R,5S)-2-phenyl-3-(2-pyridyl)-1,3-diaza-2-phosphanicyclooctan-4-one (1) and -2-phenyl-3-(2-pyridyl)-1,3-diaza-2-phosphanicyclooctane (2), have been synthesized starting from L-proline. The two ligands differ in the presence or not of a carbonyl group in the diazaphosphane ring. Their coordination chemistry towards Pd(II) was studied by reacting them with [Pd(CH3)Cl(cod)]. A different behaviour was observed: ligand 2 shows the expected bidentate chelating behaviour leading to the mononuclear Pd-complex, while ligand 1 acts as a terdentate ligand giving a dinuclear species. The corresponding cationic derivatives were obtained from the palladium neutral complexes, both as mono- and dinuclear derivatives, and tested as precatalysts for styrene dimerization, yielding E-1,3-diphenyl-1-butene regio- and stereoselectively as the sole product. A detailed analysis of the catalytic behaviour is reported.Two new chiral, enantiomerically pure, hybrid P-N ligands, namely (2 In particular, hemilable hybrid P-N multidentate ligands are able to stabilize metal ions in a variety of oxidation states and geometries. Indeed, the π-acceptor character of phosphorus atom can stabilize a metal center in a low oxidation state, while the nitrogen σ-donor ability makes the metal more susceptible to oxidative addition reactions, features that can play a crucial role in stabilizing intermediate oxidation states and/or geometries during a catalytic cycle . Af. Af1 andectively . In agreectively , both li1 and 2 were fully characterized in solution by multinuclear NMR spectroscopy Cl(cod)] . The reaction progress, monitored by 1H- and 31P-NMR spectroscopy, showed an almost instantaneous coordination of the two ligands to the metal centre: no signal of the free P-N ligand was present after 5 min from the mixing of the two species. On this basis, complexes 1a, 2a were synthesized by reacting the Pd-precursor with the P-N ligand in dichloromethane at room temperature and isolated as yellow-orange solids upon addition of n-hexane  and a downfield shift for 2a (CIS −34.0 and −20.2 ppm)  .1H- and 31P-NMR spectra of 1a and 2a suggests that from the reaction of ligands 1 and 2 with [Pd(CH3)Cl(cod)] two different complexes were obtained depending on the nature of the ligand. In particular, for ligand 2 the data indicated that the isolated species was the expected mononuclear complex [Pd(CH3)Cl(2)] (2a)  (2a) ,38. It id-P bond . 31P signals for complex 2a and the JP-CH3 values suggested that the major species was the cis isomer. This was in agreement with the complex electronic pattern: chlorido was trans to the better donor atom (phosphorus), while methyl was trans to the poorer donor (nitrogen)   38] and and 31P 1a, 2a were reacted with AgPF6 in the presence of acetonitrile, with the aim to obtain the corresponding monocationic derivatives, no clear product was isolated from the synthetic mixture. It was possible to obtain the cationic species via halogen abstraction on the neutral derivatives by using coordinating ligands stronger than acetonitrile like pyridine, 4-methylpyridine and 4-trifluoromethylpyridine  Å and the mean coordination planes formed a dihedral angle of 71.3(4)°.In fact the structural solution  provided evidence for the dinuclear nature of this complex  and also between each picoline ring and the adjacent phenyl attached to phosphorus , which stabilized the overall structure.trans-[Pd(CH3)I(PPh3)2] resulting in the exchange of the methyl group on palladium with one phenyl ring on the phosphorus to give [Pd(Ph)I(PPh3)(PPh2CH3)] as product [PF6] and the neutral derivative [Pd(CH3)Cl(2')(py)] having coordinated to Pd the pyridine in place of the nitrogen of the pyridine ring of the P-N ligand that acted as a monodentate molecule 2'.Complexes 1b-d were tested as precatalysts for CO/styrene copolymerization under standard conditions: T = 303 K, 1 atm of CO, [styrene]/[Pd] = 6,800, an excess of 1,4-benzoquinone (BQ) with respect to palladium ([BQ]/[Pd] = 40), 24 h, in 2,2,2-trifluoroethanol (TFE). No polymer was isolated at the end of the catalytic runs. After drying the reaction mixture an oil was obtained that was characterized as E-1,3-diphenyl-1-butene . By performing the catalytic reactions at T = 343 K and with [styrene]/[Pd] = 3,400 the effect of the nature of the P-N and of the pyridine-type ligand was investigated .1b. In all cases the reaction was regiospecific and stereospecific in leading to the E-1,3-diphenyl-1-butene as the sole product. The catalytic activity was affected by the nature of the P-N ligand, being all complexes with ligand 1 more active than the complex with ligand 2. For complexes with ligand 1 the nature of the pyridine-type ligand remarkably affected catalyst activity and stability. Within the first two hours of reaction the trend of the activity might be related to the Lewis basicity of the pyridine-type ligand: it increased on decreasing the Lewis basicity of the ligand, being complex 1d with the 4-CF3-pyridine the most active and 1c with the 4-CH3-pyridine the least active among the three . No codimer was formed, being E-1,3-phenyl-1-butene the unique reaction product detected, thus indicating the selective reactivity of this complex toward the vinyl arene with respect to the aliphatic alkene.Complex 7 with two cis coordination sites available for the catalysis (7 with trifluoroethanol and/or water (present in traces in the solvent). This reaction might involve benzoquinone and a rearrangement of the ligand: the amidic nitrogen might undergo an electrophilic attack with the cleavage of the related Pd-N bond followed by the coordination of the pyridine ring of the P-N ligand yielding a Pd-H monocationic species, where styrene coordination and insertion took place leading to the Pd-alkyl intermediate. On the latter the second molecule of the vinyl arene coordinated and inserted followed by β-hydrogen elimination, which led to E-1,3-phenyl-1-butene and to the Pd-H that might reenter the catalytic cycle or decompose to Pd metal , at 25 °C where not differently indicated. 31P-NMR spectra were recorded at 202.32 MHz on a Varian 500 spectrometer and automatically referenced to H3PO4. NOESY experiments were performed according to the automatic parameters of the software with a mixing time of 500 ms. Optical rotations and CD spectra were determined at 25 °C. TLC were performed on silica gel, using light petroleum - ethyl acetate or dichloromethane - methanol mixtures as the eluent. Flash chromatography was run on silica gel, 230–400 mesh using mixtures of light petroleum 40–70 °C or chloroform and ethyl acetate as the eluent. HRGC analysis were run on a SE 30 capillary column (30 m × 0.32 mm) carrier gas He 50 KPa, split 1:60. The chiral P-N ligands and the related complexes were synthesized under argon atmosphere with Schlenk technique and using freshly distilled and degassed solvents.(S)-(-)-N-(pyridine-2-yl)pyrrolidine-2-carboxamide ((S)-5). Compound (S)-4  was dissolved in dichloromethane (20 mL). The solution was cooled to 0 °C, and EDC  was added. The solution was stirred at 0 °C for 0.5 h and then 2-aminopyridine  was added in one portion. The mixture was left at room temperature and followed by TLC (EtOAc/CH3OH/NH3 8:2:0.1). After removal of the solvent under reduced pressure, the residue was purified by column chromatography on silica gel  to give Boc-(S)-5 as a white solid. [α]D –71 ; 1H-NMR : δ 1.26–1.42 , 1.69 , 1.91 , 2.24 , 3.53 , 4.32 , 7.01 , 7.68 , 8.21 , 8.28 , 9.22 ; 13C-NMR : δ 21.4, 24.8, 28.3, 47.2, 62.3, 80.96, 113.8, 119.78, 138.22, 148.0, 151.8, 155.2, 172.0; ESI-MS: 292 (M+H+), 314 (M+Na+).Boc-(S)-5  was dissolved in a trifluoroacetic acid solution  at 0 °C and the solution was stirred for 2 h at room temperature. Then the trifluoroacetic acid was neutralized by addition of Et3N  at 0 °C. The solution was washed with water, dried over anhydrous Na2SO4, and concentrated under reduced pressure obtaining (S)-5 as a yellow oil in 90% yield. [α]D –56.4 ; IR (neat) νmax: 3276, 2969, 2870, 1692, 1589, 1574, 1510, 1434, 1299, 1148, 1095, 779 cm−1; 1H-NMR : δ 1.69 , 1.97 , 2.16 , 2.54 , 3.00 , 3.84 , 6.97 , 7.64 , 8.20 , 8.24 , 10.17 ; 13C-NMR : δ 26.3, 30.9, 47.4, 61.0, 113.6, 119.7, 138.3, 148.0, 151.2, 174.4; ESI-MS: 192 (M+H+), 214 (M+Na+).(S)-(+)-N-(pyrrolidin-2-yl-methyl)pyridin-2-amine ((S)-6). To a cooled (-10 °C) and stirred solution of the amide (S)-5  in dry THF (42 mL) LiAlH4  was added portionwise, and the mixture was stirred at r.t. until almost all starting material was consumed. The mixture was concentrated and the residue diluted with CH2C12. The reaction was quenched by the careful addition of 2 M NaOH by using an ice-bath. Stirring was continued to obtain a clear organic layer and the white residue was filtered off. The two-phase mixture was separated and the aqueous layer was extracted with CH2Cl2. The combined organic phases were dried over anhydrous Na2SO4, and evaporated. The product (S)-6 was obtained as a yellow oil in 90% yield and used without further purifications. [α]D +28.9 ; IR (neat) νmax: 3287, 2961, 2870, 1606, 1518, 1488, 1291, 771, 736 cm−1. 1H-NMR : δ 1.47 , 1.75 , 1.89 , 2.52 , 2.93 , 3.14 , 3.39 , 4.94 , 6.4 , 6.53 , 7.36 , 8.04 ; 13C-NMR : δ 25.5, 29.0, 46.2, 58.3, 107.9, 112.8, 137.3, 147.7, 158.8; ESI-MS: 178 (M+H+).S)-5 or amine (S)-6 (1.05 mmol) was weighted in a Schlenk and azeotropically dried three times with distilled and degassed toluene. 5 mL of toluene were then added, and bis(diethylamino)phenylphosphine  was added dropwise under Ar at 0 °C. The reaction mixture was heated to 90 °C and stirred overnight. After cooling, the solution was evaporated in vacuo. Compound 1 was washed 7 times with distilled and degassed n-pentane, giving a white solid. Compound 2 was used as a crude reaction product (viscous orange oil) without further purifications.Amide -(-)-2-phenyl-3-(2-pyridyl)-1,3-diaza-2-phosphanicyclooctan-4-one (1). [α]D −128.9 ; 1H-NMR : δ 1.72 , 1.87 , 2.18 , 2.24 , 3.39 , 3.46 , 4.16 , 7.04 , 7.35 , 7.57 , 7.73 , 8.25 , 8.33 ; 13C-NMR : δ 26.5 (C4), 30.9 (C3), 55.4 (C5), 68.3 (C2), 114.4 (C6’), 120.1 (C4’), 128.6 (CPh-P), 129.8 (Ph), 130.0 (Ph), 138.3 (C5’), 147.7 (C3’), 152.4 (C2’), 178.1 (CO); 31P-NMR : δ 106.29; ESI-MS: 298.1 (M+H+), 320.1 (M+Na+).-(-)-2-phenyl-3-(2-pyridyl)-1,3-diaza-2-phosphanicyclooctane (2). [α]D −170.6 ; 1H-NMR : δ 1.78 , 2.07 , 3.08 , 3.31 , 3.37 , 3.55 , 4.00 , 6.61 , 6.64 , 7.30 , 7.47 , 8.10 ; 13C-NMR : δ 26.1 (C4), 31.3 (C3), 52.2 (C6), 52.6 (C5), 64.8 (C2), 108.5 (C5’), 113.7 (C3’), 128.5 (Ph), 129.2 (Ph), 129.9 (CPh-P), 137.9 (C4’), 148.7 (C6’); 31P-NMR : δ 99.71; ESI-MS: 284.1 (M+H+).3)Cl(cod)], following the procedure reported in the literature. Briefly, ligand 1 or 2 (1.50 mmol) was added to a solution of [Pd(CH3)Cl(cod)]  in freshly distilled dichloromethane (5 mL) and stirred at room temperature. After 1.5 h the reaction mixture was concentrated and the product precipitated as a yellow - orange solid upon addition of n-hexane.All complexes were prepared starting from [Pd(CH3-1)Cl]2 (1a): yield 70%; [α]D −7.6 ; IR (nujol) νmax: 1693 cm−1; 1H-NMR : δ 1.94–2.11 , 2.12 , 2.45 , 2.61 , 3.29 , 3.39 , 3.81 , 6.91 , 7.55 , 7.59 , 7.68 , 7.79 , 7.96 , 8.06 ; 13C-NMR : δ 15.3 , 25.4 , 29.7 , 49.6 (C5), 65.0 (C2), 115.2 (C3’), 117.9 (C5’), 128.8 (p-Ph), 129.6 , 132.3 , 133.0 (Ph-P), 140.5 (C4’), 144.7 (C6’), 170.3 (CO); 31P-NMR : δ 67.05 (minor isomer), 75.57 (major isomer). Isomeric ratio 9:1.[Pd(CH3)Cl(2)]D −20.4 ; 1H-NMR : δ 0.69 , 1.57 , 1.65 , 1.94 , 2.18 , 2.64–3.00 , 3.29 , 3.49 , 3.70 , 3.86 , 4.06 , 6.67 , 6.85 , 6.95 , 7.07 , 7.46 , 7.73 , 8.98 ; 13C-NMR : δ 0.69 , 18.5 , 26.2 , 32.8 , 51.6 , 52.2 , 65.9 , 109.6 , 110.4 , 116.5 , 123.4 , 127.0 , 128.6 , 130.2 , 131.5 , 137.7 , 139.9 , 148.7 ; 31P-NMR : δ 119.9 (cis isomer), 133.7 (trans isomer); Ratio cis/trans = 2:1.6 in a mixture of CH2Cl2 and pyridine or pyridine derivative. In particular, the neutral complex 1a (0.22 mmol) was dissolved in the minimal amount of CH2Cl2 under argon and kept in the dark. A solution of AgPF6 in CH2Cl2  was added, and then dropwise the pyridine derivative leading to the precipitation of AgCl. After 1.5 h, the solution was filtered over Celite and concentrated to minimal volume in vacuo. Upon addition of diethyl ether the product precipitated as a white solid.All complexes were obtained starting from the corresponding neutral derivatives upon addition of AgPF3-1)(Py)]2[PF6]2 +90.9 (c = 0.11 in CH3OH); IR (nujol) νmax: 1706, 842, 557 cm−1; 1H-NMR : δ 2.09 , 2.32 , 2.38 , 2.92 , 3.21 , 5.03 , 6.72 , 6.87 , 7.33 , 7.56 , 7.62 , 7.81 , 8.66 , 8.85 ; 31P-NMR : δ 50.73.3-1)(4-CH3Py)]2[PF6]22[PF6]2  +120.5 (c = 0.22 in CH3OH); 1H-NMR : δ 2.13 , 2.36 , 2.40 , 2.95 , 3.22 , 5.06 , 6.77 , 7.08 , 7.22 , 7.31 , 7.40  7.66 , 7.85 , 8.97 , 9.18 ; 31P -NMR : δ 50.73.6 was dissolved in pyridine (or in 4-picoline) and the solution was added to the reaction mixture.The synthesis of complexes 2b-c was performed following a procedure analogous to that of 1b-d, with the unique difference that AgPF2)(CH3)(Py)][PF6][PF6][Pd( (2c): yield% [α]D −12.3 ; 1H-NMR : δ 0.58 , 1.05 , 1.37 , 1.70 , 1.77 , 2.07 , 2.28 , 2.32 , 2.41 , 2.77 , 3.07 , 3.38 , 3.59 , 3.67 , 3.77 , 4.15 , 6.86 , 7.05 , 7.14 , 7.24 , 7.31 , 7.40 , 7.54 , 7.74 , 7.83 , 7.88 , 8.05 , 8.44 , 8.50 ; 31P-NMR : δ 122.2 (trans isomer), 135.5 (cis isomer). Ratio cis/trans 2:1.vacuo. IR (neat) νmax: 3081, 3059, 3025, 2964, 2928, 2870, 1945, 1873, 1806, 1599, 1492, 1450, 965 cm−1; 1H-NMR : δ 1.47 , 3.65 , 6.42 , 7.19-7.48 ; 13C-NMR : δ 21.2, 42.5, 126.1, 126.2, 127.0, 127.3, 128.5, 135.1, 137.5, 145.6.All catalytic experiments were carried out in a three-necked, thermostated, 75 mL glass reactor equipped with a magnetic stirrer and connected to a temperature controller. After establishment of the reaction temperature, the precatalyst, 1,4-benzoquinone, styrene and TFE were placed inside. The system was stirred at the same temperature for 24 h. At the end of the reaction time the two layers formed were separated (dimer being the lower density phase), the Pd(0) residue was filtered over Celite and washed with dichloromethane. The product was dried under 1b (1.3·× 10−5 mol), styrene (10 mL), TFE (20 mL), connected to the ethylene tank and pressurized at the desired pressure. The reactor was placed in an oil bath and warmed up to 343 K. The mixture was stirred for 24 h. The reactor was vented, the solution was filtered over Celite to remove palladium black and evaporated under vacuum obtaining a yellow oil.A 50 mL Büchi Tinyclave glass reactor was used for this reaction. The Tinyclave was charged with 1,4-benzoquinone, 1 having an amidic nitrogen led to a Pd-dinuclear species with the concomitant transfer of the methyl group from the metal centre to the phosphorus atom, while ligand 2 gave the expected mononuclear derivative. This different behavior might be related to the different nature of the nitrogen atom deriving from 2-aminopyridine: in ligand 1 this nitrogen atom is 2sp hybridized enforcing a planar, more strained, conformation of the N-P-N heterocycle, while in ligand 2 this nitrogen atom is 3sp hybridized and thus the related heterocycle should result more flexible and, as a consequence, more stable than in ligand 1.In this work we report the synthesis of two new, chiral, enantiomerically pure, hybrid P-N ligands, together with the study of their coordination chemistry towards palladium and the catalytic behavior of the synthesized complexes in styrene dimerization reactions. Starting from the same Pd precursor, complexes of different nature were obtained depending on the nature of the ligand. In particular, ligand E-1,3-phenyl-1-butene. Despite the optical activity of the precatalysts no asymmetric induction was observed, and the product was obtained as a racemic mixture. It was noted that 1,4-benzoquinone plays a fundamental role for the catalytic reaction, being involved in catalyst activation. Further investigations are required to clarify these specific reactions.The monocationic palladium complexes were found to generate catalysts with modest activity for styrene dimerization reaction leading, regio- and stereospecifically, to


Cissus assamica, including 14 benzenoids, 11 triterpenes, nine steroids, five tocopherols, five chlorophylls, four flavonoids, two benzoquinones, two tannins, and three other compounds. Their structures were constructed by 1D and 2D nuclear magnetic resonance (NMR) and mass spectral data, and were also identified by a comparison of their spectral data with those reported in the literature. Among these isolates, 1,2-bis-(5-γ-tocopheryl) ethane (51) was reported for the first time from natural sources. Some purified compounds were examined for their anti-inflammatory and anticancer bioactivities. The results indicated that betulinic acid (16) exhibited strong inhibition of superoxide anion generation with IC50 value of 0.2 ± 0.1 μM, while betulinic acid (16) and pheophytin-a (47) inhibited elastase release with IC50 value of 2.7 ± 0.3 and 5.3 ± 1.0 μM, respectively. In addition, betulinic acid (16) and epi-glut-5(6)-en-ol (18) exhibited potential cytotoxicity to non-small-cell lung carcinoma (NCI-H226) and colon cancer (HCT-116) cell lines with IC50 values in the range of 1.6 to 9.1 μM.Fifty-five compounds were isolated from the fresh stems of  Cissus assamica L. belong to the Vitaceae family and is distributed in mainland China, Vietnam, India, Thailand, Indonesia, the southern part of Taiwan, and Lanyu Island [C. assamica can activate the circulation to remove blood stasis and treat bruises, fractures, and rheumatoid arthritis [Cissus genus [C. sicyoides showed moderate cytostatic activity against HEp-2 cells [C. assamica L. is scarce. Only a few reported the effects of antagonizing the vasoconstriction induced by endothelin-1 [C. assamica.u Island . Traditirthritis . Moreoveus genus ,9,10,11.-2 cells  and a si-2 cells . In addi-2 cells ,24,25,26thelin-1 ,24,25,26C. assamica L. were extracted with methanol and refluxed for 8 h. The filtrate was concentrated under reduced pressure to yield a dark brown syrup. The crude extract was suspended in water and then partitioned with chloroform and n-butanol successively to afford chloroform, n-butanol, and water layers, respectively. Purification of the three layers by column chromatography yielded a mixture of β-sitosterol (1) and stigmasterol (2) [3) [4) and 3β-hydroxystigmast-5, 22-dien-7-one (5) [6) [7) [8) [9) [10) [11) [12) [13) [14) [15) [16) [17) [epi-glut-5(6)-en-ol (18) [19) [epi-friedelinol (20) [21) [22) [23) and β-amyrin (24) [25) [p-hydroxybenzaldehyde (26) [27) [28) [29) [30) [31) [trans-cinnamic acid methyl ester (32) and 4-hydroxy-cis-cinnamic acid methyl ester (33) [trans-ferulate (34) and octadecyl-cis-ferulate (35) [36) [37) [38) [39) [40) [O-methyl-ellagic acid (41) [O-methylellagic acid (42) [43) [S)-pheophorbide-a (44) and methyl-21-hydroxy-(21R)-pheophorbide-a (45) [S)-pheophorbide-b (46) [47) [48) [49) [50) [51) [52) [53) [54) [55) [51) (The fresh stems of erol (2) , β-sitos (2) [3) , a mixtu-one (5) , a mixtu (5) [6)  and stig [6) [7) , 6β-hydr [7) [8) , ergoste [8) [9) , 3,5,7,4[9) [10) , 3′,4′,310) [11) , 3′,4′,511) [12) , 4′,5,6,12) [13) , a mixtu13) [14)  and urso14) [15) , betulin15) [16) , friedel16) [17) , epi-glu-ol (18) , taraxernol (20) , glutino20) [21) , lup-28-21) [22) , a mixturin (24) , bergeni24) [25) , p-hydroyde (26) , vanilli26) [27) , methyl 27) [28) , gallic 28) [29) , 4-metho29) [30) , vanilliter (33) , a mixtuate (35) , 1-(4-me36) [37) , hexadec37) [38) , 2-hydro38) [39) , 2,6-dim39) [40) , 3,3′,4-cid (41) , 3,3′,4,cid (42) , methyl 42) [43) , a mixtue-a (45) , methyl-e-b (46) , pheophy46) [47) , α-tocop47) [48) , tocophe48) [49) , tocophe49) [50) , 1,2-bis50) [51) , α-tocos51) [52) , 5,6-dim52) [53) , 3-methy53) [54) , and met54) [55) , respect55) [51)  is repor51) was isolated as a light yellow syrup. Its UV spectrum had an absorption maximum at 294 nm. The IR spectrum suggested the presence of hydroxyl (3444 cm−1) and an aromatic conjugated double bond (1458 and 1377 cm−1). The 13C-NMR and DEPT spectra exhibited a benzene ring partial structure that has two oxygenated substituents at δ 117.0 (s), 122.1 (s), 123.4 (s), 124.1 (s), 145.5 (s), and 146.3 (s). The 1H-NMR spectrum of 51 exhibited signals for two methyl and one methylene groups attached to a benzene ring at δ 2.13, 2.18, and 2.73. Comparing all the 1H- and 13C-NMR spectral signals carefully, the structure of 51 was similar to that of α-tocopherol (48) [H 2.73, δC 26.7) in 51, instead of the methyl group  found in 48 , nuclear Overhauser enhancement spectroscopy (NOESY), heteronuclear multiple quantum correlation (HMQC), and heteronuclear multiple bond correlation (HMBC) experiments were conducted. In the HMBC experiment, the correlations observed for H-5a (δH 2.18)/C-4, C-5, C-6 and H-6a (δH 2.13)/C-1, C-5, C-6 indicate that two methyl groups are located in the ortho position on the benzene ring. Moreover, the correlation of the methylene proton at δH 2.73 with C-3 in HMBC spectrum suggests that the location C-3a in the dimerization of alpha-tocopherol forms the dimer 51. Conclusively, the structure of 51 was assigned as 1,2-bis-(5-γ-tocopheryl)ethane, which had been reported by synthesis [1,2-Bis-(5-γ-tocopheryl)ethane (rol (48) . It indind in 48 . To estaN-formyl-l-methionyl-phenylalanine/cytochalasin B (fMLP/CB). Only compound 16  was examined in human neutrophils using an LDH release assay  and epi-glut-5(6)-en-ol (18)  exhibited powerful inhibitory activity against NCI-H226 and HCT116 with IC50 values of 2.0 and 1.6 μM, respectively. Our study suggested the stem extracts of C. assamica and the purified compounds are potential candidates for the development of anti-cancer drugs. The preliminary growth inhibitory activity of all the tested compounds is presented in In order to evaluate the growth inhibitory activity of the purified compounds against cancer cells, this study selected three different cell lines from malignant tumors including human nasopharyngeal carcinoma (NPC-TW01), non-small-cell lung carcinoma (NCI-H226), and colon cancer cell lines (HCT116). The results showed that betulinic acid (-ol (18)  exhibiteo 9.1 μM . Moreove1H- and 13C-NMR spectra were measured using Bruker Avance-300, AMX-400, and AV-500 spectrometers  with TMS as the internal reference, and chemical shifts are expressed in δ (ppm). Silica gel  and Spherical C18 100 Å reversed phase silica gel  were used for column chromatography (CC), and silica gel 60 F254 and RP-18 F254S thin-layer chromatography (TLC) plates  were used for preparative TLC, respectively.UV spectra were obtained with a Hitachi UV-3210 and UV-3010 spectrophotometer , and IR spectra were measured with a Shimadzu FTIR Prestige-21 spectrometer . Optical rotations were measured with a HORIBA SEPA-300 digital polarimeter in a 0.5 dm cell . The ESIMS and HRESIMS were taken on a Bruker Daltonics APEX II 30e spectrometer . C. assamica L. were collected from Taitung Hsien, Taiwan, in October 2009 and verified by Prof. Chang-Sheng Kuoh . A voucher specimen (TSWu 20091016) has been deposited in the Herbarium of School of Pharmacy, National Cheng Kung University, Tainan, Taiwan. The fresh stems of C. assamica L. (15 kg) were extracted with methanol (15 × 20 L) and refluxed for 8 h. The filtrate was evaporated under reduced pressure to yield a dark brown syrup (418 g). The residue was suspended in water and then partitioned with chloroform (5 × 2 L) and n-butanol (5 × 2 L) successively to afford chloroform (63 g), n-butanol (145 g) and water (210 g) soluble fractions respectively.The fresh stems of n-hexane/acetone (9:1) to afford seven fractions, on the basis of TLC monitoring. Fraction 1 was subjected to silica gel column chromatography eluted with n-hexane/acetone (79:1) to yield a mixture of β-sitosterol (1) and stigmasterol (2) (3.1 g), 3,5,7,4′-tetramethoxyflavone , 3′,4′,3,6,7-pentamethoxyflavone , 3′,4′,5,6,7-pentamethoxyflavone , 4′,5,6,7-tetra-methoxyflavone , friedelin , taraxerol , glutinone  and methyl linoleate .The chloroform soluble extracts were fractionated via silica gel column chromatography eluting with epi-glut-5(6)-en-ol , α-tocopherol , tocopherol trimer IVa , tocopherol trimer IVb , 1,2-bis-(5-γ-tocopheryl)- ethane , α-tocospirol B  and 5,6-dimethoxy-3-methyl-2-cyclohexa-2,5-dien-1,4-dione .Purification of fraction 2 by column chromatography with silica gel was eluted by a gradient of benzene/ethyl acetate (79:1) to afford n-hexane/ethyl acetate (9:1) yielded a mixture of 3β-hydroxystigmast-5-en-7-one (4) and 3β-hydroxystigmast- 5,22-dien-7-one , a mixture of β-sitostenone (6) and stigmasta-4,22-dien-3-one , 6β-hydroxy-β-sitostenone , ergosterol peroxide , epi-friedelinol , lup-28-al-20(29)-en-3-ol , a mixture of α-amyrin (23) and β-amyrin , a mixture of 4-hydroxy-trans-cinnamic acid methyl ester (32) and 4-hydroxy-cis-cinnamic acid methyl ester , a mixture of octadecyl-trans-ferulate (34) and octadecyl-cis-ferulate , hexadecyl ferulate  and 3-methyl-8-hydroxy-3,4-dihydroisocoumarin .Separation of fraction 3 by column chromatography with silica gel eluted by 14) and ursolic acid , betulinic acid , 4-methoxybenzoic acid , 1-(4-methoxyphenyl)undecan-1-one  and pheophytin-a .Fraction 4 was chromatographed over silica gel eluted with a benzene/acetone gradient (49:1) to give a mixture of oleanolic acid , methyl pheophorbide-a , a mixture of methyl-21-hydroxy-(21S)-pheophorbide-a (44) and methyl-21-hydroxy-(21R)-pheophorbide-a .Purification of fraction 5 by column chromatography with silica gel was eluted by a gradient of chloroform/acetone (49:1) to afford 2-hydroxybenzoquinone , 3,3′,4-tri-O-methylellagic acid , 3,3′,4,4′-tetra-O-methylellagic acid  and methyl-21-hydroxy-(21S)-pheophorbide-b .Separation of fraction 6 by column chromatography with a silica gel eluted by chloroform/acetone (49:1) yielded 3, 700 mg), vanillin , vanillic acid , 3-hydroxy-4-methoxybenzoic acid , 2, 6-dimethyoxybenzoquinone (40) (3.5 mg).Fraction 7 was subjected to silica gel column chromatography eluted with chloroform/methanol (49:1) to yield β-sitosteryl glucoside . Fraction 2 was chromatographed on Sephadex LH-20 eluted with gradient of water/methanol to afford bergenin . Fraction 4 was chromatographed on Sephadex LH-20 with water/methanol to give methyl gallate .The 51): light yellow syrup; UV λmax (MeOH) nm (log ε) 294; IR (KBr) νmax cm−1 3444, 2920, 2850, 1458, 1377, 1257, 1087; 1H- and 13C-NMR data, see 1,2-Bis-(5-γ-tocopheryl)ethane  who provided written informed consent before blood was drawn. Briefly, neutrophils were isolated by dextran sedimentation, Ficoll‒Hypaque gradient centrifugation, and hypotonic lysis of the erythrocytes .c and elastase substrate, methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide, respectively, as described previously [c (0.6 mg/mL) or elastase substrate (100 μM) at 37 °C and treated with DMSO or tested compounds for 5 min. The cells were then activated using fMLF (0.1 μM)/cytochalasin B  and the change of absorbance was continually measured at 550 nm and 405 nm by a spectrophotometer  to determine the superoxide anion generation and elastase release, respectively.The superoxide anion generation and elastase release were measured using the reduction of ferricytochrome eviously ,69,70. HHuman neutrophils were treated with DMSO or tested compounds and incubated at 37 °C for 15 min. The supernatant was assayed to detect the released LDH using CytoTox 96 non-radioactive cytotoxicity assay . The results are presented in 2.Human cancer cell lines, non-small cell lung carcinoma (NCI-H226) and colon cancer cell line (HCT116) were obtained from the American Type Culture Collection . A nasopharyngeal carcinoma (NPC-TW01) cell line was purchased from Food Industry Research and Development Institute . Tumor cells were maintained in proper medium supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere of 5% COThe evaluation of cell growth and survival was carried out according to Hansen et al.  with somC. assamica, including 14 benzenoids, 11 triterpenes, nine steroids, five tocopherols, five chlorophylls, four flavonoids, two benzoquinones, two tannins, and three other compounds. Among these isolates, 1,2-bis-(5-γ-tocopheryl)ethane was reported for the first time from natural sources. Furthermore, the inhibitory activity on superoxide anion generation and elastase release and the cytotoxicity on three cancer cells were analyzed. The present study suggests that the stems of C. assamica and several compounds of its isolation could be further developed as candidates for the treatment or prevention of cancer and various inflammatory diseases. Thus, the detailed mechanism of action of these compounds appears worthy of follow-up investigation.In summary, 55 compounds were characterized from the fresh stems of


Their structures were elucidated on the basis of spectroscopic methods, including IR, ESIMS, 1D- and 2D-NMR experiments, as well as by comparison of their spectral data with those of related compounds. All compounds were evaluated in vivo for their effects on the mean arterial blood pressure (MABP) and heart rate (HR) of spontaneously hypertensive rats (SHR) and also in vitro for their capacity to inhibit the human platelet aggregation. None of the isolated flavonoids 1–3 showed cardiovascular effects on SHR and among the isolated triterpenoids 4–9 only 5 and 6 produced a significant reduction in MABP  and an elevation in HR . Compounds 3, 4, 5 and 6 were able to inhibit human platelet aggregation induced by ADP, collagen and arachidonic acid with different selectivity profiles.Three new lupane-type triterpenoids: 6β,30-dihydroxybetulinic acid glucopyranosyl ester ( Licania have been used in South America for various medicinal purposes such as the treatment of inflammation [Licania cruegeriana Urb. and structure elucidation of three new lupane-type triterpenoids: 6β,30-dihydroxybetulinic acid gluco-pyranosyl ester (4) 6β,30-dihydroxybetulinic acid (5) and 6β-hydroxybetulinic acid (6), along with six known compounds. Additionally, all isolated compounds were evaluated for their effects on the mean arterial blood pressure (MABP) and heart rate (HR) of spontaneously hypertensive rats (SHR) and also for their capacity to inhibit the human platelet aggregation in vitro.The genus Licania  consists of more than 200 species of trees and shrubs, which are mainly distributed in tropical regions of America and Africa ,2. The sammation , diabeteammation , stomachammation . Previouammation . In the in vacuo to yield 89.0 g of ethanolic extract (EE). Two fractions were obtained from EE partition in methanol-water (1:1): A red solution that was then evaporated in vacuo to yield a red residue (16.7 g), named methanol-water soluble fraction (MWSF), and a green residue (71.2 g) named methanol-water insoluble fraction (MWIF). A portion of MWSF (5.0 g) was three times extracted with acetone to obtain a brownish residue and a yellowish solution that was then concentrated to dryness yielding a yellowish residue named AF (4.7 g). AF (1 g) was fractionated on Sephadex LH-20 column chromatography (CC) using methanol as eluent to give three fractions named I–III. Myricetin  was separated from fraction III by CC on RP-18, eluting with a mixture methanol-water (3:2). From fraction II myricetin 3-O-α-rhamnoside  and dihydromyricetin-3-O-α-rhamnoside  were separated by low-pressure CC with a mixture of acetonitrile-water (2:3). MWIF (2 g) was subjected to low-pressure CC with a mixture of methanol-water (7:3) as eluent to afford 6β,30-dihydroxybetulinic acid glucopyranosyl ester , 6β,30-dihydroxybetulinic acid , 6β-hydroxybetulinic acid , alphitolic acid , betulinic acid  and lupeol . The structure of compounds 1–3 and 7–9  were extracted by percolation with ethanol for a week. The solvent was evaporated  and 7–9  were estresidue 1.7 g, nam5 and 6 were assigned as C30H48O5 and C30H48O4, respectively, from their ESIMS and NMR data. The analysis of their 1H- and 13C-NMR spectra indicated that these compounds are lupane-type triterpenoids.The molecular formulae of compounds 20,29-functionality of the lupene skeletons were inferred from the resonances of the sp2 carbons at C-29  at δC 106.9 and δC 110.2 ppm and C-20 (quarternary carbon) at δC 156.3 and δC 152.0 ppm of 5 and 6 respectively [13C-NMR spectra is was deduced that these triterpenoids have a hydroxyl group at C-6 by the shifts of the 13C-NMR signals at δC 19.7 ppm (C-6) of betulinic acid [C 69.3 ppm in 5 and 6. In both compounds the axial orientation of the hydroxyl group at C-6 was inferred by the shifts of the 13C-NMR signal at δC 16.4 ppm (C-24) in 6α-hydroxybetulinic acid [C 24.8 ppm, thus compound 6 was deduced as 6β-hydroxybetulinic acid. The Δectively . From thnic acid  to δC 6913C-NMR spectra of 5 and 6 are similar, except those at C-30 and those corresponding to the proton coupling patterns. The shifts of the signals at δC 19.6 (C-30) in 6 to δC 65.2 in 5 suggest an additional hydroxyl substituent at this position which was confirmed in the HMBC spectrum of 5 given the correlations between H-29a/H29b  and the carbon signal at δC 65.2 ppm and also between H-30 at δH 4.0 ppm and C-20 at δC 156.3 ppm . The comparison of 1H- and 13C-NMR spectroscopic data of 4 with those of 5 indicated that 4 is the glycosylated derivative of 5. The NMR spectra of 4 showed an anomeric proton at δH 5.48 ppm  with the corresponding carbon at δC 95.2 ppm. Comparison of NMR data with those reported in the literature suggested a β-oriented glucopyranoside moiety on the basis of the large 3JH1',H2' coupling constant [C 176.2) indicated that the β-glucopyranosyl unit was attached to the carboxyl group. Thus, the structure of 4 was determined to be 6β,30-dihydroxybetulinic acid glucopyranosyl ester. To the best of our knowledge this is the first report of compounds 4, 5 and 6 from Nature.Compound constant ,15,16,17L. cruegeriana, all of them were intravenously administered to pentobarbital-anaesthetized SHR over thirty seconds and the MABP and the HR were monitored continuously during forty-five minutes as described in Experimental 5 and 6 induced changes in MABP and HR of SHR as shown in 5 and 6 produced significant reductions in MABP  and induced elevation in HR . These cardiovascular effects exhibit a time of peak effect of three minutes and were recorded for more than forty-five minutes without recovering baseline levels. The widely used antihypertensive drug losartan (an AT1 receptor antagonist) served as a positive control [p < 0.05). The reduction in MABP lasted longer than forty-five minutes and showed a time of peak effect near ten minutes.To determine the cardiovascular effects of the isolated compounds from  control ,19,20. I5 and 6 and their structurally related compounds 4, 7, 8 and 9 , collagen and adenosine 5'-diphosphate (ADP). In 3 was the only isolated flavonoid that exhibited antiplatelet effects causing total inhibition against the action of AA, a significant decrease against collagen and near middle inhibition against ADP. Among the isolated triterpenoids, compound 4 significantly inhibited the aggregation of platelets induced by collagen and had an average effect against the actions of AA, and compound 5 significantly decreased the ADP and collagen-induced platelet aggregations, while compound 6 exhibited significant antiplatelet effect against collagen. The mode of action by which these compounds exert their effects on platelet aggregation needs to be further studied.Compound ® Rf Reversed-phase C18 was used for low pressure CC and silica gel 60 RP18 F254 (E. Merck) for TLC on glass (Merck). Dimethylsulfoxide (DMSO), adenosine 5'-diphosphate sodium salt (ADP) and Sephadex®LH-20 were obtained from Sigma Aldrich . 1H- and 13C-NMR spectra were obtained using a Bruker DRX 500 (500 MHz for 1H and 125 MHz for 13C) and Bruker Avance 300 (300 MHz for 1H and 75 MHz for 13C) in CD3OD. Measurements of electrospray ionization mass spectra were acquired in negative ion mode on an Ion Trap mass spectrometer . Infrared (IR) spectra (KBr discs) were recorded using a FTIR spectrophotometer . Collagen and arachidonic acid were from Helena Laboratories . All solvents used were of HPLC grade quality, obtained commercially from Sigma. The purity of compounds 4, 5 and 6 was confirmed by using an HPLC-MS Agilent 1260 series LC/MSD trap, SL model (Bruker) equipped with an electrospray interface (ESI), a quaternary pump, degasser, autosampler and a thermostatted column compartment. A column XBridgeTM C18 4.6 × 75 mm, 2.5 μm  was used and kept at 25 °C in the column compartment. Nitrogen was used as nebulizing and drying gas at 220 °C. The ESI source was operated in negative ion mode. Complete system control, data acquisition and processing were done using the HyStar 3.2 for LC/MSD trap software from Bruker. The injection volume was 5 μL. The mobile phase was delivered in isocratic mode and consisted of a mixture of methanol/water (70:30). The chromatograms were recorded in full scan mode for compounds 4, 5 and 6 and only MS/MS mode for compound 4 (in negative mode MW = 649). Full-scan spectra were acquired over a scan range of m/z 70–2200 at 32.5 m/z/s.RediSepLicania cruegeriana Urb. were collected in August 2009 at the Parque Nacional Henri Pittier, Aragua, Venezuela. This specimen was identified by Dr. Alfonso Cardozo and a voucher specimen (AC2706) was deposited in the herbarium of Facultad de Agronomía, UCV, Maracay.The leaves of 3β,6β,30-Trihydroxy-20(29)-lupen-28-O-β-glucopyranosyl ester (4). A white powder, IR (KBr) υmax 2947, 2871, 1705, 1651, 1386, 1187, 1059 cm−1; 1H-NMR : δ 5.48 , 4.96 , 4.85 , 4.32 , 4.04 , 3.82 , 3.70 , 3.45 , 3.37 , 3.31, 3.25  , 2.88 , 2.37 , 2.03 , 1.8 , 1.6 , 1.25 , 1.20 , 1.18 , 0.99 , 0.97 ; 13C-NMR : δ 176.2 (C-28), 156.2 (C-20), 107.1 (C-29), 95.2 (C-1'), 78.7 (C-5'), 78.5 (C-3), 78.3 (C-3'), 74.0 (C-2'), 71.1 (C-4'), 69.3 (C-6), 65.3 (C-30), 62.3 (C-6'), 58.0 (C-17), 52.2 (C-5), 51.2 (C-9), 50.1 (C-18), 43.9 (C-19), 43.7 (C-14), 42.8 (C-7), 41.3 (C-8), 39.4 (C-4), 38.4 (C-13), 38.0 (C-10), 37.3 (C-21), 36.8 (C-21), 33.2 (C-16), 32.6 (C-22), 30.9 (C-15), 28.9 (C-23), 28.1 (C-2), 26.3 (C-12), 24.8 (C-24), 22.1 (C-11), 18.0 (C-25), 17.3 (C-26), 15.4 (C-27). Negative mode ESI-MS: m/z 649.4.3β,6β,30-Trihydroxy-20(29)-lupen-28-oic-acid (5). A white powder, IR (KBr) υmax 2946, 2870, 1704, 1645, 1452, 1397, 1202, 1058 cm−1; 1H-NMR : δ 4.96 , 4.33 , 4.04 , 3.25 , 2.90 , 1.26 , 1.20 , 1.19 , 1.0 , 0.97 ; 13C-NMR : δ 180.0 (C-28), 156.3 (C-20), 106.9 (C-29), 78.5 (C-3), 69.3 (C-6), 65.2 (C-30), 57.5 (C-17), 52.1 (C-5), 51.1 (C-9), 50.2 (C-18), 44.1 (C-19), 43.7 (C-14), 42.8 (C-7), 41.2 (C-8), 39.4 (C-4), 38.7 (C-13), 38.0 (C-10), 37.9 (C-1), 36.8 (C-21), 33.4 (C-16), 33.1 (C-22), 31.0 (C-15), 28.9 (C-23), 28.1 (C-2), 26.3 (C-12), 24.8 (C-24), 22.1 (C-11), 18.0 (C-25), 17.3 (C-26), 15.4 (C-27). Negative mode ESI-MS: m/z 487.5.3β,6β-Dihydroxy-20(29)-lupen-28-oic-acid (6). A white powder, IR (KBr) υmax 2920, 2870, 1700, 1642, 1452, 1394, 1180, 1064 cm−1; 1H-NMR : δ 4.71 and 4.59 , 4.33 , 3.25 , 3.05 , 1.69 , 1.26 , 1.20 , 1.19 , 0.97 ; 13C-NMR : δ 180.1 (C-28), 152.0 (C-20), 110.2 (C-29), 78.5 (C-3), 69.3 (C-6), 57.5 (C-17), 52.1 (C-5), 50.5 (C-9), 50.2 (C-19), 48.4 (C-18), 43.8 (C-14), 42.8 (C-7), 41.2 (C-8), 39.3 (C-4) 38.8 (C-13), 38.1 (C-10), 38.0 (C-1), 36.8 (C-22), 33.1 (C-15), 31.7 (C-21), 30.9 (C-16), 28.9 (C-23), 27.0 (C-2), 26.3 (C-12), 24.8 (C-24), 22.0 (C-11), 19.6 (C-30), 18.1 (C-26), 17.3 (C-25), 15.4 (C-27). Negative mode ESI-MS m/z: 471.5.L. cruegeriana, an i.v. line was placed in the femoral vein using a polyethylene catheter. Once the basal conditions remained constant for more than 45 min, the changes in MABP and HR induced by the L. cruegeriana samples were recorded for at least 45 min after injection. Samples, at the indicated doses, were injected as a single bolus of 0.1 mL  over 30 s. The doses of isolated compounds from L. cruegeriana used in this study were the minimal doses that after inducing a significant hypotensive effect allowed the survival of rats for at least 2 h. This protocol was evaluated and approved by the Bioethics Commission for Investigations in Animals (COBIANIM) at the Venezuelan Institute for Scientific Research (IVIC) , in accordance with the Code on Bioethics and Biosecurity (2008) established by the Bioethics Commission National Fund on Science and Technology (FONACIT), under the national legislation .Spontaneously Hypertensive Rats (SHR), male (250–300 g) were used for all experiments and were obtained from the animal care service of IVIC. Animals were housed under conditions of controlled temperature (21 ± 2 °C) and lighting (lights on 06:00–18:00 h). In addition, they had free access to food  and tap water. All animal procedures were approved by the bioethical committee of IVIC (number 201417). SHR were anesthetized by an intraperitoneal (i.p.) injection of sodium pentobarbital (40 mg/kg). The trachea was exposed and cannulated with a polyethylene catheter to avoid ventilation disturbances. Arterial blood pressure was recorded from the femoral artery through a catheter connected to a blood pressure transducer  and a bridge amplifier  from which MABP and HR were continuously recorded using a 4/20 High Performance Data Recording System (PowerLab). To facilitate the intravenous (i.v.) administration of isolated compound from Human platelets were obtained from blood of healthy volunteers who did not take any drugs during previous two weeks and gave informed consent before taking part in this study. The written informed consent form and this protocol were evaluated and approved by the Bioethics Commission for Investigations involving Human Subjects of the Venezuelan Institute for Scientific Research (IVIC) , in accordance with the Code on Bioethics and Biosecurity (2008) established by the Bioethics Commission National Fund on Science and Technology (FONACIT), under the national legislation . Platelet rich plasma (PRP) was prepared and used in platelet aggregation assays as described earlier . InhibitL. cruegeriana led to the isolation and identification of myricetin, two myricetin glycosides and six triterpenoids with lupane moieties (4), 6β,30-dihydroxybetulinic acid (5) and 6β-hydroxybetulinic acid (6) have never been reported before in the literature. The myricetin derivates found in L. cruegeriana seem to follow a similar glycosylation pattern to those reported for the Licania genus and other species of the Chrysobalanaceae family [4–9 with lupane skeletons identified in L. cruegeriana only betulinic acid, lupeol and betulin have previously been reported in species of Licania [L. cruegeriana leaves.The present chemical investigation of the leaves of moieties . This co Licania . 6β-HydrL. cruegeriana had cardiovascular effects in SHR and only two triterpenoids 5 and 6 showed significant reduction in MABP (60.1% and 17.2% respectively) inducing an elevation in HR (11.0% and 41.2% respectively) as shown in 3) and three triterpenoids  were able to inhibit the aggregation of human platelets induced by AA, collagen and ADP as shown in 4, 5 and 6 the addition of one hydroxyl group at C-30 in compound 4 and 5 appears to increase their antiplatelet properties against collagen stimulation. Additionally, the glycosylation at C-28 in compound 4 seems to decrease the anti-platelet effect against ADP with respect to compound 5. None of these triterpenoids have significant effect on the AA-induced platelet aggregation.Pharmacological studies of some plants growing in Venezuela are being conducted by our research group in order to identify secondary metabolites as potential therapeutic agents for cardiovascular diseases. In the present study, none of the flavonoids isolated from L. cruegeriana leaves is a novel contribution to the current acknowledge of the phytochemistry of Licania genus, being remarkable that triterpenoids with a lupane-type skeletons and flavonoids, particularly myricetin and their glycosides are the characteristic chemotaxonomic markers for this genus [Taken together, the present phytochemical study of is genus . Additio


Larrea nitida Cav., using interleukin-6 (IL-6) inhibitory assay in human mast cells (HMC-1), led to the isolation of three new compounds with an unprecedented skeleton in nature (1–3) and three known compounds (4–6). Their structures were elucidated through extensive spectroscopic analysis. The three new compounds were elucidated as two new spiroketones, nitidaones A (1), and B (2) and one new biphenyl analog, nitidaol (3). The known compounds were identified as nordihydroguaiaretic acid (4), 7,3′,4′-tri-O-methylquercetin (5) and ayanin (6). All the isolates were tested for their inhibitory activity against IL-6 production in HMC-1 cells. Of them, compounds 1, 3–6 showed potent anti-inflammatory activity, with IC50 values of 12.8, 17.5, 14.9, 22.9, and 17.8 µM, respectively.Bioactivity-guided fractionation for the stems of leaves of  Larrea (Zygophyllaceae) has been used for ethnobotanical purposes by the native peoples of northwest, central, and southeast Argentina. This genus comprises five species seen in North and South America: L. ameghinoi Speg., L. nitida Cav., L. divaricata Cav., L. mexicana Moric., and L. tridentata (DC.) Coville + at m/z 343.1918 . The 1H NMR disclosed the presence of a para-substituted benzene ring at δH 7.28 , and 6.89 , four olefinic protons at δH 7.19 , 7.10 , 6.32  and 6.29 , an acetyl group at δH 2.00 , and a methoxy group at δH 3.76   .H 3.76) and two aromatic protons . A 2,3-dimethylbutoxy group was confirmed by the 1H and COSY NMR signals at δH 4.03 , 3.85 , 2.29 , 1.95 , 1.77 , 1.45 , 0.93  and 0.89 . The COSY NMR spectrum showed correlations between H-11 and H-8, between H-12 and H-9 and sequential correlations from H-7 to H-10.From the HMBC NMR spectrum, the correlations between both H-3 and H-5 and the carbonyl carbon at δc 188.6 (C-1) and between both H-2 and H-6 and the quaternary carbon at δc 50.5 (C-4) suggested a quinoid-type skeleton. The 4-methoxyphenyl group was suggested by the HMBC correlations between one quaternary aromatic carbon at δc 133.4 (C-1′) and H-3′,5′ and between another quaternary aromatic carbon at δc 160.6 (C-4′) and both methoxy protons (δC 50.5) and between protons (H-7a and 7b) on a 2,3-dimethylbutoxy group and both C-4 and C-1′ enabled us to put these groups together via a spiral carbon at C-4, as depicted in C 173.1 and both H-10 and methyl protons (δH 2.00). Therefore, the structure of compound 1 was determined to be 4-(1-(4-methoxyphenyl)-4-oxocyclohexa-2,5-dienyl)-2,3-dimethylbutyl acetate, named nitidaone A.The link between these groups was confirmed by the HMBC correlations . The cor+ at m/z 345.2069  of compound 2 suggested its molecular formula as C21H28O4. The 1H NMR data of 2  and δH 2.24–2.14 (2H) were observed in the spectrum of 2, assignable to H-5 and H-6. Furthermore, the HMBC correlations of H-7 with C-4 (δC 44.7) and C-1′ (δC 137.0) and H-2′,6′ with C-4 confirmed its spiral skeleton (A protonated molecular ion [M + H]ata of 2  were simskeleton . Taken t*) in the 340 nm region if the ring has a half-chair conformation with a pseudo-axial disposition of an aryl group  in perature b. TherefR configuration. From the UV and CD spectrum, the negative exciton-split Cotton effect at 220 nm was observed due to the π→π* intramolecular charge-transfer transition of a 4-methoxyphenyl chromophore and a 2-cyclohexenone chromophore  to yield 3′,4′,7-trimethylquercetin , 3,7,3′-trimethylquercetin , and 2 . From LN11-13 (118 mg), compound 3  was purified using sequentially connected two columns [Luna C18  + YMC-packed ODS-AM ] with isocratic elution of 70% MeCN in H2O.Further HPLC separation for LC8-9  yielded nordihydroguiaretic acid : white amorphous solid; c 0.5, MeOH); UV (MeOH) λmax (log ε) 256 (3.60), 272 (3.41) nm; 1H and 13C NMR data, see m/z 343.1918 [M + H]+ .Nitidaone B (2): white amorphous solid; c 0.1, MeOH); UV (MeOH) λmax (log ε) 208 (3.79), 212 (3.82) nm; CD (MeOH) λmax (Δε) 208 (−0.05), 216 (0.08), 233 (−0.30), 339 (−0.04); 1H and 13C NMR data, see m/z 345.2069 [M + H]+ .Nitidaol (3): white amorphous solid; c 0.2, MeOH); UV (MeOH) λmax (log ε) 257 (3.63), 281 (3.60) nm; 1H and 13C NMR data, see m/z 343.1913 [M + H]+ .6/mL per well in 24-well tissue culture plates and pretreated with the indicated concentration of compounds for 30 min before stimulation by PMA (50 nM) + A23187 (1 µM). After 24 h, the supernatant was decanted into a new micro-centrifuge tube, and the amount of interleukin-6 (IL-6) was determined using an enzyme-linked immunosorbent assay (ELISA) kit according to the procedures described by the manufacturer . All subsequent steps took place at room temperature, and all standards and samples were assayed in duplicate [Cells were seeded at 1 × 10uplicate .1, 2) and one new biphenyl analog (3), together with three known compounds, were isolated from the stems and leaves of L. nitida. Three new compounds (1–3) have unprecedented skeletons in nature. The absolute configuration of C-4 (compound 2) was demonstrated by ECD analysis, but that of C-8 and C-9 (compounds 1–3) could not be identified. All the isolates were tested against IL-6 production inhibitory activity in the HMC-1 cells induced by PMA+A23187. It was found that nitidaone A (1), nitidaol (3), nordihydroguaiaretic acid (4), 7,3′,4′-trimethylquercetin (5), and 3,7,3′-trimethylquercetin (6) remarkably downregulated the PMA+A23187-induced synthesis of interleukin-6 (IL-6) in HMC-1 cells without cytotoxicity.Two new spiroketones (


Ocimum species: O. americanum L., O. basilicum L, O. basilicum var purpurascens Benth, O. basilicum var. minimum L, O. gratissimum L, O. micranthum Willd, O. selloi Benth. and O. tenuiflorum L. grown in Northeastern Brazil. In these Ocimum species, UA was detected in different yields, with O.tenuiflorum showing the highest content (2.02%). This yield is very significant when compared with other sources of UA.Ursolic acid is a very important compound due to its biological potential as an anti-inflammatory, trypanocidal, antirheumatic, antiviral, antioxidant and antitumoral agent. This study presents the HPLC analysis of ursolic acid (UA) content in eight different  Chemical shifts were recorded in (ppm) relative to residual solvent (2.49 ppm for 1H-NMR and 39.5 ppm for 13C-NMR). Multiple-pulse experiments  were performed using the standard Bruker programs.HPLC analysis was performed on a Shimadzu LC-10AD pump system equipped with a Shimadzu SPD-M10A photodiode array detector with the detection wavelength set at 206 nm. Melting points were determined using a Microquímica MQAPF-301 melting point apparatus and NMR spectra were recorded on a Bruker DRX 500 [500 MHz . Voucher specimens were deposited in the Prisco Bezerra Herbarium of the UFC under numbers presented in Leaves of the eight O. gratissimum (4.1 kg) were extracted with ethanol and the solvent was removed under reduced pressure given a solid which was submitted to a chromatographic silica gel column, sequentially eluted with hexane, dichloromethane, ethyl acetate and methanol. The ethyl acetate fraction was chromatographed on a silica gel column to yield ursolic acid (35.2 mg) which was identified by melting point and 1H- and 13C-NMR spectroscopy including a 2D sequence, comparing the UA assignments with literature data [Ocimum species was carried out by HPLC: leaves (5 g) were dried using a microwave oven for nine minutes (three times) then they were powdered and extracted with methanol (125 mL) using a Soxhlet apparatus for three hours. Methanol extracts were concentrated and the extracts were diluted with methanol to 10 mL. The afforded solution was filtered through a 0.45 μm syringe filter prior to HPLC use.The air-dried leaves of Ursolic acid (3β-hydroxy-urs-12-en-28-oic acid) (1): White powder, 1H-NMR (DMSO-d6) ppm: 5.14 ; 3.01 ; 2.12 ; 1.92 ; 1.53 ; 1.58 ; 1.56 ; 1.54 ; 1.52 ; 1.47 ; 1.43 ; 1.31 ; 1.29 ; 1.29 ; 1.27 ; 1.05 ; 1.01 ; 0.92 , 0.90 ; 0.87 ; 0.82 ; 0.69 , 0.68 , 0.66 ; 13C-NMR (DMSO-d6) ppm: 178.23 (C-28); 138.18 (C-13); 124.54 (C-12); 76.82 (C-3); 54.77 (C-5); 52.37 (C-18); 47.00 (C-9); 46.81 (C-16); 41.62 (C-14); 39.09 (C-8); 38.48 (C-20); 38.42 (C-19); 38.35 (C-4); 38.22 (C-1); 36.51 (C-10); 36.29 (C-22); 32.69 (C-15); 30.77 (C-7); 28.23 (C-23); 27.52 (C-21); 26.79 (C-2); 23.80 (C-11); 23.24 (C-27); 22.82 (C-16); 21.10 (C-30); 17.97 (C-6); 16.97 (C-29); 16.90 (C-24); 16.03 (C-25); 15.18 (C-26).3PO4 aqueous; A:B = 86:14 (v/v), with a detection wavelength set at 206 nm and 20 μL injection [HPLC was performed using a reversed-phase column (Shimpack CLC-ODS (M) 4.6 mm x 15 cm – particle size 5 μm) eluted at a rate of 0.5 mL/min with an A:B solvent system (A-acetonitrile; B- 1.25% H


Penicillium species collected in Vietnam yielded ten secondary metabolites, including sporogen AO-1 (1), 3-indolecarbaldehyde (2), 2-[methoxy]ethanol (3), 2-[(2R-hydroxypropanoyl)amino]benzamide (4), 4-hydroxybenzandehyde (5), chrysogine (6), 3-acetyl-4-hydroxycinnoline (7), acid 1H-indole-3-acetic (8), cyclo (Tyr-Trp) (9), and 2’,3’-dihydrosorbicillin (10). Their structures were identified by the analysis of 1D and 2D NMR data. Among the isolated compounds, 2-[methoxy]ethanol (3) showed a strong inhibitory effect against Enterococcus faecalis with a minimum inhibitory concentration value of 32 µg/mL. Both 2-[(2R-hydroxypropanoyl)amino]benzamide (4) and 4-hydroxybenzandehyde (5) selectively inhibited E. coli with minimum inhibitory concentration values of 16 and 8 µg/mL, respectively. 2’,3’-Dihydrosorbicillin (10) potentially inhibited α-glucosidase activity at a concentration of 2.0 mM (66.31%).Marine microorganisms are an invaluable source of novel active secondary metabolites possessing various biological activities. In this study, the extraction and isolation of the marine sediment  Streptococcus pneumoniae, Staphylococcus aureus, and Enterococcus faecium, as well as Gram-negative pathogens such as Escherichia coli and Pseudomonas aeruginosa benzamide (4) + , that could be assigned to the molecular formula of 7 is C10H8N2O2. In the 1H-NMR spectrum, four methine signals of a 1,2-disubstituted aromatic ring at δH 8.36 , 7.63 , 7.85 , and 7,89 , one methyl group at δH 2.77 , and an OH signal at δH 9.95  were observed. The 13C-NMR and distortionless enhancement by polarization transfer (DEPT) spectrum showed that this metabolite has 10 carbon signals. Among them, a methyl signal at δC 24.0 (CH3-10), a ketone carbon at δC 194.1 (C-9), four methine carbon of aromatic ring, and four quaternary carbons at δC 145.3 (C-3), 160.6 (C-4), 123.5 (C-4a), and 147.7 (C-8a) were identified. In the heteronuclear multiple bond correlation (HMBC) spectrum, the long-range correlation signals between H-5 and C-8a and C-4, H-8, and C-4a were identified  were tested for their antibacterial activity toward Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Salmonella enterica (ATCC 12228), Enterococcus faecalis (ATCC 13124), Staphylococcus aureus (ATCC 25923), Bacillus cereus (ATCC 13245), and anti-yeast activity against Candida albicans (ATCC 1023). As shown in 3) showed strong inhibitory effect against E. faecalis and C. albicans with MIC values of 32 and 64 µg/mL, respectively. In addition, 2-[(2R-hydroxypropanoyl)amino]benzamide (4) and 4-hydroxybenzandehide (5) selectively suppressed E. coli with the MIC value of 16 µg/mL and 8 µg/mL, respectively. These compounds showed stronger inhibitory effects than the reference compound, streptomycin, that presented an MIC value of 32 μg/mL (7) and 1H-indole-3-acetic (8) exhibited moderate antibacterial ability against E. faecalis with MIC value of 64 µg/mL.All of the isolated secondary metabolites  potentially inhibited α-glucosidase activity at a concentration of 2.0 mM (66.31%) (10) against α-glucosidase. This result suggested that 2’,3’-dihydrosorbicillin (10) may have potential use in the treatment of type 2 diabetes through α-glucosidase inhibition.Among the tested compounds, 2’,3’-dihydrosorbicillin ((66.31%) . InteresThe ESI-MS was measured on Agilent 6120 series single quadrupole LC/MS systems . NMR spectra were recorded on a Bruker AM500 FT-NMR spectrometer using transcranial magnetic stimulation (TMS) as an internal standard. Column chromatography (CC) was performed using a silica gel . Thin layer chromatography (TLC) used pre-coated silica gel 60 F254 .−3. At the final dilution step, aliquots of 50 µL were spread on medium A1  supplemented with 50 µg/mL polymycin B to inhibit Gram-negative bacteria. The plates were incubated at 28 °C for seven days. The colony of fungus M30 was transferred onto a new Petri dish of medium A1 for purification.The marine sediment samples were collected at 14 m depth with geographic coordinates 2105′11″–107050′57″, water at temperature 26 °C in the Co To Island, Vietnam. The samples were collected into 15 mL or 50 mL sterile Falcon tubes and preserved in an ice-box and processed within 24 h. Briefly, 0.5 g of the sample was suspended in 4.5 mL of sterile distilled water, homogenized by vortexing for 1 min, and the suspension was treated using a wet-heat technique (60 °C for 6 min). Next, 0.5 mL of this suspension was transferred to 4.5 mL sterile distilled water, and this step was repeated to set up a ten-fold dilution series to 102, 0.5 µL of 10 mM deoxynucleotide (dNTP’s), 0.2 µL of Taq polymerase, 1.0 µL for both 0.05 mM of NS3F (5′-GCAAGTCTGGTGCCAGCAGCC 3′) and 0.05 mM of NS8R (5′-TCCGCAGGTTCACCTACGGA 3′) primers and 2.0 µL of genomic DNA. The reaction tube was then put into an MJ Thermal cycler, which had been programmed to preheat at 94 °C for 3 min, followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 62 °C for 30 s, and elongation at 72 °C for 45 s before a final extension of 72 °C for 10 min. The estimated PCR product size was about 1300 bp. PCR products were purified by the DNA purification kit (Invitrogen) then sequenced by a DNA Analyzer . Gene sequences were handled by BioEdit v.2.7.5 and compared with bacterial 18S rRNA sequences in the GeneBank database by the National Center for Biotechnology Information (NCBI) Blast program.Genomic DNA was extracted with the Gen Elute Bacterial Genomic DNA kit . Sequences of 18S rRNA was used for identification of the strain. The gene amplifications were performed in a 25.0 µL mixture containing 16.3 µL of distilled water, 2.5 µL of 10X polemerase chain reaction (PCR) buffer, 1.5 µL of 25 mM MgClPenicillium sp.) was cultured in high-nutrient medium  for seven days at 25 °C while stirring at 200 rpm in a bioreactor . The culture solution (30 L) was directly extracted with EtOAc (15 L × 3 times). The EtOAc extract solution was concentrated to dryness by a rotary evaporator .Strain M30 (E. coli (ATCC25922), P. aeruginosa (ATCC27853), S. enterica (ATCC12228), E. faecalis (ATCC13124), S. aureus (ATCC25923), B. cereus (ATCC13245), and C. albicans (ATCC1023). Stock solutions of samples were prepared in DMSO, and the antimicrobial assays were carried out in 96-well microtiter plates against the microbial strains (5 × 105 CFU/mL) using a modification of the published method. After incubation for 24 h at 37 °C, the absorbance at 650 nm was measured using a microplate reader. Streptomycin and nystatin were used as reference compounds.The antimicrobial assay was carried out using α-glucosidase inhibitor. The α-glucosidase inhibitory activity was calculated according to the equation below:control is the absorbance of the control and Asample is the absorbance of the tested samples.The inhibition of α-glucosidase activity was determined using the modified, published assay . Briefly2. The tubes containing the substrate solution were boiled for 5 min and then pre-incubated at 37 °C for 5 min. A total of 100 µL of each sample and 100 µL of substrate solution and 50 µL of porcine pancreatic amylase in Tris–HCl buffer (2 units/mL) were incubated at 37 °C for 10 min. Then, 250 µL of acetic acid 50% was added in each tube to stop the reaction. After, the reaction tubes were centrifuged at 3000 rpm for 5 min at 4 °C, the absorbance of the resulting supernatant was measured at 595 nm using a microplate reader .The assay was carried out following the standard protocol with slight modifications . Starch control is the absorbance of the control and Asample is the absorbance of the tested samples.The α-amylase inhibitory activity was expressed as the percentage of inhibition and was calculated as follows:2Cl2–MeOH to afford six fractions (E1–E6). Fraction E2 (5 g) was chromatographed over silica gel using n-hexane-acetone to get six subfractions (E2.1–E2.6). Fraction E3 (10 g) was subjected to silica gel CC and eluted with CH2Cl2–MeOH to get seven subfractions (E3.1–E3.7). Subfraction E3.1 (0.6 g) was purified by CC on Sephadex LH-20 using MeOH/CH2Cl2  as an eluent to get sĩx subfractions (E3.1.1–E3.1.6). Fraction E3.4 (170 mg) was separated by CC on Sephadex LH-20 using MeOH to get four subfractions (E3.4.1–E3.4.4). Fraction E4 was chromatographed on an RP-18 column eluting with MeOH: water to give seven subfractions, E4.1–E4.7. Subfraction E4.2 (1.99 g) was separated over Sephadex LH-20 eluted with MeOH to get seven subfractions (E4.2.1–E4.2.7). Sub-fraction E2.2 (100 mg) was chromatographed on silica gel eluted with n-hexane-acetone to yield 10 (7.0 mg). Sub-fraction E3.1.2 (120 mg) was handled similar to E2.2 to yield 2.0 mg of 4 and 3.0 mg of 5. By using CC (silica gel), sub-fraction E3.1.1, E4.2.5, and E4.2.7 was eluted individually with n-hexane-EtOAc, TLC CH2Cl2/EtOAc , and TLC (CH2Cl2/MeOH: 9/1) gradient to yield 2, 8, and 9 . Subfraction E3.2 (0.1 g) was purified by CC on silica gel using MeOH as an eluent to yield 3 (7.4 mg). Subfraction E3.3 (90 mg) was purified by CC on Sephadex LH-20 (MeOH), followed by preparative TLC (n-hexane/EtOAc/MeOH: 7/3/0.1) to furnish 6 (4 mg). Subfraction E3.4.3 (100 mg) was chromatographed on silica gel eluted with CH2Cl2–MeOH gradient, consequently gathered and checked by TLC (CH2Cl2/MeOH: 9/1), where 1 (2.3 mg) and 7 (1.6 mg) were obtained.Sterilized Amberlite XAD-16 resin (2 kg) was added to fermentation broth (50 L) to absorb the extracellular secondary metabolites. The culture medium and resin were shaken for 10 h and filtered using a cheesecloth to remove the resin. The resin, cell mass, and cheesecloth were extracted with methanol overnight, concentrated under vacuum, and partitioned between water and ethyl acetate. The organic layer was dried under vacuum to afford 26.4 g of extract. The organic layer (26.4g) was separated by column chromatography (CC) eluted successively with CH1 (Sporogen AO-1)Compound : White amorphous powder; 1H-NMR  δH (ppm): 5.76 , 5.12 , 5.10 , 3.62 , 3.33 , 2,51 , 2.33 , 2.13-2,17 , 1.87 , 1.80-1.83 , 1.42-1.49 , 1.26 , 1.23 ; 13C-NMR  δC (ppm): 192.8 (C-8), 162.9 (C-10), 139.1 (C-11), 121.2 (C-9), 114.5 (C-12), 71.0 (C-3), 68.3 (C-6), 64.5 (C-7), 44.4 (C-4), 41.0 (C-5), 35.2 (C-2), 31.0 (C-1), 19.8 (C-13), 18.8 (C-15), 11.3 (C-14).2 (3-indolecarbadehyde):Compound  White amorphous powder; 1H-NMR  δH (ppm): 7.32 , 7.33 , 7.45 , 7.85 , 8.32 , 10.1 (CHO); 13C-NMR  δC (ppm): 111.4 (C-7), 120.1 (C-7a), 122.0 (C-4), 123.1 (C-5), 124.0 (C-3), 124.5 (C-6), 135.1 (C-2), 136.9 (C-3a), 185.1 (CHO).3 methoxy)ethanol):Compound  Colorless oil; IR (KBr): 3454, 2866, 1456, 1373, 1350, 1296, 1251, 1105 cm−1; MS-ESI: m/z 199.09 [M + Na]+ ; 1H-NMR  δH (ppm): 3.59 , 3.63 , 3.62 , 3.35 , 3.47 , 3.56 , 3.58 , 3.59 , 3.59 , 1.10 ; 13C-NMR  δC (ppm): 71.2 (C-2), 69.2 (C-3), 75.5 (C-5), 75.7 (C-6), 71.5 (C-1’), 71.4 (C-3’), 71.2 (C-4’), 17.7 (CH3-5).4 (2-[(2R-hydroxypropanoyl)amino]benzamide):Compound  White amorphous powder; [α]D28 +21.2 ; ESI-MS: m/z 231.07[M + Na]+; 1H-NMR  δH (ppm): 1.45 , 4.25 , 7.18 , 7.51 , 7.53 , 8.54 ; 13C-NMR  δC (ppm): 21.1 (C-3’), 69.8 (C-2’), 122.2 (C-3), 122.8 (C-1), 124.5 (C-5), 129.4 (C-6), 133.3 (C-4), 139.6 (C-2), 173.3 (C-7), 176.6 (C-1’).5 :Compound  White amorphous powder; 1H-NMR  δH (ppm): 6.95 , 7.80 , 9.87 .6 (Chrysogine):Compound  Yellow powder, 1H-NMR  δH (ppm): 8.26 , 7.76 , 7.67 , 7.47 , 4.87 , 1.65 . 13C-NMR  δC (ppm): 162.8 (C-1), 157.9 (C-8), 148.6 (C-7), 134.9 (C-4), 127.2 (C-6), 126.9 (C-5), 126.5 (C-3), 121.0 (C-2), 67.5 (C-9), 22.5 (C-10).7 (3-Acetyl-4-hydroxycinnoline):Compound  Yellow powder; IR νmax 3412 and 1669 cm−1; HR-ESI-MS m/z 189.0657 [M+H]+ ; 1H-NMR  δH (ppm): 9.95 , 8.36 , 7.89 , 7.85 , 7.63 , 2.77 ; 13C-NMR  δC (ppm): 24.0 (C-10), 123.5 (C-4a), 126.9 (C-5), 129.2 (C-8), 129.4 (C-6), 134.9 (C-7), 145.3 (C-3), 147.7 (C-8a), 160.6 (C-4), 194.1 (C-9).8 (1H-Indole-3-acetic acid):Compound  White amophous powder; 1H-NMR  δH (ppm): 3.62 , 6.98 , 7.06 , 7.15 , 7.31 , 7.62 .9 (Cyclo (Tyr-Trp)):Compound  White amophous powder; 1H-NMR  δH (ppm): 1.48 , 2.57 , 2.76 , 3.05 , 3.87 ethanol (3), 2-[(2R-hydroxypropanoyl)amino]benzamide (4), 4-hydroxybenzandehyde (5), chrysogine (6), acid 1H-Indole-3-acetic (8), cyclo (Tyr-Trp) (9), and 2’,3’-dihydrosorbicillin (10). Among the isolates, 2-[ methoxy]ethanol (3) showed a strong inhibitory effect against E. faecalis with a MIC value of 32 µg/mL. Both of 2-[(2R-hydroxy propanoyl) amino] benzamide (4) and 4-hydroxybenzandehyde (5) selectively killed E. coli with MIC values of 16 and 8 µg/mL, respectively. 2’, 3’-Dihydrosorbicillin (10) potentially inhibited activity at a concentration of 2.0 mM (66.31% α-glucosidase). This study highlights the value of Penicillium sp. derivation from sediment as a source of antimicrobial, antifungal, and anti-α-glucosidase compounds with the potential to combat afflicting pathogens and diabetic diseases.In recent years, ordinary natural-derived


Copaifera spp. are Amazonian species widely studied and whose oleoresins are used by local people for various medicinal purposes. However, a detailed study of the activity of the main phytochemical components of these oleoresins remains to be done. Here, we studied the cytotoxicity and in vitro anti-inflammatory effects of six diterpene acids: copalic, 3-hydroxy-copalic, 3-acetoxy-copalic, hardwickiic, kolavic-15-metyl ester, and kaurenoic, isolated from the oleoresins of Copaifera spp. The diterpenes did not show cytotoxicity in normal cell lines, nor did they show significant changes in viability of tumoral line cells. The 3-hydroxy-copalic was able to inhibit the enzyme tyrosinase (64% ± 1.5%) at 250 µM. The kolavic-15-metyl ester at 200 µM showed high inhibitory effect on lipoxygenase (89.5% ± 1.2%). Among the diterpenes tested, only kaurenoic and copalic acids showed significant hemolytic activities with 61.7% and 38.4% at 100 µM, respectively. In addition, it was observed that only the copalic acid (98.5% ± 1.3%) and hardwickiic acid (92.7% ± 4.9%) at 100 mM inhibited nitric oxide production in macrophages activated by lipopolysaccharide. In this assay, the diterpenes did not inhibit tumor necrosis factor-α production. The acids inhibited the production of IL-6, 3-acetoxy-copalic (23.8% ± 8.2%), kaurenoic (11.2% ± 5.7%), kolavic-15-methyl ester (17.3% ± 4.2%), and copalic (4.2% ± 1.8%), respectively, at 25 µM. The kaurenoic, 3-acetoxy-copalic and copalic acids increased IL-10 production. This study may provide a basis for future studies on the therapeutic role of diterpenic acids in treating acute injuries such as inflammation or skin disorders. Copaifera species, known in Brazil as “copaíbeiras” or “copaíba”, are Amazonian plants best known and used by traditional Amazonian people for the pharmacological activity of the oleoresins that they contain. The species of the Copaifera genus are among the most studied in the world, due to their economic and ecological importance. Copaíba oleoresin is exuded from the tree trunks of certain Copaifera species and has been demonstrated to be primarily composed of sesquiterpenes and diterpenes. The oleoresin has widespread cosmetic and medicinal purposes due to its emollient, bactericide, anti-inflammatory, and anti-melasma properties , 301, 285 m/z; 1H-NMR (CDCl3) δ (ppm) J (Hz): 0.70 ; 0.78 ; 1.01 ; 1.09 ; 1.17 ; 1.35–1.45 ; 1.51–1.60 ; 1.61–1.68 ; 1.70–1.75 ; 1.76–1.80 ; 1.93–2.03 ; 2.17 ; 2.27–2.37 ; 2.38–2.45 ; 3.27 ; 4.52 ; 4.83–4.90 ; 5.68 . 13C-NMR (CDCl3) δ (ppm) J (Hz):37.05 (C-1); 27.86 (C-2); 78.78 (C-3); 39.12 (C-4); 54.57 (C-5); 23.98 (C-6); 38.09 (C-7); 147.59 (C-8); 55.82 (C-9); 39.39 (C-10); 21.61 (C-11); 39.94 (C-12); 163.66 (C-13); 114.81 (C-14); 171.14 (C-15); 19.18 (C-16); 14.47 (C-17); 28.27 (C-18); 15.39 (C-19); 106.8 (C-20). 3-Acetoxy-copalic acid (2). White solid. ESI-MS: 361 [M], 319, 301, 285 m/z; 1H-NMR (CDCl3) δ (ppm) J (Hz): 0.72 ; 0.81–0.90 ; 0.90–0.99 ; 1.22–1.29 ; 1.51–1.60 ; 1.61–1.69 ; 1.72–1.82 ; 1.95–2.03 ; 2.06 ; 2.12–2.22 ; 2.27–2.37 ; 2.38–2.47 ; 4.51–4.61 ; 4.84–4.93 ; 5.63–5.73 . 13C-NMR (CDCl3) δ (ppm) J (Hz):36.7 (C-1); 24.24 (C-2); 80.63 (C-3); 37.96 (C-4); 54.67 (C-5); 23.8 (C-6); 38.02 (C-7); 147.39 (C-8); 55.78 (C-9); 39.25 (C-10); 21.89 (C-11); 39.93 (C-12); 163.55 (C-13); 114.85 (C-14); 171.55 (C-15); 19.19 (C-16); 14.53 (C-17); 28.22 (C-18); 16.52 (C-19); 106.95 (C-20); 21.51 (C-21); 171.17 (C-22).Copalic acid (3). ESI-MS: 303 [M], 285, 259, 99 m/z; 1H-NMR (CDCl3) δ (ppm) J (Hz):0.69 ; 0.81 ; 0.88 ; 0.98–1.07 ; 1.10 ; 1.15–1.23 ; 1.26–1.35 ; 1.36–1.43 ; 1.49–1.55 ; 1.56–1.61 ; 1.67–1.77 ; 1.94–2.04 ; 2.18 ; 2.29–2.36 ; 2.37–2.42 ; 4.45–4.54 ; 4.86 ; 5.31 ; 5.68 . 13C-NMR (CDCl3) δ (ppm) J (Hz):40.09 (C-1); 19.37 (C-2); 42.11 (C-3); 33.58 (C-4); 55.5 (C-5); 24.44 (C-6); 38.29 (C-7); 148.28 (C-8); 56.16 (C-9); 39.71 (C-10); 21.51 (C-11); 39.07 (C-12); 164.08 (C-13); 114.74 (C-14); 171.85 (C-15); 19.22 (C-16); 106.3 (C-17); 33.58 (C-18); 21.71 (C-19); 14.46 (C-20). Kolavic acid-15-metyl ester (4). ESI-MS: 347 [M], 315, 303, 271 m/z; 1H-NMR (CDCl3) δ (ppm) J (Hz): 0.74–0.84 ; 1.10–1.19 ; 1.25 ; 1.29 ; 1.37–1.45 ; 1.46–1.51 ; 1.60 ; 1.86 ; 1.99–2.08 ; 2.19 ; 2.27 ; 2.37 ; 2.68–2.78 ; 3.70 ; 5.69 ; 6.82 . 13C-NMR (CDCl3) δ (ppm) J (Hz): 16.82 (C-1); 28.61 (C-2); 142.24 (C-3); 137.53 (C-4); 40.32 (C-5); 36.21 (C-6); 24.38 (C-7); 37.84 (C-8); 36.32 (C-9); 45.46 (C-10); 36.79 (C-11); 34.46 (C-12); 161.39 (C-13); 114.95 (C-14); 172.82 (C-15); 19.16 (C-16); 15.87 (C-17); 167.24 (C-18); 18 (C-19); 33.38 (C-20); 50.8 (C-21).Kaur-9-en-16-oic acid kaurenoic acid (5). White powder. ESI-MS: 301 [M] m/z; 1H-NMR (CDCl3) δ (ppm) J (Hz): 0.06–0.03 ; 0.70–0.78 ;0.86–0.92 ; 0.88 ; 0.93–1.02 ; 1.07 ; 1.12–1.22 ; 1.17 ; 1.34–1.42 ; 1.47–1.55 ; 1.75–1.83 ; 1.90–2.00 ; 2.03–2.13 ; 2.51–2.61 ; 4.67 ; 4.73 . 13C-NMR (CDCl3) δ (ppm) J (Hz): 41.28 (C-1); 19.09 (C-2); 37.82 (C-3); 44.23 (C-4); 55.12 (C-5); 21.83 (C-6); 39.70 (C-7); 18.43 (C-8); 55.12 (C-9); 39.7 (C-10); 18.43 (C-11); 33.1 (C-12); 43.86 (C-13); 40.71 (C-14); 48.97 (C-15); 155.88 (C-16); 102.98 (C-17); 28.96 (C-18); 183.93 (C-19); 15.6 (C-20).Hardwickiic acid (6). Yellow solid. ESI-MS: 315 [M], 271, 255, 223 m/z. 1H-NMR (CDCl3) δ (ppm) J (Hz): 0.76–0.79 ; 0.80–0.86 ; 0.88 ; 0.97–1.06 ; 1.15–1.22 ; 1.23–1.28 ; 1.40–1.42 ; 1.44–1.46 ; 1.47–1.55 ; 1.56–1.60 ; 1.64–1.73 ; 2.15–2.25 ; 2.17 ; 2.30–2.39 ; 2.40–2.46 ; 6.80–6.90 . 13C-NMR (CDCl3) δ (ppm) J (Hz): 17.45 (C-1); 27.28 (C-2); 140.3 (C-3); 141.46 (C-4); 37.59 (C-5); 35.82 (C-6); 27.49 (C-7); 36.25 (C-8); 38.82 (C-9); 46.69 (C-10); 38.64 (C-11); 18.18 (C-12); 125.58 (C-13); 110.97 (C-14); 142.7 (C-15); 138.38 (C-16); 15.96 (C-17); 172.45 (C-18); 20.54 (C-19); 18.3 (C-20). 2 in RPMI-1640 medium containing 10% fetal bovine serum (FBS), 50 U/mL penicillin and 50 μg/mL streptomycin . Lipopolysaccharide (LPS) was prepared as a 1 mg/mL stock solution in sterile water and stored at −20 °C. The diterpene compounds were added along with treatment with LPS.The murine macrophage cell line J774 was kindly provided by Dr Leda Quercia Vieira  and were cultured at 37 °C in a humidified incubator with 5% COCytotoxicity assay. The cytotoxicity of diterpene compounds to the murine macrophage cell line J774 was determined by the Alamar Blue method as described by Nakayama et al. [3 cells/well) were grown in 96-well tissue culture plates and exposed to substances (2.5 to 10 µg/mL) for 24, 48 and 72 h. After incubation, the Alamar Blue solution (10 μL of 0.4% Alamar Blue (resazurin) in PBS) was added and the cells were incubated for 3 h at 37 °C. Fluorescence was measured (excitation at 545 nm and emission at 595 nm) and expressed as a percentage of the cells in the control after background fluorescence was subtracted. As a positive control of cell death, doxorubicin (5 µg/mL) was used. The assays were performed in triplicate. For the assays with tumoral cells, an Alamar Blue assay was performed using the following cell lines: AGP01—gastric; HCT116—colorectal; MCF07—breast cancer; NIHOVCAR—ovary; SKAMELL4—melanoma; SF295—human glioblastoma, which were seeded in 96-well plates (0.5 × 104 cells per well) and incubated in a CO2 atmosphere at 5% and 37 °C. After 24 h, diterpenes at 20 µM were added to each well and incubated for 72 h. Doxorubicin (10 μM) was used as a positive control and DMSO (0.1%) as a negative control. Before the end of the incubation, 10 µL of Alamar Blue (0.02%) was added to each well. The fluorescent signal was monitored using a multiplate reader using a 530–560 nm excitation wavelength and 590 nm emission wavelength. The fluorescent signal generated from the assay was proportional to the number of live cells in the sample, according to the manufacturer. The IC50 values and their 95% confidence intervals were obtained by nonlinear regression using the GRAPHPAD program.a et al. . Briefly2 10 mM) by centrifugation at 700 g for 10 min. The pellet of erythrocyte was re-suspended in saline solution for preparation of erythrocyte suspension −2% (SE). A total of 100 µL of the samples of diluted diterpenes was added to 96-well microplates in different concentrations (6.25 to 800 µM); 100 µL of SE was then added; and the samples were incubated for 60 min at 37 °C under constant agitation. The release of hemoglobin was determined after centrifugation (700 g for 10 min) by photometric analysis of the supernatant at 540 nm. Complete hemolysis was achieved using 0.2% Triton X-100 yielding the 100% control value. All procedures used during the experiments were maintained in accordance with internationally accepted principles for laboratory animal use and the experimental protocols were approved (n° 066/2011) by the Ethical Committee of the Animal Research of the Federal University of Amazonas, Manaus, Brazil.Blood was collected in heparinized-tubes from Wistar rats, and washed three times with saline solution (NaCl 0.85% + CaCl• production by J774 cells was assayed by measuring the accumulation of nitrite in the culture medium using a Griess reaction [6 cells/mL) with diterpene compounds in different concentrations from 6.25 to 100 μM, cells were incubated for 24 h with LPS (1 μg/mL), at 37 °C in a 5% CO2 incubator. Nitric oxide was measured as NO2 in culture supernatant by reaction with Griess reagent. Absorbance of the reaction product was determined at 560 nm using a microplate reader . Sodium nitrite was used as a standard to calculate nitrite concentrations.NOreaction . Briefly6 cells/mL) were incubated with different concentrations of diterpene compounds (12.5 to 50 μM), and then stimulated with 1 μg/mL of LPS. The culture supernatants were collected after 24 h of LPS stimulation. The levels of cytokines in the culture media were measured by flow cytometry (BD Cytometric Bead Array—CBA—Mouse Inflammation kit) according to the manufacturer’s instructions.Macrophage cells  followed by the Tukey Copaifera spp. oleoresin. These isolated compounds did not show toxicity to normal or tumor cell lines. The compound 3-hydroxy-copalic acid showed higher inhibitory activity for tyrosinase enzyme and NO• production in LPS stimulated macrophages. Kolavic acid 15-methyl ester showed high lipoxigenase inhibition activity when compared to standard quercertin. Kaurenoic, 3-acetoxy-copalic, kolavic-15-methyl ester and copalic acids inhibited the production of IL-6 and kaurenoic, 3-hydroxy-copalic and copalic acids induced production of interleukin-10. Thus, we conclude that Copaifera spp. oleoresins contain components such as diterpenic acids, which are responsible for contributing to anti-inflammatory effects and other biological activities. However, the development of pharmaceutical formulations of copaíba oleoresin are needed in order to perform clinical trials aimed at demonstrating its application in the treatment of acute skin lesions and/or chronic inflammatory diseases.In this study, we obtained six diterpenic acids, three labdanes , two clerodanes (hardwickiic acid and kolavic acid 15-methyl ester), and one kaurane (kaurenoic acid) from


Taxus yunnanensis and determined to be a member of Talaromyces. The extracts from the solid fermentation of Talaromyces sp. T1BF were purified and obtained three azaphilones, including a new one. They were identified on the basis of spectral data as 6α-hydroxy-7β-methyl-8-oxo-3-((E)- prop-1-en-1-yl)-5,6,7,8-tetrahydro-1H-isochromen-7-yl-4'-hydroxy-2'-methoxy-6'-methyl- benzoate, named as kasanosin C (1), entonaemin A (2) and (+)-mitorubrin (3).The strain T1BF was isolated from the old bast tissue of  Taxus brevifolia . After cultivation for two weeks at 28 Kasanosin C (1): Yellow amorphous solid.  : = + 151 . UV (MeOH) λmax (log ε): 205 (5.30), 284 (2.39), 374 (3.66). 1H and 13C-NMR data  see M + Na]+; HR-ESI-MS: 423.1424 [M + Na]+ .Entonaemin A (2): Yellow powder. : = + 132 . 1H and 13C-NMR data  see M + Na]+. (+)-Mitorubrin (3): Yellow powder. : = 144 . 1H and 13C-NMR data  see M + Na]+.Talaromyces fungus sp. The new compound was determined to be kasanosin C by spectroscopic methods, including HR-ESI-MS and 2D-NMR experiments, in combination to the comparison with known compounds. A new azaphilone as well as two known azaphilones were isolated from an endophytic


Reaction of 12a,b with formamide, formic acid and triethyl orthoformate give the pyrazolopyrimidine, pyrazolopyrimidin-4(3H)one and 5-ethoxymethylene-aminopyrazole-4-carbo-nitrile derivatives 13-15a,b, respectively. Compounds 15a,b reacted with benzhydrazide and hydrazine hydrate to afford pyrazolotriazolopyrimidine and pyrimidin-5-yl]amine derivatives 16a,b and 17a,b. Reactions of compounds 17a,b with triethyl orthoformate and carbon disulfide give the corresponding pyrazolo-triazolopyrimidine derivatives 18a,b and 19a,b, respectively.Hydrazonyl bromides  To the resulting solution the appropriate hydrazonyl bromide 4-Benzoyl-3-(4-fluorophenyl)-1-(4-nitrophenyl)-5-phenylpyrazole (5a). Off-white solid; mp. 185-8 °C (from acetic acid).4-Benzoyl-3--1-(4-nitrophenyl)-5-phenylpyrazole (5b). Pale yellow solid; mp. 159-61 °C (from ethanol); lit. mp. 150 °C pyrimidin-4-yl]amines13a,b12a,b (0.005 mol) and formamide (10 mL) was refluxed for 2-3 h. The solution was cooled, then poured onto water. The solid that precipitated was collected and crystallized to give pyrimidin-4-yl]amine pyrimidin-4-yl]amine .A mixture of 5-amino-3-aryl-1-(4-nitrophenyl)pyrazole-4-carbonitrile Synthesis of 3-aryl-1-(4-nitrophenyl)-1,4-dihydro-5H-pyrazolopyrimidin-4-ones14a,b12a,b (0.005 mol) and formic acid (20 mL) was refluxed for 1 h. The solution was cooled, and then poured on water. The solid that precipitated was collected and crystallized from the indicated solvent to give 3-(4-fluorophenyl)-1-(4-nitrophenyl)-1,4-dihydro-5H-pyrazolopyrimidin-4-one pyrimidin-4-one .A mixture of 5-amino-3-aryl-4-1-(4-nitrophenyl)pyrazole-4-carbonitrile Synthesis of 3-aryl-5-ethoxymethyleneamino-1-(4-nitrophenyl)pyrazole-4-carbonitriles15a,b12a,b (0.005 mol), triethyl orthoformate (3 mL) and acetic anhydride (20 mL) was heated under reflux for 5 h. After cooling the precipitated solid was filtered off and recrystallized from the indicated solvent to give 5-ethoxy-methyleneamino-3-(4-fluorophenyl)-1-(4-nitrophenyl)pyrazole-4-carbonitrile  or 5-ethoxymethyleneamino-3--1-(4-nitro-phenyl)pyrazole-4-carbonitrile .A mixture of 5-amino-3-aryl-1-(4-nitrophenyl)pyrazole-4-carbonitrile Synthesis of 9-aryl-7-(4-nitrophenyl)-2-phenyl-7H-pyrazolotriazolopyrimidines16a,b15a,b (0.01 mol) and benzhydrazide  in THF (30 mL) was refluxed for 12 h. The mixture was cooled and the solvent was removed under reduced pressure and the resulting precipitate was purified by crystallization from the indicated solvent to give 9-(4-fluorophenyl)-7-(4-nitrophenyl)-2-phenyl-7H-pyrazolotriazolopyrimidine  or 9--7-(4-nitrophenyl)-2-phenyl-7H-pyrazolo-triazolopyrimidine .A mixture of 3-aryl-5-ethoxymethyleneamino-1-(4-nitrophenyl)pyrazole Synthesis of pyrimidin-5-yl]amines17a,b15a,b (0.01 mol) in tetrahydrofuran (40 mL). The reaction mixture was stirred at room temperature for 1 h. The precipitate which formed was filtered off, washed with water, dried in air and crystallized from the indicated solvent to give pyrimidin-5-yl]amine  or pyrimidin-4-yl]amine .Hydrazine hydrate (8 mL) was added to a suspension of 5-ethoxymethyleneamino-3-aryl-1-(4-nitrophenyl)pyrazole-4-carbonitriles Synthesis of 9-aryl-7-(4-nitrophenyl)-7H-pyrazolotriazolopyrimidines18a,b17a,b (0.005 mol) with (15 ml) of triethyl orthoformate was refluxed for 1 h. After cooling, the precipitated product was collected by filtration and crystallized from the indicated solvent to give 9-(4-fluorophenyl)-7-(4-nitrophenyl)-7H-pyrazolotriazolopyrimidine  or 9--7-(4-nitrophenyl)-7H-pyrazolo-triazolopyrimidine .A mixture of [3-aryl-4-imino-1-(4-nitrophenyl)pyrazole-5-yl]amine Synthesis of 9-aryl-7-(4-nitrophenyl)-7H-pyrazolotriazolopyrimidine-2(3H)-thiones19a,b17a,b (0.01 mol) with carbon disulfide  and potassium hydroxide  in ethanol (15 mL) was heated under reflux for 10 h. After removal of ethanol, water was added and the resulting alkaline solution was acidified with acetic acid and the precipitate formed collected by filtration, washed with water, dried and crystallized from the indicated solvent to afford 9-(4-fluorophenyl)-7-(4-nitrophenyl)-7H-pyrazolotriazolopyrimidine-2(3H)-thione  or 9--7-(4-nitrophenyl)-7H-pyrazolo-triazolopyrimidine-2(3H)-thione .A mixture of [3-aryl-4-imino-1-(4-nitrophenyl)pyrazol-5-yl]amine


Then α-O-4 phosphorylation and β-O-4 phosphorylation follow a two-step reaction mechanism in the acid treatment step, respectively. However, since phosphorylation of α-O-4 is more energetically accessible than phosphorylation of β-O-4 in phosphoric acid, the phosphorylation of α-O-4 could be controllably realized under certain operational conditions, which could tune the electron and hole transfer on the right side of β-O-4 in the H2PO4− functionalized lignin. The results provide a fundamental understanding for process-controlled modification of lignin and the potential novel applications in lignin-based imprinted polymers, sensors, and molecular devices.The study focused on the structural sensitivity of lignin during the phosphoric acid–acetone pretreatment process and the resulting hydrolysis and phosphorylation reaction mechanisms using density functional theory calculations. The chemical stabilities of the seven most common linkages (β Lignin is a complex polymer of aromatic alcohols mostly derived from wood ,2 and ac3PO4, then the dissolved cellulose sediments in acetone while the lignin dissolves in the acetone [Recently, the phosphoric acid–acetone pretreatment process represents one of the most efficient methods to separate lignin and cellulose from lignocellulose, where bonds between lignin and cellulose break and cellulose dissolves into H acetone ,12,13,143PO4 is a weak triprotic acid, non-toxic and inexpensive compared with other mineral acids. Cellulose can be dissolved in phosphoric acid more simply, faster and at lower temperatures. The hydrogen ions from H3PO4 could be easily diffused into cellulose [3PO4 to form cellulose phosphate, and a competition of hydrogen-bond formation among the remaining hydroxyl groups on cellulose chains, a hydrogen ion and a water molecule; the second case is that the pretreated cellulose changes into amorphous cellulose after phosphoric acid pretreatment [3PO4 pretreatment can accelerate the enzyme adsorption rate and enhance the subsequent hydrolysis process. However, limited attention has been played to the behavior of lignin in the phosphoric acid-acetone pretreatment process. The detailed interaction between lignin and solution, the related reaction mechanism and the resulting modified properties of the lignin remain unknown.In these processes, Hellulose . Two phereatment . The amoTo address these fundamental questions, we used models for the seven most common linkages in lignin (accounting for approximately 87% of linkages in softwood lignin ) to dete-O-4, α-O-4, 5-5, β-1, β-5, β-β, and 4-O-5) were built in reference to the work of Beste [2O, CH3COCH3, and H3PO4 solutions. The reactions start from the optimized geometries of β-O-4, α-O-4, 5-5, β-1, β-5, β-β, and 4-O-5 in these different solutions, respectively. The energy barrier (Ea) for β-O-4 decomposition in H2O, CH3COCH3, and H3PO4 solutions is 55.2 kcal/mol, 60.4 kcal/mol, and 55.5 kcal/mol, respectively, while the reaction energy (Er) is 54.1 kcal/mol, 53.544 kcal/mol, and 55.2 kcal/mol. Ea and Er for α-O-4 decomposition in H2O, CH3COCH3, and H3PO4 solutions is 42.8 kcal/mol and 12.2 kcal/mol, 53.2 kcal/mol and 42.5 kcal/mol, 44.1 kcal/mol and 19.8 kcal/mol, respectively. Ea and Er for 5-5 decomposition in H2O, CH3COCH3, and H3PO4 solutions is 138.6 kcal/mol and 131.9 kcal/mol, 140.1 kcal/mol and 139.2 kcal/mol, 138.3 kcal/mol and 138.0 kcal/mol, respectively. Ea and Er for β-1 decomposition in H2O, CH3COCH3, and H3PO4 solutions is 133.0 kcal/mol and 132.4 kcal/mol, 133.1 kcal/mol and 132.2 kcal/mol, 130.7 kcal/mol and 129.6 kcal/mol, respectively. Ea and Er for β-5 decomposition in H2O, CH3COCH3, and H3PO4 solutions is 100.3 kcal/mol and 97.5 kcal/mol, 97.4 kcal/mol and 93.3 kcal/mol, 95.8 kcal/mol and 92.2 kcal/mol, respectively. Ea and Er for β-β decomposition in H2O, CH3COCH3, and H3PO4 solutions is 134.1 kcal/mol and 116.1 kcal/mol, 95.6 kcal/mol and 85.6 kcal/mol, 103.2 kcal/mol and 96.4 kcal/mol, respectively. Ea and Er for 4-O-5 decomposition in H2O, CH3COCH3, and H3PO4 solutions is 88.2 kcal/mol and 84.9 kcal/mol, 82.2 kcal/mol and 82.0 kcal/mol, 85.4 kcal/mol and 82.4 kcal/mol, respectively. The results imply that energetically 5-5, β-1, β-5, β-β, and 4-O-5 are rather chemically stable during the phosphoric acid–acetone pretreatment process, which can maintain the structural integrity and the yield of lignin during the pretreatment process [-O-4 and α-O-4, and as mentioned before that β-O-4 is one of the main cleavage modes [-O-4 is small  [-O-4 linkages resulted in relatively permanent damage to lignin, since it is relatively difficult for the reverse process of the decomposition to happen due to the higher activation energy  . Interesm et al. .i.e., initial state (IS), transition state (TS), and final state (TS) of the decomposition reaction of β-O-4, α-O-4, 5-5, β-1, β-5, β-β, and 4-O-5, respectively, is further discussed. The different density isosurfaces for the β-O-4, α-O-4, 5-5, β-1, β-5, β-β and 4-O-5 linkage are symmetric at IS. However, at TS and FS, the electron density is enriched at the O atom of the β-O-4, α-O-4 and 4-O-5 linkage, respectively, while the electron density is depleted at the corresponding Cβ, Cα, and C4 atom; for the β-1 and β-5 linkages, the electron density is enriched at one Cβ atom and depleted at the C1 atom and C5 atom, respectively; also for the 5-5 and β-β linkages, the electron density is enriched at one C atom and depleted at the other C atom, respectively. The partial electron transfer through the reaction resulted in an unsymmetrical electron density population, which implies that the decomposition of β-O-4, α-O-4, 5-5, β-1, β-5, β-β, and 4-O-5 in these solutions will proceed via a heterolytic, unzipping mechanism, as described in the work of Sturgeon et al. [The charge density difference of the system at the critical points, n et al. .-O-4 and α-O-4. The one-step reaction channel starts from the optimized adsorption geometries of H3PO4 on β-O-4 and α-O-4, respectively.Further, we focused on the phosphorylation mechanism of lignin. 3PO4 to the O site  and the lowest unoccupied molecular orbit (LUMO) of lignin.Hydrolysis and phosphorylation of lignin occur during the phosphoric acid-acetone pretreatment process, which can modify the properties of lignin. For example, -OH groups provide an opportunity to create hydrogen bonds between copolymer and lignin ; incorpotal ions . Herein,-O-4 linkage and β-O-4 linkage, with the isosurface (0.01) of the related HOMO and LUMO.Further, we compared the HOMO and the LUMO of the lignin before and after hydrolysis and phosphorylation. -O-4 contributes to the LUMO of the pure lignin system. In -O-4 linkage are almost the same as those for the pure lignin in -O-4 linkage never change the HOMO and LUMO of lignin.In 2PO4-functionalized and HO-functionalized lignin at β-O-4 linkage, HO-functionalization at α-O-4 linkage changes the HOMO of the lignin system, where β-1 contributes to the HOMO  exchange-correction functional [w/w), in reference to the work of Wang [−6 Ha and gradient convergence of 1 × 10−6 Ha·Å−1. A formulation for the linear (LST) and quadratic synchronous transit (QST) methods was used to search the transition states and investigate lignin fragment decomposition [The equilibrium structure of the model lignin in different solutions was obtained by performing molecular dynamics simulations with the LAMMPS program package , using tnctional  and the nctional ,39,40 ba of Wang  and Muns of Wang . Specifi of Wang . Calculaposition .-O-4, β-β, 4-O-5, β-1, 5-5, α-O-4, and β-5) was modeled to detect the chemical stability of these linkages in H3PO4, CH3COCH3, and H2O solutions present during the phosphoric acid–acetone pretreatment process. 5-5, β-1, β-5, β-β, and 4-O-5 are chemically stable to maintain the relative structural integrity of lignin, while partial structural damage will happen to lignin due to the relative vulnerability of β-O-4 and α-O-4. Then two-step phosphorylation reaction happened at α-O-4 linkage as the major modification to lignin in H3PO4 solution, where H2PO4− group bound to Cα, resulting in a stable configuration of H2PO3-O-lignin species. H2PO4-functionalization at α-O-4 linkage could controlled electron and hole transfer on the right ring of β-O-4 in the H2PO3-O-lignin species. Results provide a fundamental understanding for process-controlled modification of lignin and potential novel applications in lignin-based imprinted polymers, sensors, and molecular devices.We theoretically studied the lignin hydrolysis and phosphorylation mechanism during the phosphoric acid–acetone pretreatment process of lignin using density functional theory calculations. The lignin fragment with the seven most common linkages (β


Their structures were elucidated on the basis of their spectroscopic analysis, and the absolute configuration of compounds was confirmed by CD and X-ray crystallographic analysis using anomalous scattering of Cu Kα radiation. Compounds 1 and 3 showed moderate inhibitory activities against NO production in lipopolysaccharide-induced (LPS) RAW 264.7 macrophages in vitro.Three 18(4→3) Tripterygium wilfordii, also called “leigongteng”, is a member of the Celastracrae family + , indicating eight degrees of unsaturation. The 1H NMR spectrum data of 1 ], two aromatic methines [δH 6.10  and 6.12 ], an oxygenated methylene [δH 4.77], and two oxygenated methines [δH 3.96]. The 13C NMR and DEPT spectra demonstrated 20 carbons resonances, which contained a carbonyl, seven oxygenated carbons , two aromatic quaternary carbons, and two aromatic methenes. According to this information, the skeleton of 1 was recognized to be an 18(4→3)-abeo-abietane  to C-18(δC 173.5) and C-3(δC 122.8). Structure of rings A and B could be determined by the 1H, 1H COSY correlations of H-1/H-2 and H-5/H-6/H-7, and the HMBC correlation of H3-20 [δH 1.00 ]/C-1 (δC 27.5), C-5 (δC 35.1), C-9 (δC 76.0), and C-10 (δC 40.2). The HMBC Correlations from H-12 to C-9, C-13 (δC 82.7), C-14 (δC 77.6), and C-15 (δC 70.5); from H-14 [δH 3.99 ] to C-8 (δC 75.9) and C-13 corroborated the ring C. The four hydroxy groups were assigned to C-8, C-9, C-14, and C-15, indicated by the HMBC correlations of 8-OH [δH 4.86 ]/C-7 (δC 74.3) and C-8, 9-OH [δH 4.03 ]/C-10, C-11 (δC 134.0), and C-12 (δC 131.1), 15-OH [δH 4.15 ]/C-13, C-15, C-16 (δC 24.3), and C-17 (δC 27.4); the 1H, 1H COSY correlation from H-14 to 14-OH [δH 4.90 ]. Finally, the fifth ring was an oxide bridge between C-7 and C-13 (furan ring) to meet with the HRESIMS data.Tripordolide A .NOESY analysis was used to deduce the relative configuration of 1 . Orientaadiation . Accordi2) was obtained as a colorless oil and showed a [M + Na]+ ion peak at m/z 399.1052  in the HRESIMS, which corresponded to the molecular formula C19H20O8. Some obvious signals, a ring of α, β-unsaturated-γ-lactone  and an oxygen isopropyl group , indicated 2 was also an 18(4→3)-abeo-abietane derivative ]/C-4, C-5 (δC 128.7) and C-9 (δC 60.7); of H-2 [δH 7.70 ]/C-4, C-10 (δC 138.5) and C-18 (δC 69.7) and C-13 (δC 74.9). Furthermore, compared to the tripterlide F, the oxygen isopropyl group of 2 also lead to the lower field of C-15 (δC 73.6), C-16 (δC 25.2), and C-17 (δC 26.8). The relative configuration was established by the NOESY correlations of H-12/H-1 and of H-7/H-14/H-12  was obtained as a colorless oil and had the molecular formula C20H26O7, deduced from the [M + Na]+ ion peak at m/z 401.1574 . Its 1H and 13C NMR characteristics -abeo-abietane derivative. Its planar structure has been confirmed by an analysis of a two-dimensional NMR ]/C-15 (δC 69.6), C-16 (δC 27.4), and C-17 (δC 26.1); 1H, 1H COSY correlations of 7-OH [δH 5.34 ]/H-7 [δH 4.22 ], 11-OH [δH 5.02 ]/H-11[δH 4.45 ], and 14-OH [δH 4.86 ]/H-14 [δH 4.47 ] suggested that the four hydroxy groups were located at C-7, C-11, C-14, and C-15. HMBC correlations from H3-20 [δH 1.05 ] to C-5 (δC 40.8), C-10 (δC 35.6), and C-11 (δC 62.0), and from H-14 [δH 4.47 ] to C-8 (δC 131.7), C-9 (δC 137.8), C-12 (δC 55.9), and C-13 (δC 61.9) indicated a double bond between C-8 and C-9. Moreover, a trisubstituted epoxide between C-12 and C-13 was formed to meet with its indices of hydrogen deficiency, which also led the chemical shift of C-12 (δC 55.9) and C-13 (δC 61.9) to upfield. The relative configuration was established by the NOESY correlations of H-12/H-11/H-20 and of H-6β/H-7/H-14-OH  RAW 264.7 macrophages in vitro. Compounds 1 and 3 inhibited NO production in mouse peritoneal macrophage 48.4 ± 5.7% and 53.5 ± 4.8% at a concentration of 10 μM, while the positive control dexamethasone gave an inhibitory ratio of 63.1 ± 4.6% at an identical concentration.Three 18(4→3)1. A JASCO P-2000 polarimeter , JASCO V650 , spectrophotometer, and XT-5B micro melting point apparatus  were used to collect optical rotations, UV spectra, and melting points, respectively. VNS-600 and Bruker-500  spectrometers were used to collected NMR spectra. The Agilent 1100 series LC/MSD ion trap mass spectrometer  was used to collect HRESIMS spectra. Preparative HPLC was performed on a Shimadzu LC-6AD instrument with a SPD-20A detector , using a YMC-Pack ODS-A column . Column chromatography was performed with silica gel , polyamide , and ODS . A Bruker APEX DUO diffractometer with Cu Kα radiation  was used to collect the X-ray data of compound T. wilfordii were collected in Taining, Fujian, China, in September 2009 and identified by Professor Lin Ma from the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College. A voucher specimen (No. 20090034) is deposited at the herbarium of the Institute of Materia Medica, Chinese Academy of Medical Sciences, and Peking Union Medical College, China.The leaves of Tripterygium wilfordii (100 kg) were extracted two times with 80% EtOH (800 L) at 80 °C for 2 h. After filtration and evaporation ethanol under reduced pressure at 50 °C, the aqueous residue was diluted with H2O and then partitioned three times with ethyl acetate (30 L). The remaining water extract was subjected to passage over polyamide by elution with water and 30%, 60%, and 95% EtOH-H2O. Then the water elution was subjected to passage over D101 macroporous resin by elution successively with H2O, 30%, 60%, and 95% EtOH-H2O (Fractions B1-B4). Fraction B3 (338.5 g) was subjected to passage over a bergmeal column eluting successively with ethyl acetate, ethanol, and methanol (Fractions C1–C3). Fraction C2 (151.3 g) was chromatographed on a silica gel column eluting successively with a solvent gradient system  to afford 7 fractions (D1−D7). Fraction D4 (3.36 g) was passed over an PRP-512A macroporous resin column with MeOH-H2O  to give four fractions (E1–E4), and the fraction E3 (0.45 g) was purified by preparative HPLC  to give 1 (5.3mg), 2 (1.3 mg), and 3 (1.0 mg).The air-dried and powdered leaves of 20H26O7 were recrystallized from MeOH mounted in inert oil and transferred to the cold gas stream of the diffractometer.Single crystals of C1): C20H26O7, M = 378.41, monoclinic, a = 7.7259(3) Å, b = 7.3200(3) Å, c = 15.4365(5) Å, β = 98.081(3)°, U = 864.33(5) Å3, T = 105(2), space group P21 (no. 4), Z = 2, μ(Cu Kα) = 0.912, 6799 reflections measured, 3061 unique (intR = 0.0282) which were used in all calculations. The final wR(F2) was 0.0901 . Flack parameter was −0.02(11).Crystal data of tripordolide A (1) have been deposited in the Cambridge Crystallographic Data Centre (deposition numbers: CCDC 1844652). Copies of the data can be obtained free of charge via www.ccdc.cam.ac.uk.Crystallographic data for the structure of tripordolide A (Tripordolide A (1): colorless needle; [α]c 0.1 MeOH); UV (MeOH) λmax (log ε) 220 (3.53) nm; 1H NMR  and 13C NMR , see m/z 379.17532 [M + H]+ . (.17513). .Tripordolide B (2): colorless oil (MeOH); [α]c 0.1, MeOH); IR (microscope) νmax 3394, 2921, 1757, 1646, 1419, 1323, 1050, and 1027 cm−1; 1H NMR  and 13C NMR , see m/z 399.1052 [M + Na]+ . (9.1050). .Tripordolide C (3): colorless oil (MeOH); [α]c 0.1, MeOH); UV (MeOH) λmax (log ε) 220 (4.02) nm; 1H NMR  and 13C NMR , see m/z 401.1574 [M + Na]+ . (1.1571). .The inhibitory effects of three compounds on NO production in LPS-induced RAW264.7 macrophage were evaluated. Dexamethasone was used a positive control. Concrete operation method was according to described in the literature .abeo-abietanes were isolated from the plant. Phytochemical studies on T. wilfordii have led to three abeo-abietanoids, a new natural product, and two new compounds, named tripordolides A–C (1–3). Tripordolide A (1) was reported in a patent as a synthetic product, but none of the spectra data was used as reference [Kα radiation. Notably, tripordolide A (1) was the first example of 18(4→3)-abeo-abietanes, which possessed 7/13 oxygen bridges in the natural products. Compounds 1 and 3 showed moderate inhibitory activities against NO production in LPS-induced RAW 264.7 macrophage in vitro.In this research, no more than 30 18(4→3)-