Isolation of secondary metabolites from Hortia oreadica (Rutaceae) leaves through high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) with a two-phase solvent system (hexane–ethanol–acetonitrile–water 10:8:1:1, v/v) was applied to examine the leaves of Hortia oreadica, which afforded the known limonoid guyanin (1), the alkaloids rutaecarpin (2) and dictamnine (6), the dihydrocinnamic acid derivatives methyl 5,7-dimethoxy-2,2-dimethyl-2H-1-benzopyran-6-propanoate (3), 5,8-dimethoxy-2,2-dimethyl-2H-1-benzopyran-6-propanoic acid (4), together with the new E-3,4-dimethoxy-α(3-hydroxy-4-carbomethoxyphenyl)cinnamic acid (5). The recovery of compounds 1–6 was determined by comparison with LC-atmospheric pressure chemical ionization MS/MS data: 66.2%, 93.1%, 102.5%, 101.2%, 99.0% and 84.9%, respectively. Compound 3 showed IC₅₀ of 23.6 μM against Plasmodium falciparum and 15.6 μM against Trypanosoma brucei rhodesienses and was not toxic to KB cells (IC₅₀ > 100 μM).     

Reaction of hydroxyflavones with secondary phosphine chalcogenides in the CCl4/Et3N system: synthesis of a new family of phosphorylated flavonoids

Flavonoids (3-hydroxy-, 3-hydroxy-4′-methoxy-, 3-hydroxy-7-methoxy-, and 5,7-dihydroxyflavones) react with secondary phosphine chalcogenides in the CCl₄/Et₃N redox system (50–52 °C, 3–5 h) to afford chemoselectively the corresponding chalcogenophosphinates of the hydroxyflavones in 65–82% yield.     

Using the cationic surfactants N-cetyl-N-methylpyrrolidinium bromide and 1-cetyl-3-methylimidazolium bromide for sweeping–micellar electrokinetic chromatography

This paper describes a sweeping–micellar electrokinetic chromatography (sweeping–MEKC) technique for the determination of seven benzodiazepines, using, as sweeping carriers, the ionic liquid-type cationic surfactants 1-cetyl-3-methylimidazolium bromide (C₁₆MIMBr) and N-cetyl-N-methylpyrrolidinium bromide (C₁₆MPYB). These surfactants resemble the commonly employed cationic surfactant cetyltrimethylammonium bromide (CTAB), but they provide different separation efficiencies. We optimized the separation and sweeping conditions, including the pH, the concentrations of organic modifier and surfactant, and the sample injection volume. Adding C₁₆MIMBr or C₁₆MPYB to the background electrolyte enhanced the separation efficiency and detection sensitivity during the sweeping–MEKC analyses of the benzodiazepines. C₁₆MIMBr enhanced the sensitivity for each benzodiazepine 31–59-fold; C₁₆MPYB, 86–165-fold. In the presence of C₁₆MPYB, the limits of detection for the seven analytes ranged from 4.68 to 9.75 ng/mL. We adopted the sweeping–MEKC conditions optimized for C₁₆MPYB to satisfactorily analyze a human urine sample spiked with the seven benzodiazepines. To minimize the matrix effects, we subjected this urine sample to off-line solid phase extraction (SPE) prior to analysis. The recoveries of the analytes after SPE were satisfactory (ca. 77.0–88.3%). Our experimental results reveal that the cationic surfactant C₁₆MPYB exhibits superior sweeping power relative to those of C₁₆MIMBr and CTAB and that it can be applied in sweeping–MEKC analyses for the on-line concentrating and analyzing of benzodiazepines present in real samples at nanogram-per-milliliter concentrations.     

Organocatalytic asymmetric Friedel–Crafts reaction of 1-naphthols with isatins: an enantioselective synthesis of 3-aryl-3-hydroxy-2-oxindoles

An organocatalytic enantioselective Friedel–Crafts reaction of 1-naphthols with isatins has been developed employing bifunctional thiourea–tertiary amine organocatalysts. A variety of isatin derivatives react well with 1-naphthols in the presence of Cinchona derived thiourea 1a to provide biologically important chiral 3-aryl-3-hydroxy-2-oxindoles (3a–zg) in good yield (70–84%) and moderate to good enantioselectivity (37–83%).     

Sequential determination of fat- and water-soluble vitamins in Rhodiola imbricata root from trans-Himalaya with rapid resolution liquid chromatography/tandem mass spectrometry

A rapid method was developed to determine both types of vitamins in Rhodiola imbricata root for the accurate quantification of free vitamin forms. Rapid resolution liquid chromatography/tandem mass spectrometry (RRLC–MS/MS) with electrospray ionization (ESI) source operating in multiple reactions monitoring (MRM) mode was optimized for the sequential analysis of nine water-soluble vitamins (B₁, B₂, two B₃ vitamins, B₅, B₆, B₇, B₉, and B₁₂) and six fat-soluble vitamins (A, E, D₂, D₃, K₁, and K₂). Both types of vitamins were separated by ion-suppression reversed-phase liquid chromatography with gradient elution within 30 min and detected in positive ion mode. Deviations in the intra- and inter-day precision were always below 0.6% and 0.3% for recoveries and retention time. Intra- and inter-day relative standard deviation (RSD) values of retention time for water- and fat-soluble vitamin were ranged between 0.02–0.20% and 0.01–0.15%, respectively. The mean recoveries were ranged between 88.95 and 107.07%. Sensitivity and specificity of this method allowed the limits of detection (LOD) and limits of quantitation (LOQ) of the analytes at ppb levels. The linear range was achieved for fat- and water-soluble vitamins at 100–1000 ppb and 10–100 ppb. Vitamin B-complex and vitamin E were detected as the principle vitamins in the root of this adaptogen which would be of great interest to develop novel foods from the Indian trans-Himalaya.     

Binaphthyl-based chiral macrocyclophanes with variously sized cavities: Dn-symmetrical structure constructed from unidirectionally-inclined rod segments

As new and chiral macrocyclophanes with unique structures, variously sized Pn and Mn (n=2–7=number of ‘rod’ segments) with D₂–D₇ symmetry were constructed by alternating connection of axially chiral binaphthyls and linear biphenyls via –CH₂O– moieties, so that the macrocycle consists of multiple rod-like naphthalene–biphenyl–naphthalene units linked together at the binaphthyl bonds. The dihedral angle of the two naphthalene rings of binaphthyl is restricted to around 90°, and the calculated values of strain energy difference per naphthalene–biphenyl unit in P2–P7 are almost independent of the macrocycle size, presumably owing to the flexibility of the –CH₂O– connectors.     

Hexaphenylbenzene end-capped tri(p-phenylenevinylenes): synthesis and properties

Three new hexaphenylbenzene end-capped tri(p-phenylenevinylenes) were synthesized by the Horner–Wadsworth–Emmons reaction in good yields (86–76%). All compounds have bright fluorescent emissions in the blue to blue–green region in solution (λ_max = 445–492 nm in tetrahydrofuran) and also high emission efficiency (Φ_fl = 0.51–0.72 in tetrahydrofuran). They exhibit good electrochemical reversibility, high thermal stability, and have high HOMO value.     

Completed preparative separation of alkaloids from Cephaltaxus fortunine by step-pH-gradient high-speed counter-current chromatography

Cephalotaxine-type alkaloids are the anti-cancer components in twigs, leaves, roots and seeds of Cephalotaxus fortunine. It is very important to use the limited resource by finding an efficient purification technology of the alkaloids. Separation of cephalotaxine-type alkaloids in Cephalotaxus fortunine by step-pH-gradient high-speed counter-current chromatography (step-pH-gradient HSCCC) was studied in this paper. The step-pH-gradient HSCCC was performed on a HSCCC instrument equipped with a 400-mL column, using the upper phase of ethyl acetate–n-hexane–water, with added 0.01% trifluoroacetic acid (TFA) as stationary phase, and the lower phase of ethyl acetate–n-hexane–water, with added 2% NH₄OH, 0.2% NH₄OH and 0.05% TFA as mobile phase. For each separation, 800 mg of extract of cephalotaxine-type alkaloids was separated to yield 9.3 mg of drupacine, 15.9 mg of wilsonine, 130.4 mg of cephalotaxine, 64.8 mg of epi-wilsonine, 12.8 mg of fortunine and 35.6 mg of acetylcephalotaxine with purities 81.2%, 85.7%, 95.3%, 97.5%, 89.1% and 96.2%, respectively. The recovery of each alkaloid was more than 90%. The structures of the six alkaloids were identified by electrospray ionization mass spectrum (ESI-MS) and ¹H and ¹³C NMR.     

Crystal structure,spectroscopy and magnetism of a dinuclear strongly antiferromagnetically coupled N-oxido-bridged Cu(II) compound with 2,2′-bipyridine-1,1′-dioxide (bpdo) as a ligand; [Cu2(bpdo)2Br4]

A new dinuclear compound, [Cu₂(bpdo)₂Br₄], (in which bpdo = 2,2′-Bipyridine-1,1′-dioxide), has been synthesized and fully characterized, including the X-ray and the magnetic susceptibility. Each copper(II) ion in the dinuclear compound has a distorted square pyramidal geometry with the basal plane formed by two oxygen atoms of two ligand molecules which are bridging between the Cu ions with Cu–O distances of 2.021(2) and 2.039(2) Å and two bromide atoms with Cu–Br distances of 2.3577(6) and 2.3665(7) Å. The fifth position is occupied by a non bridging oxygen atom of a ligand with a Cu–O distance of 2.197(2) Å. The distance between the Cu ions is 3.334 Å, while the Cu–O–Cu angle is 110.37(9)°. The magnetic susceptibility measurements (from 5 to 350 K) agree with a very strong antiferromagnetic interaction with a large singlet–triplet splitting (J) of −905 cm⁻¹. At high T (above 250 K) a triplet powder EPR is observed.     

Rapid Legionella pneumophila determination based on a disposable core–shell Fe3O4@poly(dopamine) magnetic nanoparticles immunoplatform

A novel amperometric magnetoimmunoassay, based on the use of core–shell magnetic nanoparticles and screen-printed carbon electrodes, was developed for the selective determination of Legionella pneumophila SG1. A specific capture antibody (Ab) was linked to the poly(dopamine)–modified magnetic nanoparticles (MNPs@pDA-Ab) and incubated with bacteria. The captured bacteria were sandwiched using the antibody labeled with horseradish peroxidase (Ab-HRP), and the resulting MNPs@pDA-Ab-Legionella neumophila-Ab-HRP were captured by a magnetic field on the electrode surface. The amperometric response measured at −0.15 V vs. Ag pseudo-reference electrode of the SPCE after the addition of H₂O₂ in the presence of hydroquinone (HQ) was used as transduction signal. The achieved limit of detection, without pre-concentration or pre-enrichment steps, was 10⁴ Colony Forming Units (CFUs) mL⁻¹. The method showed a good selectivity and the MNPs@pDA-Ab exhibited a good stability during 30 days. The possibility of detecting L. pneumophila at 10 CFU mL⁻¹ level in less than 3 h, after performing a membrane-based preconcentration step, was also demonstrated.     

One-pot three-component reaction for the synthesis of biologically important spiro[benzo[f]quinoline-3,3′-indoline] derivatives

A previously unknown class of highly substituted benzoquinoline–spirooxindole are easily prepared by a novel application of a mild and efficient catalyst SbCl₃ for carbon–carbon and carbon–nitrogen bond formation reaction involving isatin, alkyne (dialkyl but-2-ynedioate) with aromatic amine (2-naphthylamine) in a one-pot three-component reaction. This protocol of one-pot synthesis afforded a library of dialkyl 2′-oxo-4H-spiro[benzo[f]quinoline-3,3′-indoline]-1,2-dicarboxylate derivatives, a potential bioactive compound in very good to excellent yields.     

Ammonium salt catalyzed oxidation of organosulfides to organosulfoxydes

The selective oxidation of aromatic and aliphatic organosulfides with H₂O₂/H₂O catalyzed by metal free ammonium salts has been explored. Reactions were performed using 2.5–5 mol % of catalyst loadings, isolating the corresponding sulfoxides in good to excellent yields (75–93%) and high selectivity.     

N-Methylimidazolium ionic liquid-functionalized silica as a sorbent for selective solid-phase extraction of 12 sulfonylurea herbicides in environmental water and soil samples

A novel material for solid-phase extraction (SPE) was synthesized by chemical immobilization of a functionalized N-methylimidazolium ionic liquid on silica gel. Cartridges packed with the synthetic material were successfully applied to the pre-concentration of trace-level thifensulfuron-methyl, metsulfuron-methyl, chlorsulfuron, sulfometuron-methyl, rimsulfuron, ethametsulfuron, tribenuron-methyl, bensulfuron-methyl, prosulfuron, pyrazosulfuron, chlorimuron-ethyl and primisulfuron from environmental water and soil samples. The 12 sulfonylurea herbicides (SUs) obtained a good resolution in less than 50 min using HPLC with a UV detector. The recovery studies using the ionic liquid-functionalized silica as a sorbent were performed by three consecutive extractions of water and soil samples at two spiked levels. The average recovery for each analyte was in the range of 53.8–118.2% for the water samples and 60.9–121.3% for the soil sample, with RSDs lower than 11.3% in all cases. The ionic liquid-functionalized silica cartridges showed higher selectivity for the SUs than commercially available C₁₈ cartridges did.     

Isolation of a polysaccharide with anticancer activity from Auricularia polytricha using high-speed countercurrent chromatography with an aqueous two-phase system

Polysaccharides from a crude extract of Auricularia polytricha were separated by high-speed countercurrent chromatography (HSCCC). The separation was performed with an aqueous two-phase system of PEG1000–K₂HPO₄–KH₂PO₄–H₂O (0.5:1.25:1.25:7.0, w/w). The crude sample (2.0 g) was successfully separated into three polysaccharide components of AAPS-1 (192 mg), AAPS-2 (137 mg), and AAPS-3 (98 mg) with molecular weights of 162, 259, and 483 kDa, respectively. These compounds were tested for growth inhibition of transplanted S180 sarcoma in mice. AAPS-2 had an inhibition rate of 40.4%. The structure of AAPS-2 was elucidated from partial hydrolysis, periodate oxidation, acetylation, methylation analysis, and NMR spectroscopy (¹H, ¹³C). These results showed AAPS-2 is a polysaccharide with a backbone of (1 → 3)-linked-β-d-glucopyranosyl and (1 → 3, 6)-linked-β-d-glucopyranosyl residues in a 2:1 ratio, and has one terminal (1→)-β-d-glucopyranosyl at the O-6 position of (1→3, 6)-linked-β-d-glucopyranosyl of the main chain.     

Mercury(II) complex of inverted N-methylated porphyrin: HgPh(2-NCH3NCTPP)

The reaction of PhHgOAc with 2-NCH₃NCTPPH (2) gave a mercury(II) complex of (phenylato)(2-N-methyl-5,10,15,20-tetraphenyl-21-carbaporphyrinato-N,N′,N″)-mercury(II), [HgPh(2-NCH₃NCTPP); 7]; the coordination sphere around Hg(1) in 7 was a four-coordinate derivative with a seesaw geometry and dipole–dipole (DD) interaction governed the longitudinal relaxation rate for Hg(1)–Ph–H_2,6 protons of 7 in CDCl₃ (0.01 M) at 599.95 MHz.     

Cytosporins F–K,new epoxyquinols from the endophytic fungus Pestalotiopsis theae

Chemical investigation of an endophytic fungus, Pestalotiopsis theae, isolated from the leaves of Turraeanthus longipes (Meliaceae) collected in Cameroon, resulted in the isolation of six new epoxyquinols, cytosporins F–K (2–7), together with the known cytosporin D (1). The structures of the new compounds were unambiguously determined by analysis of the 1D, 2D NMR, and HRMS spectra. Cytosporins G–K (3–7) are the first cytosporins with a hydroxyl substituted C₇ side chain, while cytosporins F–I (2–5) contain a 13-acetoxyl group that was not reported previously. A plausible biosynthetic pathway for the cytosporin derivatives is proposed.     

Experimental and computational studies of molecules with close,non-bonded hydrogen–hydrogen contacts: common computational methods grossly overestimate some ‘through-space’ NMR scalar coupling constants

The NMR spin–spin scalar coupling constants (J_HH's) of closely contacting, but non-bonded hydrogen atoms in a series of highly strained molecules (including a new in,in-cyclophane made specifically for this study) have been examined both experimentally and computationally. The experimental J_HH's are invariably quite small (0.1–0.6 Hz), but common DFT methods with modest basis sets nearly always overestimate these values, by factors of 10–30, and even with quite large basis sets (up to cc-pVQZ) the J_HH's of two of the molecules are overestimated by a factor of 10 or more. Possible reasons for these discrepancies are discussed.     

Synthesis,crystal structure and spectroscopic characterization of new neutral and cationic (η-p-cymene)–ruthenium(II) complexes with phosphine–amide ligands

The dimeric starting material [Ru(η⁶-p-cymene)(μ-Cl)Cl]₂ reacts with the phosphino-amides o-Ph₂P–C₆H₄CO–NH–R [R = ⁱPr (a), Ph (b), 4-MeC₆H₄ (c), 4-FC₆H₄ (d)] to give the mononuclear compounds 1a–d [RuCl(η⁶-p-cymene)(o-Ph₂P–C₆H₄–CO–NH–R)]Cl. The subsequent reaction of these complexes with KPF₆ produced the cationic species 2a–d [RuCl(η⁶-p-cymene)(o-Ph₂P–C₆H₄–CO–NH–R)][PF₆] in which phosphino-amides also act as rigid P,O-chelating ligands. The molecular structures of 2b–d were determined crystallographically. Amide deprotonation is achieved when complexes 2a–d were made react with 1 M aqueous solution of KOH, affording the corresponding neutral species 3a–d [RuCl(η⁶-p-cymene)(o-Ph₂P–C₆H₄–CO–N–R)] in which a P,N-coordination mode is suggested.     

Evaluation of steviol and its glycosides in Stevia rebaudiana leaves and commercial sweetener by ultra-high-performance liquid chromatography-mass spectrometry

Stevia rebaudiana leaves contain non-cariogenic and non-caloric sweeteners (steviol-glycosides) whose consumption could exert beneficial effects on human health. Steviol-glycosides are considered safe; nonetheless, studies on animals highlighted adverse effects attributed to the aglycone steviol. The aim of the present study was to develop and validate two different ultra-high-performance liquid chromatography methods with electrospray ionization mass spectrometry (UHPLC-MS) to evaluate steviol-glycosides or steviol in Stevia leaves and commercial sweetener (Truvia^®). Steviol-glycosides identity was preliminarily established by UV spectra comparison, molecular ion and product ions evaluation, while routine analyses were carried out in single ion reaction (SIR) monitoring their negative chloride adducts. Samples were sequentially extracted by methanol, cleaned-up by SPE cartridge and the analytes separated by UHPLC HSS C₁₈ column (150 mm × 2.1 mm I.D., 1.8 μm). The use of CH₂Cl₂ added to the mobile phase as source of Cl⁻ enhance sensitivity. The LLOD for stevioside, rebaudioside A, steviolbioside and steviol was 15, 50, 10 and 1 ng ml⁻¹, respectively. Assay validation demonstrated good performances in terms of accuracy (89–103%), precision (<4.3%), repeatability (<5.7%) and linearity (40–180 mg/g). Stevioside (5.8 ± 1.3%), rebaudioside A (1.8 ± 1.2%) and rebaudioside C (1.3 ± 1.4%) were the most abundant steviol-glycosides found in samples of Stevia (n = 10) from southern Italy. Rebaudioside A was the main steviol-glycosides found in Truvia^® (0.84 ± 0.03%). The amounts of steviol-glycosides obtained by the UHPLC-MS method matched those given by the traditional LC-NH₂-UV method. Steviol was found in all the leaves extract (2.7–13.2 mg kg⁻¹) but was not detected in Truvia^® (<1 μg kg⁻¹). The proposed UHPLC-MS methods can be applied for the routine quality control of Stevia leaves and their commercial preparations.