Chemical components of the Ephedra major from Iran

Ephedra is a dioecious shrub that belongs to the Ephedraceae family of gymnosperms. Almost all commercial applications of Ephedra extracts are derived from the ephedrine alkaloids found in the evergreen stems. The purpose of this study was to compare chemical components (total alkaloid, ephedrine, total phenol, total flavonoid and tannin) of Ephedra major plants during May to October months. The seeds and stems were collected from Bojnoord altitudes in east of Iran. Total alkaloid was separated by solvent and soxhelet extraction method. The results revealed that solvent extraction method is more efficient than soxhelet extraction method. The measurement of chemical components showed significant difference during May to October months. Data from HPLC analysis revealed that while root is depleted of ephedrine, the ephedrine amount in stem organ ranged from 1.50 ± 0.15 to 2.12 ± 0.01 mg/g dry weight. The results indicate that E. major can be as a suitable source of ephedrine.     

Development of a Genus-Universal Nucleotide Signature for the Identification and Supervision of Ephedra-Containing Products

Ephedra plants generally contain ephedrine alkaloids, which are the critical precursor compounds of methamphetamine (METH). METH could cause serious physical and mental damage, and therefore Ephedra materials are strictly in supervision internationally. However, unlawful utilization of Ephedra herbs and its products still exist. Thus, it is imperative to establish a universal method for monitoring Ephedra ingredients in complex mixtures and processed products. In this study, 224 ITS2 sequences representing 59 taxa within Ephedra were collected, and a 23-bp genus-level nucleotide signature (GTCCGGTCCGCCTCGGCGGTGCG) was developed for the identification of the whole genus. The specific primers MH-1F/1R were designed, and 125 individuals of twelve Ephedra species/varieties were gathered for applicability verification of the nucleotide signature. Additionally, seven batches of Chinese patent medicines containing Ephedra herbs were used to test the application of the nucleotide signature in complex and highly processed materials. The results demonstrated that the 23-bp molecular marker was unique to Ephedra and conserved within the genus. It can be successfully utilized for the detection of Ephedra components in complex preparations and processed products with severe DNA degradation. The method developed in this study could undoubtedly serve as a strong support for the supervision of illegal circulation of Ephedra-containing products.     

Quality assessment of Radix Codonopsis by quantitative nuclear magnetic resonance

Radix Codonopsis (Dangshen) is a famous traditional Chinese medicine and has long been used for replenishing energy deficiency, strengthening the immune system, lowering blood pressure and improving appetite in China, Japan and Korea. A highly specific quantification method using ¹H NMR has been developed for the simultaneous determination of novel quaternary ammonium alkaloids codotubulosine A and B, adenosine and 5-(hydroxymethyl)furfural in Radix Codonopsis materials Codonopsis pilosula, C. pilosula var. modesta, C. tangshen, C. tubulosa, C. subglobosa, C. clematidea, C. lanceolota and Campanumoea javanica collected from different regions of China and Taiwan. A solid-phase extraction with C-18 cartridge followed by elution with water can easily remove sugars the major components that may affect the determination of target constituents. In the ¹H NMR spectrum, the signals of N-CH₃ of codotubulosine A (δ 2.75) and B (δ 2.83), H-8 of adenosine (δ 8.15), and CHO signal of 5-(hydroxymethyl)furfural (δ 9.49) are well separated from other signals in [²H₄]methanol. The quantity of the compounds was calculated by the relative ratio of the integral values of the target peaks of each compound to the known amount of internal standard pyrazine. The described NMR method is found to be relatively simple, specific, precise and accurate for the quality control of Radix Codonopsis herbs and no reference compounds are required for calibration curves, in comparison to conventional HPLC methods, for instance.     

A facile,one-pot synthesis of Ephedra-based aziridines

A series of enantiomerically and diastereomerically enriched N-sulfonylaziridines have been prepared by a single-pot process from (1R,2S)- and (1S,2R)-norephedrine and (1S,2S)-pseudonorephedrine. The cyclization process involved N-sulfonylation of the Ephedra alkaloid followed by O-sulfonylation with methanesulfonyl chloride. The bis(sulfonyl)Ephedra derivatives were treated with either hydrazine or sodium hydroxide to afford the N-sulfonylaziridines.     

Discrimination of Ephedra plants with diffuse reflectance FT-NIRS and multivariate analysis

The secondary metabolites of different Ephedra plants are various. Therefore, the discrimination of different Ephedra plants is significant. An objective, easy-to-use, rapid and pollution-free approach is proposed for discriminating Ephedra plants of different species, habitats and picking times on the basis of diffuse reflectance Fourier transform near infrared spectroscopy (FT-NIRS) measurements and multivariate analysis. The Fourier transform near infrared diffuse reflectance spectra (NIRDRS) were acquired from 37 pulverized samples of Ephedra plants put in glass vials in the near infrared (NIR) region between 10 000 and 4000 cm⁻¹, averaging 64 scans per spectrum at a resolution of 4 cm⁻¹. After spectra processing and data pre-processing, spectral data were analyzed respectively with three multivariate analysis techniques: discriminant analysis (DA), self-organizing map (SOM) and back-propagation artificial neural network (BP-ANN). The proposed method could distinguish not only the Ephedra plants of three species and two habitats but also the plants picked at different times of day without special sample treatment and the use of chemical reagents. The performance indexes of the DA model were 84.2-91.9% and the prediction accuracies of both the SOM and the BP-ANN models reached 93.3-100.0%.     

Quantitative analysis of deuterium using the isotopic effect on quaternary C NMR chemical shifts

Quantitative analysis of specifically deuterated compounds can be achieved by a number of conventional methods, such as mass spectroscopy, or by quantifying the residual ¹H NMR signals compared to signals from internal standards. However, site specific quantification using these methods becomes challenging when dealing with non-specifically or randomly deuterated compounds that are produced by metal catalyzed hydrothermal reactions in D₂O, one of the most convenient deuteration methods. In this study, deuterium-induced NMR isotope shifts of quaternary ¹³C resonances neighboring deuterated sites have been utilized to quantify the degree of isotope labeling of molecular sites in non-specifically deuterated molecules. By probing ¹³C NMR signals while decoupling both proton and deuterium nuclei, it is possible to resolve ¹³C resonances of the different isotopologues based on the isotopic shifts and the degree of deuteration of the carbon atoms. We demonstrate that in different isotopologues, the same quaternary carbon, neighboring partially deuterated carbon atoms, are affected to an equal extent by relaxation. Decoupling both nuclei (¹H, ²H) resolves closely separated quaternary ¹³C signals of the different isotopologues, and allows their accurate integration and quantification under short relaxation delays (D1 = 1 s) and hence fast accumulative spectral acquisition. We have performed a number of approaches to quantify the deuterium content at different specific sites to demonstrate a convenient and generic analysis method for use in randomly deuterated molecules, or in cases of specifically deuterated molecules where back-exchange processes may take place during work up.     

Simultaneous determination of Ephedra sinica and Citrus aurantium var. amara alkaloids by ion-pair chromatography

Ephedra sinica (Ma Huang) preparations have recently gained a lot of attention because of serious side effects associated with their prolonged consumption. Citrus aurantium var. amara is now used as an alternative, despite the fact that similar side effects are suspected. We have developed and validated the first analytical procedure for the simultaneous determination of all major alkaloids from both species. Using the ion-pairing reagent SDS, a C-18 stationary phase (3 μm material) and a pH-gradient for elution enabled the baseline separation of six alkaloids ((±)-octopamine, (±)-synephrine, tyramine, (−)-norephedrine, (+)-pseudoephedrine and (−)-ephedrine) within less than 30 min. The method is sensitive (LOD ≤ 4.6 ng and LOQ ≤ 16.2 ng on-column), selective (l-tyrosine and l-phenylalanine, two closely related amino acids did not interfere), accurate (recovery rates of spiked samples were between 97.5 and 102.0%), repeatable (σ_rel ≤ 4.6%) and precise (intra-day variation ≤7.7%, inter-day variation ≤7.0%). Without the need of a special sample treatment different matrices (plant material, commercial products) were successfully analyzed for their alkaloid content. Dominant alkaloids were (−)-ephedrine (0.9-1.6%) and/or (±)-synephrine (0.1-3.0%). Whether a product contained Ephedra-alkaloids or not could be determined in all investigated samples unambiguously.     

A mass spectrometric method to determine activities of enzymes involved in polyamine catabolism

An analytical method for the determination of three polyamines (putrescine, spermidine, and spermine) and five acetylpolyamines [N¹-acetylspermidine (N¹AcSpd), N⁸-acetylspermidine (N⁸AcSpd), N¹-acetylspermine, N¹,N⁸-diacetylspermidine, and N¹,N¹²-diacetylspermine] involved in the polyamine catabolic pathway has been developed using a hybrid tandem mass spectrometer. Heptafluorobutyryl (HFB) derivatives of these compounds and respective internal standards labeled with stable isotopes were analyzed simultaneously by TOF MS, based on peak areas appearing at appropriate m/z values. The isomers, N¹AcSpd and N⁸AcSpd were determined from their fragment ions, the acetylamidopropyl and acetylamidobutyl groups, respectively, using MS/MS with ¹³C₂-N¹AcSpd and ¹³C₂-N⁸AcSpd which have the ¹³C₂-acetyl group as an internal standard. The TOF MS method was successfully applied to measure the activity of enzymes involved in polyamine catabolic pathways, namely N¹-acetylpolyamine oxidase (APAO), spermine oxidase (SMO), and spermidine/spermine N¹-acetyltransferase (SSAT). The following natural substrates and products labeled with stable isotopes considering the application to biological samples were identified; for APAO, [4,9,12-¹⁵N₃]-N¹-acetylspermine and [1,4,8-¹⁵N₃]spermidine (¹⁵N₃-Spd), respectively; for SMO, [1,4,8,12-¹⁵N₄]spermine and ¹⁵N₃-Spd, respectively; and for SSAT, ¹⁵N₃-Spd and [1,4,8-¹⁵N₃]-N¹-acetylspermidine, respectively.     

Synthesis, characterization and antimicrobial activity of palladium(II) complexes with some alkyl derivates of thiosalicylic acids: Crystal structure of the bis(S-benzyl-thiosalicylate)-palladium(II) complex, [Pd(S-bz-thiosal)2]

S-Alkyl (R = benzyl, methyl, ethyl, propyl and butyl) derivatives of thiosalicylic acid and the corresponding palladium(II) complexes were prepared and their structures were proposed on the basis of infrared, ¹H and ¹³C NMR spectroscopy. The cis geometrical configurations of the isolated complexes were proposed on the basis of an X-ray structural study of the bis(S-benzyl-thiosalicylate)-palladium(II), [Pd(S-bz-thiosal)₂] complex.Antimicrobial activity of the tested compounds was evaluated by determining the minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC) in relation to 26 species of microorganisms. The tested ligands, with a few exceptions, show low antimicrobial activity. The palladium(II) complexes, [Pd(S-R-thiosal)₂], have statistically significant higher activity than the corresponding ligands. The complexes [Pd(S-et-thiosal)₂] and [Pd(S-pro-thiosal)₂] displayed the strongest activity amongst the all tested compounds. The palladium(II) complexes show selective and moderate antibacterial activity and significant antifungal activity. The most sensitive were Aspergillus fumigatus and Aspergillus flavus.     

NMR relaxation data for quality characterization of Balsamic vinegar of Modena

In this paper, 22 samples consisting of Balsamic vinegar of Modena and two Traditional Balsamic vinegar of Modena samples have been analyzed by means of ¹H NMR spectroscopy. Some selected resonances have been used for quantification and for T1 (spin-lattice relaxation time) measurements. Statistical protocols applied to NMR data for quantification of selected resonances revealed the possibility to differentiate the samples according to their ageing process. Additionally, T1 measurements revealed a strong correlation with the ageing process and these new data were added for improving the statistical model, which was used to predict T1 relaxation time of selected compounds for Balsamic vinegar samples. Our results indicate that the use of NMR data and statistical methods is a valid approach that can be successfully used for ageing characterization of Balsamic vinegar of Modena samples.     

H and C NMR spectroscopy of 9-acridinones

The ¹H and ¹³C NMR spectra of 9-acridinone and its five derivatives dissolved in CDCl₃, CD₃CN and DMSO-d₆ were measured in order to reveal the influence of the constitution of the compounds and features of the solvents on chemical shifts and ¹H-¹H coupling constants. Experimental data were compared with theoretically predicted chemical shifts, on the GIAO/DFT level of theory, for DFT (B3LYP)/6-31G^∗∗ optimized geometries of molecules—also for four other 9-acridinones. This comparison helped to ascribe resonance signals in the spectra to relevant atoms and enabled revelation of relations between chemical shifts and physicochemical features of the compounds. It was found that experimentally or theoretically determined ¹H and ¹³C chemical shifts of selected atoms correlate with theoretically predicted values of dipole moments of the molecules, as well as bond lengths, atomic partial charges and energies of HOMO.     

Solid-phase microextraction coupled with capillary electrophoresis to determine ephedrine derivatives in water and urine using a sol-gel derived butyl methacrylate/silicone fiber

A sensitive method for determination of ephedrine derivatives using headspace solid-phase microextraction (SPME) with a novel fiber followed by capillary electrophoresis has been developed. The co-poly(butyl methacrylate/hydroxy-terminated silicone oil) (BMA/OH-TSO) was used as stationary phases with the aid of γ-methacryloxypropyltrimethoxysilane (KH-570) as bridge in SPME using sol-gel-coating method and cross-linking technology. It has high extraction efficiency for ephedrine derivatives in comparison with commercial poly(dimethylsiloxane) and poly(acrylate)-coated fiber. The coating exhibits good thermal and solvent stability as well as long lifetime. A simple and flexible device for desorption of analytes after headspace SPME was constructed. The effect of various experimental parameters for SPME (temperature, time, pH, ionic strength, desorption solvent, etc.) were discussed. Field amplified sample injection (FASI) was applied for on-line sample concentration and a sensitivity enhancement of two orders of magnitude was achieved. Linear ranges were found to be 20-5000 ng/ml. The detection limits for (1R,2S)-ephedrine, (1R,2R)-pseudoephedrine and (1S,2S)-pseudoephedrine were 3, 5 and 5 ng/ml, respectively. Relative standard deviation (n = 6) was found to be 4.96-7.57%. The method was successfully applied to the analysis of ephedrine derivatives in human urine.     

Synthesis,characterization and antimicrobial activity of maltol functionalized chitosan derivatives

Chitin was isolated from prawn shell powder through demineralization and deproteinization process. Chitosan was synthesized from isolated chitin by deacetylation process and characterized by Fourier Transform Infrared (FTIR) spectra which showed close agreement with commercial chitosan. Physicochemical features such as moisture content, ash content, degree of deacetylation and molecular weight has been measured. The prepared chitosan was found to have comparatively higher molecular weight than the commercial chitosan. Functionalization of NH₂ group of chitosan with C?=?O group of maltol and ethyl maltol by refluxing equimolar quantities of respective ketones was performed. These synthesized derivatives of chitosan were characterized by their FTIR, ¹³C-Nuclear magnetic resonance (NMR) spectroscopy, elemental analysis, X-Ray Diffraction (XRD), Thermogravimetric analysis (TGA) and Differential thermal analysis (DTA) instrumental techniques. Antibacterial screening results of the synthesized chitosan and its derivatives indicate that these compounds are active against Escherichia coli bacteria.     

Simultaneous determination of L-arginine and 12 molecules participating in its metabolic cycle by gradient RP-HPLC method: application to human urine samples

We have developed and described a highly sensitive, accurate and precise reversed-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous determination of l-arginine and 12 molecules participating in its metabolic cycle in human urine samples. After pre-column derivatization with ortho-phthaldialdehyde (OPA) reagent containing 3-mercaptopropionic acid (3MPA), the fluorescent derivatives were separated by a gradient elution and detected by fluorescence measurement at 338 nm (excitation) and 455 nm (emission). l-Arginine (ARG) and its metabolites: l-glutamine (GLN), N^G-hydroxy-l-arginine (NOHA), l-citrulline (CIT), N^G-monomethyl-l-arginine (NMMA), l-homoarginine (HARG), asymmetric N^G,N^G-dimethyl-l-arginine (ADMA), symmetric N^G,N^G′-dimethyl-l-arginine (SDMA), l-ornithine (ORN), putrescine (PUT), agmatine (AGM), spermidine (SPERMD) and spermine (SPERM) were extracted in a cation-exchange solid-phase extraction (SPE) column and after derivatization separated in a Purospher^® STAR RP-18e analytical column. The calibration curves of analysed compounds are linear within the range of concentration: 45-825, 0.2-15, 16-225, 12-285, 0.1-32, 15-235, 0.1-12, 0.1-12, 10-205, 0.02-12, 0.1-24, 0.01-10 and 0.01-8 nmol mL⁻¹ for GLN, NOHA, CIT, ARG, NMMA, HARG, ADMA, SDMA, ORN, PUT, AGM, SPERMD and SPERM, respectively. The correlation coefficients are greater than 0.9980. Coefficients of variation are not higher than 6.0% for inter-day precision. The method has been determined or tested for limits of detection and quantification, linearity, precision, accuracy and recovery. All detection parameters of the method demonstrate that it is a reliable and efficient means of the comprehensive determination of ARG and its 12 main metabolites, making this approach suitable for routine clinical applications. The levels of analysed compounds in human urine can be successfully determined using this developed method with no matrix effect.     

Triorganotin(IV) derivatives of umbelliferone (7-hydroxycoumarin) and their adducts with 1,10-phenanthroline: synthesis, structural and biological studies

New triorganotin(IV) derivatives of the general formula R₃Sn(Umb) (where, R = Me, n-Bu and Ph; Umb = umbelliferone anion) have been synthesized using sodium salt method. Further, the adducts of the general formula R₃Sn(Umb) · phen (where R = Me and Ph; phen = 1,10-phenanthroline) have also been synthesized by the interaction of the triorganotin(IV) derivatives of umbelliferone with 1,10-phenanthroline. The bonding and coordination behavior of these derivatives are discussed on the basis of IR, NMR (¹H, ¹³C and ¹¹⁹Sn), and ¹¹⁹Sn Mössbauer spectroscopic studies. These investigations indicate that umbelliferone acts as a monoanionic bidentate ligand in R₃Sn(Umb) coordinating through O(7) and O(1) in the solid-state. These polymeric R₃Sn(Umb) derivatives (where R = Me and n-Bu) have been proposed to have a trans-trigonal bipyramidal geometry with the three R groups in equatorial positions, while the axial positions are occupied by a phenolic oxygen and the O(1) atom from the adjacent molecule. A pseudotetrahedral geometry has been suggested for Ph₃Sn(Umb). A distorted octahedral geometry around tin has been proposed for R₃Sn(Umb) · phen, in which umbelliferone anion acts as a monodentate ligand coordinating through phenolic oxygen O(7). The newly synthesized derivatives have been assayed for their anti-inflammatory, cardiovascular and anti-microbial activities. The average LD₅₀ values >1000 mg kg⁻¹ of these derivatives indicate their safety margin. Among all the compounds tested, Ph₃Sn(Umb) · phen has been found to show potent anti-inflammatory activity with low mammalian toxicity and mild hypotensive activity.     

Synthesis and Stereochemistry of New N-Substituted Cytisine Derivatives

A series of new N-substituted cytisine derivatives was synthesized. The ¹ H and ¹³ C NMR spectra of certain compounds exhibit a doubled set of signals. This is explained by formation of diastereomeric pairs in compounds containing an asymmetric center in the substituents. The signal splitting in -COHC=CHCO ₂ H and HC=O (formyl) derivatives is explained by the existence of Z and E invertomers. Their stereochemical features are discussed. Amide conjugation is confirmed by temperature experiments.     

The Chemical Constituents from Fruits of Catalpa bignonioides Walt. and Their α-Glucosidase Inhibitory Activity and Insulin Secretion Effect

Catalpa pod has been used in traditional medicine for the treatment of diabetes mellitus in South America. Studies on the constituents of Catalpa species have shown that it is rich in iridoids. In the present study, three previously undescribed compounds (2–4), including two secoiridoid derivatives along with twelve known compounds, were isolated from the fruits of Catalpa bignonioides Walt. In addition, fully assigned ¹³C-NMR of 5,6-dihydroxy-7,4’-dimethoxyflavone-6-O-sophoroside (1) is reported for the first time in the present study. The structures of compounds were determined on the basis of extensive spectroscopic methods, including UV, IR, 1D, and 2D NMR, mass spectroscopy, and CD spectroscopic data. All the isolated compounds were evaluated for α-glucosidase inhibitory activity. Among the tested compounds, compounds 2, 3, and 9 exhibited significant inhibitory activity against α-glucosidase enzyme assay. Meanwhile, the effect of compounds 2, 3, and 9 on glucose-stimulated insulin secretion (GSIS) was measured using pancreatic β-cells. Compounds 2, 3, and 9 exhibited non-cytotoxicity-stimulated insulin secretion in INS-1 cells. The expression levels of proteins associated with β-cell function and insulin secretion such as phosphorylation of total insulin receptor substrate-2 (IRS-2), phosphatidylinositol 3-kinase (PI3K), Akt, activated pancreatic duodenal homeobox-1 (PDX-1), and peroxisome proliferator-activated receptor-γ (PPAR-γ) were increased in INS-1 cells after treatment with compounds 2, 3, and 9. The findings of the present study could provide a scientific warrant for their application as a potential antidiabetic agent.     

18O同位素标记定量肽段串联体蛋白质结合同位素稀释-多反应监测质谱的蛋白质绝对定量新方法

建立了定量肽段串联体蛋白质(concatamers of Q peptides, QconCATs)结合¹⁸O同位素标记-多反应监测质谱的蛋白质绝对定量新方法。首先对QconCAT重组蛋白质进行了纯度表征,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)表征结果表明重组蛋白质的纯度在99%以上,相对分子质量约为63.4 kDa。对QconCAT重组蛋白质酶切后的肽段混合物进行质谱分析,并经pFind和pLabel软件处理,验证了目标肽段。还考察了QconCAT重组蛋白质的酶切效率和¹⁸O标记效率,并对QconCAT蛋白质结合¹⁸O标记-同位素稀释-多反应监测质谱方法进行了评价。实验结果表明,采用该方法对腾冲嗜热厌氧菌(Thermoanaerobacter tengcongensis, TTE)中选定蛋白质的肽段进行绝对含量测定时,相对标准偏差小于20%,准确度较高,说明该方法可用于复杂生物样本中蛋白质的绝对定量。更重要的是所建方法不仅解决了细胞培养氨基酸稳定同位素标记(SILAC)技术的重标试剂价格昂贵的问题,也为定量蛋白质组学提供了一种新的方法。     

Quantification of isoorientin and total flavonoids in Passiflora edulis fruit pulp by HPLC-UV/DAD

A method is reported for the quantification of isoorientin (using a standard addition method) and total flavonoids (expressed as rutin, using the external standard method) in passion fruit pulp (Passiflora edulis Sims f. flavicarpa Degener, Passifloraceae). Extraction of flavonoids was optimized by experimental design methodology, and quantitative analysis was performed by high-performance liquid chromatography with photo-diode array detection (HPLC-UV/DAD). The method was developed and validated according to ICH requirements for specificity, linearity, accuracy, precision (repeatability and intermediate precision), LOD and LOQ. Rutin was chosen as standard for the quantification of total flavonoids in order to propose a HPLC method feasible for routine analysis of the flavonoids in the passion fruit pulp. The passion fruit pulp contained 16.226 ± 0.050 mg L^− 1 of isoorientin and 158.037 ± 0.602 mg L^− 1 of total flavonoid, suggesting that P. edulis fruits may be comparable with other flavonoid food sources such as orange juice or sugarcane juice.