti-AD activities in vitro. Bavachalcone was found to be the most potent inhibitor which exerted comparable effects as curcumin, the well-known neuroprotective agent, on LPS-induced neuroinflammation, H


Tibouchina pulchra (Cham.) Cogn. is a plant native to Brazil whose genus and family (Melastomataceae) are poorly studied with regards to its metabolite profile. Phenolic pigments of pink flowers were studied by ultra-performance liquid chromatography with a photodiode array detector and electrospray ionization quadrupole time-of-flight mass spectrometry. Therein, twenty-three flavonoids were identified with eight flavonols isolated by preparative high-performance liquid chromatography and analysed by one- and two-dimensional nuclear magnetic resonance. Kaempferol derivatives were the main flavonols, encompassing almost half of the detected compounds with different substitution patterns, such as glucoside, pentosides, galloyl-glucoside, p-coumaroyl-glucoside, and glucuronide. Concerning the anthocyanins, petunidin p-coumaroyl-hexoside acetylpentoside and malvidin p-coumaroyl-hexoside acetylpentoside were identified and agreed with previous reports on acylated anthocyanins from Melastomataceae. A new kaempferol glucoside was identified as kaempferol-(2′′-O-methyl)-4′-O-α-d-glucopyranoside. Moreover, twelve compounds were described for the first time in the genus with five being new to the family, contributing to the chemical characterisation of these taxa. Tibouchina Aubl., the most representative genus within Melastomataceae, has approximately 460 species +, in which fragmentation resulted in m/z 625.1552 [M − 174]+, m/z 491.1176 [M − 308]+ and m/z 317.0665 [M − 482]+, corresponding to the neutral loss of acetylpentoside and p-coumaroylhexoside from a petunidin. The other anthocyanin, malvidin p-coumaroylhexoside acetylpentoside (24), exhibited a fragment of m/z 813.2243 [M + H]+, whose fragmentation resulted in m/z 639.1716 [M − 174]+, m/z 505.1336 [M − 308]+ and m/z 331.0812 [M − 482]+ consistent with the loss of acetylpentoside and p-coumaroylhexoside from malvidin -β-d-glucopyranoside -β-d-glucopyranoside -β-d-glucopyranoside -β-d-glucopyranoside -4′-O-α-d-glucopyranoside , δ 6.21 , and δ 6.46 , corresponding to a meta-coupling of these protons which were attributed to the flavonoid A-ring; and another three doublets with ortho-coupling constants at δ 6.89 , δ 6.93  and δ 8.04 , suggesting two para-substituted B-rings of flavonoids. The anomeric protons appeared at δ 5.46  and δ 4.51 . The smaller coupling constant of the latter suggests an α-linked carbohydrate. Signals between δ 3.09 and δ 3.58 were attributed to the hydrogens of the sugar moiety. The presence of a methyl group was confirmed by signals at δ 3.26  and δ 54.74 (OMe). Furthermore, the heteronuclear multiple bond correlation (HMBC) spectrum showed two relevant correlations: the first between the anomeric protons at δ 5.46 with C3 (δ 133.63) from the flavonoid C-ring, confirming the position of the sugar moiety in kaempferol 3-O-β-d-glucopyranoside , and the second one, the anomeric proton (δ 4.51) with the methyl group at δ 54.74, suggesting the presence of 2-methoxyglycosyl moiety  and C3 (δ 136.10) positions of the flavonoid, which is consistent with the presence of a free hydroxyl group at C3 +m/z 601.1183. 1H NMR : δH 12.52 , 10.87 , 10.06 , 7.94 , 6.92 , 6.77 , 6.45 , 6.21 , 5.45 , 4.28 , 4.17 , 3.51 -3.48 . 13C NMR : δC 165.36 (C-7′′′), 164.50 (C-7), 161.90 (C-5), 157.26 (C-9), 157.15 (C-2), 150.58 (C-4′); 146.11 , 138.94 (C-4′′′), 133.52 (C-3), 131.19 , 121.04 (C-1′), 120.00 (C-1′′′), 116.46 , 109.70 , 104.66 (C-10), 102.17 (C-1′′), 99.58 (C-6), 94.91 (C-8), 76.51 (C-3′′), 74.60 (C-5′′), 74.52 (C-2′′), 69.89 (C-4′′), 63.15 (C-6′′). Signal assignments were performed by comparison to similar data from the literature +m/z 601.1183. 1H NMR : δH 8.04 , 6.90 , 6.89/6.93 , 6.45 , 6.21 , 5.47 , 3.62/3.38 , 3.51 -3.48 . 13C NMR : δC 163.30 (C-7), 162.11 (C-7′′′), 160.0 (C-5), 158.40 (C-4′), 155.50 (C-9), 155.46 (C-2), 145.96 , 138.86 (C-4′′′), 133.22 (C-3), 129.54 , 124.0 (C-1′′′), 120.90 (C-1′), 114.66 , 108.45 , 102.70 (C-10), 100.38 (C-1′′), 98.02 (C-6), 93.05 (C-8), 76.74 (C-5′′), 75.87 (C-3′′), 73.68 (C-2′′), 69.48 (C-4′′), 60.68 (C-6′′). Signal assignments were performed by comparison to similar data from the literature +m/z 449.1079 and m/z 463.0865. 1H NMR  and 13C NMR  are shown in in The mixture of kaempferol 3-O-glucuronide-6′′-O-methylester (23) is a pale-yellow liquid (yield of 0.5 mg). UV λmax = 268, 320 nm. [M + H]+m/z 477.1031. 1H NMR  δH 8.02 , 6.89 , 6.45 , 6.23 , 5.47 , 3.57 . The HMBC spectrum showed three relevant correlations: the anomeric hydrogen (δ 5.47) with C3 (δ 133.58) from the flavonoid C-ring, the H5′′ (δ 3.73) from the sugar moiety and the methoxyl group (δ 3.57) both with C6′′ (δ 169.52) from the carboxyl group in the glucuronic acid. Analyses of HSQC and HMBC spectra confirmed the assignment of the carbon signals aggressed with previously reported data +m/z 611.1393. 1H NMR  δH 7.66 , 7.52 , 7.37 , 7.36 .6.83 , 6.78 , 6.38 , 6.14 , 6.13 , 5.42  and 4.12 . HSQC spectrum allowed assignment of carbon signals in agreement with data from the literature +m/z 595.1418. 1H NMR  δH 8.05 , 7.34 , 6.86 , 6.78 , 6.40 , 6.14 , 6.11 , 5.41 , 4.10 . The HMBC spectrum showed one relevant correlation confirming the p-coumaroyl position: the H6′′ (δ 4.10) from the sugar moiety with the C9′′′ from the p-coumaroyl group. HSQC and HMBC spectra reinforced the assignment of the carbon signals in accordance to the literature +m/z 287.0546. 1H NMR : δH 12.48 , 10.83 , 10.13 , 8.05 , 6.94 , 6.46 , 6.21 . 13C NMR : δC 176.36 (C-4), 164.38 (C-7), 161.15 (C-5), 159.67 (C-4′), 156.63 (C-9), 147.27 (C-2), 136.11 (C-3), 129.95 , 122.12 (C-1′), 115.91 , 103.49 (C-10), 96.68 (C-6), 93.95 (C-8) [Kaempferol (95 (C-8) ,51,63,64