Identification of Iridoids,Flavonoids and Triterpenes from the Methanolic Extract of Melampyrum bihariense A. Kern. and the Antioxidant Activity of the Extract

Melampyrum bihariense A. Kern. (Scrophulariaceae), a plant species used in traditional medicine for the treatment of rheumatic disorders and skin infections, was investigated with regard to its antioxidant activity and identification of its bioactive chemical constituents. The crude methanolic extract of the aerial parts of M. bihariense was examined by the spectrophotometric DPPH (1,1-diphenyl-2-picrylhydrazyl) and ferric reducing antioxidant power methods. The free radical scavenging capacity (SC₅₀) of the extract was found by the DPPH method to be 27.10 mg mL⁻¹, and the ferric reducing ability equivalent to ascorbic acid at 50 mg mL⁻¹ was 0.709 μg mL⁻¹. The chemical composition of this highly effective in the methanolic extract was analysed, and the main compounds were isolated through solvent–solvent partition, and multiple chromatographic separations, including column chromatography, vacuum liquid chromatography, centrifugal planar chromatography and preparative thin-layer chromatography. The structures were established by one- and two-dimensional NMR and liquid chromatography-mass spectrometry. The iridoids aucubin (1), 8-epi-loganin (2) and mussaenoside (3), the flavones apigenin and luteolin and the triterpene acids ursolic acid and oleanolic acid were identified; components 2, 3, ursolic acid and oleanolic acid for the first time in this species. The present study reveals that M. bihariense exerts antioxidant activity, and the iridoids, flavonoids and triterpene acids may be the main bioactive constituents of its methanolic extract.

     

Novel biotransformation of pentacyclic triterpenoid acids by Nocardia sp. NRRL 5646

Six pentacyclic triterpene acids, ursolic acid, oleanolic acid, betulinic acid, 23-hydroxybetulinic acid, glycyrrhetinic acid, and senegenin, were metabolized by the microbe Nocardia sp. NRRL 5646 to selectively furnish their corresponding 28-methyl esters. Notably, ursolic acid (1) was converted to oleanolic acid methyl ester (4) via two intermediates, oleanolic acid (2), and ursolic acid methyl ester (3), which are formed by participation of ‘retro-biosynthetic’ methyl migration from C-19 to C-20. Senegenin (11) was selectively converted to a nortriterpene methyl ester, senegenic acid methyl ester (12), via an unprecedented C-C bond cleavage. The stereochemical assignments of compounds 11 and 12 were made unambiguously for the first time using 2D NMR spectroscopy.     

LC Method for Analysis of Three Flavonols in Rat Plasma and Urine after Oral Administration of Polygonum aviculare Extract

A high-performance liquid chromatographic method has been developed for the simultaneous analysis of the flavonols myricitrin (1), avicularin (2), and juglanin (3) in rat plasma and urine after oral administration of the total flavonoids from Polygonum aviculare. Samples were prepared by solid-phase extraction then separated on a C₁₈ reversed-phase column by use of a mobile-phase gradient prepared from methanol and aqueous formic acid solution. The flow rate was 1 mL min^?1. Detection was performed at 254 nm. The calibration range was 11–1,100 μg mL^?1 for both 2 and 3 in plasma; in urine the calibration ranges for 1, 2, and 3 were 32–1,600, 11–1,100, and 22–1,100 μg mL^?1, respectively. Intra-day and inter-day RSD were less than 4.33 and 3.62% for 2 and 3, respectively, in plasma, and no more than 4.03 and 2.22% for all the analytes in urine. The analytical sensitivity and selectivity of the assay enabled successful application to pharmacokinetic studies of flavonols 1–3 in rats.     

Comparison Between Methyl and Trimethylsilyl Ester Derivatives in the Separation and GC Quantification of Triterpene Acids in Eugenia brasiliensis Leaf Extract

This study details a method to characterize the triterpene acid-rich extract obtained from the defatted leaves of Eugenia brasiliensis (Myrtaceae) via extraction with 2 % NaOH in ethanol at room temperature. The crude extract (yield 2.35 %) was submitted to analysis by gas chromatography coupled to mass spectrometry (GC–MS) confirming ursolic acid as its major compound. The optimal conditions for the separation of oleanolic, betulinic and ursolic acids were assayed by GC with flame ionization detection (GC–FID) using two different columns (DB-5 and DB-17HT) and by applying two distinct derivatizing protocols. The use of a DB-17HT column led to the best results, with a shorter runtime and a better resolution (Rs) between the oleanolic and betulinic signals for both the bis-trimethylsilyl (Rs 2.84) and methyl ester derivatives (Rs 2.47). A DB-5 column also gave satisfactory results for the TMS ester, with a runtime of 30 min and Rs 2.14. Ursolic acid in the crude extract was quantified by comparison to two individual standard curves determined using commercial ursolic as its TMS derivative on the DB-5 column and its methyl ester on the DB-17HT column. Good linearity was achieved in both cases (r ² = 0.9776 and 0.9953, respectively), and the amounts of ursolic acid in the extracts were calculated to be 144.7 and 147.9 mg·g^?1, respectively. These results showed no significant differences when compared using Tukey’s HSD test. Total triterpene acids amounted to 0.52 % in E. brasiliensis dry leaves.     

Phytochemical Study of the Rhizome of Pinellia ternata and Quantification of Phenylpropanoids in Commercial Pinellia Tuber by RP-LC

Four phenylpropanoids, (E)-p-coumaryl alcohol (1), 3,4-dihydroxycinnamyl alcohol (2), sachaliside 1 (3), and coniferin (4) have been isolated from the rhizome of Pinellia ternata. Their structures were elucidated by spectroscopic methods. Compounds 1–3 were isolated from the genus Pinellia for the first time. Compound 4 was isolated from this plant for the first time. A rapid, sensitive, and accurate reversed-phase high-performance liquid chromatographic method with UV detection at 260 nm was established for simultaneous separation and determination of the four phenylpropanoids in nineteen batches of dried rhizomes of P. ternata. Compounds were separated on a 250 × 4.6 mm C₁₈ column with methanol–acetonitrile–water–phosphoric acid, 20:5:75:0.3, as mobile phase. The amounts of 1–4 in the rhizome of P. ternata could be easily determined within 30 min. The linear calibration ranges for 1–4 were 0.05–137.50, 0.66–1050.00, 0.06–30.00, and 0.05–67.50 μg mL^?1, respectively. Recovery of 1–4 was 97.43–103.73%, with RSD from 0.12 to 1.62%. Limits of quantification for 1–4 were 50, 660, 60, and 50 ng mL^?1, respectively. The method was successfully used for phytochemical analysis of phenylpropanoids from the rhizome of P. ternata.     

Preparative Isolation and Purification of Two Phenylpropanoids from Daphne giraldii Nitsche by HSCCC

Preparative high-speed counter-current chromatography (HSCCC) was successfully applied to purify phenylpropanoids from the stem and root bark of Daphne giraldii Nitsche, a traditional Chinese medicine. Their structures were identified on the basis of ¹H NMR and ¹³C NMR technology. The two-phase solvent system composed of n -hexane–ethyl acetate–methanol–water (2: 3: 0.5: 4, v/v/v/v) was selected for HSCCC. A total of 8.0 mg woonenoside XI (1) and 18.0 mg daphnetin (2) were obtained in one-step separation from 200 mg of the crude extract with purity of 96.0 and 99.1%, respectively, as determined by LC. And the major compound (2) showed antithrombotic activity in vitro.     

Four new dimeric triterpene glucosides from Sanguisorba officinalis

In search for bioactive compounds from the roots of Sanguisorba officinalis L. (Rosaceae), four new dimeric triterpene glucosides, namely sanguidioside A, B, C, and D (1-4) were isolated. Alkaline hydrolysis of 1-2 afforded the corresponding dimeric aglycones (1a and 2a). Meanwhile, a ready intra-molecular transesterification was observed, providing dimeric triterpenes 1b and 2b. Alkaline hydrolysis of the crude dimmeric saponin also provided a new dimeric triterpene, sanguidiogenin (9). The structures of all these compounds are elucidated via spectroscopic and chemical methods, and are further confirmed by the X-ray diffraction analysis of the dimeric aglycone 2a. Compound 3 represents the first dimeric saponin of an oleanolic acid and an ursonic acid derivative, while compound 4 is the first dimeric saponin of oleanolic acid derivatives.     

Domoic acid at trace levels in lagoon waters: assessment of a method using internal standard quantification

Domoic acid (DA) is a neurotoxin produced by different algae, including pennate diatoms, principally from the genus Pseudo-nitzschia, and it is the main cause of amnesic shellfish poisoning. Determination of this toxin in seawater samples is fundamental to define the real contamination risks for aquatic species. We have developed two very sensitive instrumental methods using hydrophilic interaction liquid chromatography coupled using tandem mass spectrometry in positive and negative polarity modes. Instrumental detection limits were 9 pg mL^?1 for positive and 19 pg mL^?1 for negative ionisation. A procedural method based on solid-phase extraction for the determination of dissolved DA present in seawater has been developed, and an extraction procedure was employed for the determination of the toxin in the particulate fraction. DA quantification was performed using the internal standard method to account for signals fluctuations and random errors during sample treatment. To our knowledge, this is the first study to use this quantification method for DA determination. Trueness, extraction yield, matrix effects, repeatability and procedural detection limits were evaluated during method validation. Procedural detection limits of 0.3 pg mL^?1 (positive mode) and 0.6 pg mL^?1 (negative mode) were found for the dissolved fraction, and absolute limits of 0.4 pg (positive mode) and 6.0 pg (negative mode) for particulate samples were obtained. The most sensitive method in positive mode was applied to define DA occurrence in the Venice Lagoon. Trace concentrations of domoic acid ranging from 1.5 to 16.2 pg mL^?1 were found for the first time in the Venetian environment.

Naphthologs of overcrowded bistricyclic aromatic enes: (E)-bisbenzo[a]fluorenylidene

(E)-11H-Bisbenzo[a]fluorenylidene (E-6) was synthesized by Barton’s double extrusion diazo-thione coupling method from 11H-benzo[a]fluoren-11-thione (11) and 11-diazo-11H-benzo[a]fluorene (13). The reaction is probably thermodynamically controlled; in the event that the less stable Z -6 is also formed, it would rapidly undergo Z → E diastereomerization to give E -6. The B3LYP/6-311G(d,p) calculated diastereomerization barrier for Z -6 → E -6 is ΔG ₂₉₈ = 57.0 kJ/mol (13.6 kcal/mol). The calculated equilibrium constant K _eq(E -6 → Z -6) = 92:8 (at 298 K) is indicative of a marked diastereoselectivity of the reaction leading to E -6. The structure of E-6 was established by ¹H-NMR and ¹³C-NMR spectroscopies and by X-ray analysis. PAE E-6 crystallizes in the monoclinic space group C2/c. The unit cell of the crystal structure E -6 contains eight molecules, arranged as four pairs of enantiomers. PAE E -6 adopts a twisted conformation with the pure twist of the central C¹¹=C^11′ bond ω = 39°. The dihedral angle ν in E -6 is 60.6°, which is significantly higher than the respective dihedral angle in PAEs Z -6, 2, E -7, Z -7, 14, and 15. The large syn-pyramidalization angles at C¹¹ and C^11′ (χ = 12.6° and 14.8°) of E-6 indicates the enhanced strain in the fjord regions of the molecule. The enhanced twist is primarily attributed to the double benzo[a]annelation of the bifluorenylidene moiety at the fjord regions. The B3LYP/6-311G(d,p) calculated structure of E -6 is in a very good agreement with the experimental X-ray structure. PAE E -6 adopts a twisted conformation in solution, with the downfield chemical shift of H¹/H^1′ (8.31 ppm); H¹⁰/H^10′ (δ = 7.20 ppm) and H⁹/H^9′ (δ = 6.86 ppm) in E -6 are positioned above the planes of the opposing naphthalene rings. PAEs E -6 and Z -6 are significantly higher in energy than their corresponding benzo[b]annelated isomers E -7 and Z -7.     

Metabolite Identification of Myricetin in Rats Using HPLC Coupled with ESI-MS

Myricetin, a naturally occurring flavonol, shows multifarious pharmacological activities, e.g., antidiabetic, antioxidant, anti-inflammatory, antitumor, and liver protection effects. In order to obtain an understanding of the myricetin’s metabolism in vivo, a rapid and sensitive method by high-performance liquid chromatography coupled with electrospray-ionization mass spectrometry (HPLC-MS ⁿ ) techniques was employed to investigate the biotransformation in rats after oral administration of myricetin. Recognition and structural exposition of the metabolites were operated by comparing the changes in molecular mass (ΔM) and MS ⁿ spectra with the parent drug. As a result, the parent compound and seven metabolites were found in rat plasma, urine, and feces. In addition, besides 3,5-dihydroxyphenylacetic acid (M1) and 3,4,5-trihydroxyphenylacetic acid (M2), five other compounds were first discovered in the metabolite research of myricetin. These results indicated that, besides ring-fission, there were methylate (M3, M4, M5) and glucuronide (M6, M7) biotransformations of myricetin occurring in vivo.     

Solubilization of cholesterol in aqueous solution by two β-cyclodextrin dimers and a negatively charged β-cyclodextrin derivative

Two βCD dimers (linked by succinic acid, 2, or ethylenediaminetetraacetic acid, EDTA, 3, bridges) and a negatively charged monomer derivative of βCD, 1, have been synthesized and their ability to solubilize cholesterol in aqueous solution was studied. The three compounds exhibit a great capacity in solubilizing cholesterol as, for instance, concentrations up to 6 mM of cholesterol were measured in the presence of 25 mM of 3. The phase-solubility diagrams of the two dimers exhibit A _L type profiles while the monomer 1 follows an A _P isotherm. The cholesterol/dimer complexes have 1:1 stoicheiometries while monomer 1 forms two complexes with molar ratios of 1:1 and 1:2 (cholesterol/1). The equilibrium constants are K _1:1 = (5.9 ± 0.3) × 10⁴ M^?1 and K _1:1 = (8.8 ± 0.2) × 10⁴ M^?1 for 2 and 3, respectively, and K _1:1 = 73 ± 19 M^?1 and K _1:2 = 204 ± 65 M^?1 for 1. The comparison of K _1:1(3) with the product K _1:1 × K _1:2 (1) reveals that a chelate effect in binding the cholesterol by 3 exists. The structure of the cholesterol/3 complex was studied by ROESY experiments and by molecular dynamics simulations.     

Evaluation of ultrahigh-performance liquid chromatography columns for the analysis of unmodified and antisense oligonucleotides

Ultrahigh-performance liquid chromatography has been used for the separation and analysis of unmodified and modified antisense oligonucleotides. For this reason, we tested various columns of low particle sizes in our analysis of unmodified and phosphorothioate oligonucleotides. The influence of both the type and concentration of ion-pair reagent on the retention of the studied biomolecules was tested. The developed methods were used for separation of unmodified oligonucleotides and to determine antisense oligonucleotides in human serum samples. The results proved that octadecyl and phenyl columns are the most selective in the resolution of oligonucleotides which differ in the position of single nucleotides in the sequence. The phenyl column was selected and applied for the analysis of phosphorothioate oligonucleotides in serum samples. The calibration plots showed good linearity within the test concentration ranges. The intra-day CV of the calibration curve slopes was in the range of 1.6 to 4.2 %. The limits of detection (LODs) were in the range of 0.11–0.16 μg mL^?1, while the limit of quantification (LOQ) values were between 0.35 and 0.51 μg mL^?1. Figure

Determination of antisense phosphorothioate oligonucleotides in serum     

Complexation of the cesium cation with lithium ionophore VIII: extraction and DFT study

From extraction experiments and γ-activity measurements, the extraction constant corresponding to the equilibrium Cs⁺(aq) + 1·Na ⁺ (nb) = 1·Cs⁺(nb) + Na⁺(aq) taking place in the two-phase water-nitrobenzene system (1 = lithium ionophore VIII; aq = aqueous phase, nb = nitrobenzene phase) was evaluated as log K _ex (Cs⁺, 1·Na⁺) = ?0.5 ± 0.1. Further, the stability constant of the 1·Cs⁺ complex in nitrobenzene saturated with water was calculated for a temperature of 25 °C: log β _nb (1·Cs⁺) = 4.8 ± 0.2. Finally, by using quantum mechanical DFT calculations, the most probable structure of the cationic complex species 1·Cs⁺ was derived. In the resulting complex, the “central” cation Cs⁺ is bound by six bond interactions to the corresponding six oxygen atoms of the parent ligand 1.     

Synthesis,antifungal and insecticidal activity of novel [1,2,4]triazolo[4,3-<Emphasis Type="Italic">a</Emphasis>]pyridine derivatives containing a sulfide substructure

A series of [1,2,4]triazolo[4,3-a]pyridine derivatives bearing a sulfide substructure was designed, synthesized and characterized via ¹H·NMR, ¹³C·NMR, IR and elemental analyses. Bioassay Results indicated some of the derivatives displayed good fungicidal activity on Rhizoctonia cerealis, moderated insecticidal activity against Plutella xylostella and good insecticidal activity on Helicoverpa armigera. The inhibitory effects of compounds 4g and 4u against Rhizotonia cerealis were 70.9% at 50 μg mL^?1; the IC₅₀ values of compounds 4d and 4s against Plutella xylostella were 43.87 and 50.75 μg mL^?1, respectively. And the IC₅₀ values of compounds 4d, 4q, and 4s on Helicoverpa armigera were 58.3, 77.14 and 65.31 μg mL^?1, respectively, which were better than that of commercial chlorpyrifos (103.77 μg mL^?1).     

Anion-directed organized assemblies of protonated pyrazole-based ionic salts

The reactions of 3(5)-(4-methoxyphenyl)-5(3)-phenyl-1H-pyrazole (L ¹ ) with nitric acid and 5-(4-benzyloxyphenyl)-3-(furan-2-yl)-1H-pyrazole(L ² ) with hydrochloric acid produced [HL ¹ · NO₃] (Salt-1) and [HL ² · Cl] (Salt-2). The structures of Salt-1 and Salt-2 were determined by single crystal X-diffraction. In Salt-1, HL ¹ showed [2 + 2] binding of NO₃ ^? ions in the solid state to form dimer architecture with R ₁ ² (4) and R ₄ ⁴ (14) graph sets. An anion directed one-dimensional anion-assisted helical chain with active participation of the chloride ion and protonated pyrazole via N–H···Cl hydrogen bonding in Salt-2. In addition, the protonated HL ² molecules interacted with each other through weak C–H···π interactions resulting in the formation of another one-dimensional helical chain.     

Experimental and theoretical study on the complexation of Ca2+ with beauvericin

From extraction experiments and γ-activity measurements, the exchange extraction constant corresponding to the equilibrium Ca²⁺(aq) + 1·Sr²⁺(nb) ? 1·Ca²⁺(nb) + Sr²⁺(aq) taking place in the two-phase water–nitrobenzene system (1 = beauvericin; aq = aqueous phase, nb = nitrobenzene phase) was evaluated as log K _ex(Ca²⁺, 1·Sr²⁺) = 1.1 ± 0.1. Further, the stability constant of the 1·Ca²⁺ complex in nitrobenzene saturated with water was calculated for a temperature of 25 °C: log β _nb(1·Ca²⁺) = 10.1 ± 0.2. Finally, by using quantum mechanical density functional level of theory calculations, the most probable structures of the non-hydrated 1·Ca²⁺ and hydrated 1·Ca²⁺·H₂O complex species were predicted.     

Syntheses and antibacterial studies of some 2-[5-(Aryl)-[1,3,4]oxadiazole-2-ylsulfanyl] alkanoic Acids

Some new 2-[5-(aryl)-[1,3,4]oxadiazole-2-ylsulfanyl]alkanoic acids were synthesized and studied for their antibacterial activity. These compounds were prepared from aromatic carboxylic acid hydrazides. Aromatic carboxylic acid hydrazides 1 on refluxing with carbon disulfide and methanolic potassium hydroxide and then on subsequent acidification with hydrochloric acid furnish 5-aryl-1,3,4-oxadiazole-2-thiones 2. 2-Chloro alkanoic acids react with 2 in alkaline media and on acidification yield the title compounds 3. These compounds were characterised by CHN analyses, IR, mass and ¹H NMR spectral data. All the compounds were evaluated for their in vitro antibacterial activity against two Gram negative strains (Escherichia coli and Pseudomonas aeruginosa) and two Gram positive strains (Bacillus subtilis and Staphylococcus aureus) and their minimum inhibitory concentration (MIC) were determined.     

Synthesis and thermal behavior study of complexes of the type [Pd(μ-X)(4-eb-p-phen)]2 (X = Cl,Br, I,N3, NCO,SCN) and 4-eb-p-phen [bis(4-ethylbenzyl)p-phenylenediimine]

The Schiff base bis(4-ethylbenzyl) p-phenylenediimine, 4-eb-p-phen (1), and six new dimeric Pd(II) complexes of the type [Pd(μ-X)(4-eb-p-phen)]₂ {X = Cl (2), Br (3), I (4), N₃ (5), NCO (6), SCN (7)} have been synthesized and characterized by elemental analysis, IR spectroscopy, and ¹H and ¹³C{¹H}-NMR experiments. The thermal behavior of the complexes 2–7 has been investigated by means of thermogravimetry and differential thermal analysis. From the final decomposition temperatures, the thermal stability of the complexes can be ordered in the following sequence: 3 > 4 > 7 > 2 ≈ 5 > 6. The final products of the thermal decompositions were characterized as metallic palladium by X-ray powder diffraction (XRD).     

Molecularly imprinted solid-phase extraction monolithic capillary column for selective extraction and sensitive determination of safranine T in wolfberry

A method was developed to sensitively determine safranine T in wolfberry by molecularly imprinted solid-phase extraction (MISPE) coupled with high-performance liquid chromatography and laser-induced fluorescence detection (HPLC-LIF). The MISPE capillary monolithic column was prepared by water-bath in situ polymerization, using safranine T, methacrylic acid (MAA), and ethylene dimethacrylate (EDMA) as template, functional monomer, and cross-linker, respectively. The properties of the homemade MISPE capillary monolithic column, including capacity and specificity, were investigated under optimized conditions and the morphologies of inner polymers were characterized by scanning electron microscopy (SEM). The mean recoveries of safranine T in wolfberry ranged from 91.2 % to 92.9 % and the intraday and interday relative standard deviation (RSD) values all ranged from 3.4 % to 4.2 %. Good linearity was obtained over 0.001–1.0 μg mL^–1 (r?=?0.9999) with a detection limit (S/N?=?3) of 0.4 ng g^–1. Under the selected conditions, enrichment factors of over 90-fold were obtained and the extraction on the monolithic column effectively cleaned up the wolfberry matrix. The results demonstrated that the proposed MISPE-HPLC-LIF method could be applied to sensitively determine safranine T in wolfberry.

Extraction and theoretical study on the complexation of the strontium cation with beauvericin

From extraction experiments and γ-activity measurements, the extraction constant corresponding to the equilibrium Sr²⁺(aq) + 2A^?(aq) +1(nb) ? 1·Sr²⁺(nb) + 2A^?(nb) taking place in the two-phase water–nitrobenzene system (A^? = picrate, 1 = beauvericin; aq = aqueous phase, nb = nitrobenzene phase) was evaluated as log K _ex(1·Sr²⁺,2A^?) = ?0.6 ± 0.1. Further, the stability constant of the 1·Sr²⁺ complex in nitrobenzene saturated with water was calculated for a temperature of 25 °C: log β _nb(1·Sr²⁺) = 8.5 ± 0.1. Finally, by using quantum-mechanical DFT calculations, the most probable structure of the resulting cationic complex 1·Sr²⁺ was derived.