High-performance Liquid Chromatography Coupled To A Diode Array Detector Research Articles

Comparative study of sample preparation procedures to determine the main compounds in ayahuasca beverages by QuEChERS and high-performance liquid chromatography analysis.

Ayahuasca is a psychoactive drink originally consumed by indigenous people of the Amazon. The lack of regulation of this drink leads to uncontrolled consumption, and it is often consumed in religious contexts. The aim of this work is to compare three miniaturised extraction techniques for extracting the main ayahuasca compounds from beverages. Three sample pretreatment techniques were evaluated (dispersive liquid-liquid microextraction [DLLME], microextraction by packed sorbent [MEPS] and QuEChERS [Quick, Easy, Cheap, Effective, Rugged and Safe]) for the simultaneous extraction of N,N-dimethyltryptamine (DMT), tetrahydroharmine (THH), harmine, harmaline, harmol and harmalol from ayahuasca beverage samples. Then, the most promising technique (QuEChERS) was chosen to pre-concentrate the analytes, subsequently detected by high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD). The procedure was optimised, with the final conditions being 500 μL of extractor solvent, 85 mg of primary secondary amine (PSA) and 4s of vortexing. The analytical method was validated, showing to be linear between 0.16 and 10μg/mL for β-carbolines and between 0.016 and 1μg/mL for DMT, with coefficients of determination (R2) between 0.9968 and 0.9993. The limit of detection (LOD) and lower limit of quantification (LLOQ) were 0.16 μg/mL for all compounds, except for DMT (0.016 μg/mL) and extraction efficiencies varied between 60.2% and 88.0%. The analytical methodology proved to be accurate and precise, with good linearity, LODs and LLOQs. This method has been fully validated and successfully applied to ayahuasca beverage samples.

Characterization and antioxidant capacity of phenolic compounds of jackfruit genotypes from Nayarit, Mexico

Jackfruit is a rich source of phytochemicals, thus offering a broad perspective in developing value-added healthy nutritional foods. This research compares the content of phenolic compounds and antioxidant capacity in different jackfruit genotypes. In the results of total soluble phenols (TSP), the genotype "Agüitada" showed the highest values at the end of storage life (1.43 mg of gallic acid equivalent (GAE)/g in dry weight (DW)), as well as in 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay with 7.82 mmol of trolox equivalent (TE)/g DW. The "Licenciada" genotype obtained the highest ferric reducing antioxidant power (FRAP) and 2,2′-Azino-bis-(3-Ethylbenzothiazoline-6-Sulfonic Acid) (ABTS•+) assays values, with 244.29 and 12.08 mmol TE/g DW, respectively. Phenolic profiling by high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD) quantified 28 phenolic compounds, with shikimic acid being the most abundant in all genotypes; flavonoids were also identified, with epigallocatechin standing out. The results obtained in phenolic profile, TSP and antioxidant capacity showed sufficient evidence of differences among all genotypes. This information will generate interest among consumers and will revalue this developing crop.

The potential of Acmella oleracea as a nutraceutical source for the symptomatic treatment of Burning Mouth Syndrome

This study analysed the phytochemical profile of Acmella oleracea extract and the molecular interactions of its main compounds with TRPV1 and CB2, target receptors in the Burning Mouth Syndrome (BMS) pathogenesis. The phytochemical profile of A. oleracea’s floral buds extract treated with activated charcoal (TCEE) was analysed by High-Performance Liquid Chromatography (HPLC) coupled to Mass Spectrometry (LC-MS). The quantification of spilanthol was analysed by HPLC coupled to a Diode-Array Detector (HPLC-DAD). The phytochemical analysis revealed the presence of nine alkylamides and phenolic compounds. The TCEE showed a significant increase in spilanthol content compared to the crude extract (CEE), going from 28.33 mg/g to 117.96 mg/g. The molecular docking indicated a behaviour of the alkylamides as partial TRPV1 agonists and CB2 agonists and, for the first time, indicates the action of these compounds in the symptomatic management of BMS.

Homogeneous immunoassay for cyclopiazonic acid based upon mimotopes and upconversion-resonance energy transfer

Strains of Penicillium spp. are used for fungi-ripened cheeses and Aspergillus spp. routinely contaminate maize and other crops. Some of these strains can produce toxic secondary metabolites (mycotoxins), including the neurotoxin α-cyclopiazonic acid (CPA). In this work, we developed a homogeneous upconversion-resonance energy transfer (UC-RET) immunoassay for the detection of CPA using a novel epitope mimicking peptide, or mimotope, selected by phage display. CPA-specific antibody was used to isolate mimotopes from a cyclic 7-mer peptide library in consecutive selection rounds. Enrichment of antibody binding phages was achieved, and the analysis of individual phage clones revealed four different mimotope peptide sequences. The mimotope sequence, ACNWWDLTLC, performed best in phage-based immunoassays, surface plasmon resonance binding analyses, and UC-RET-based immunoassays. To develop a homogeneous assay, upconversion nanoparticles (UCNP, type NaYF4:Yb3+, Er3+) were used as energy donors and coated with streptavidin to anchor the synthetic biotinylated mimotope. Alexa Fluor 555, used as an energy acceptor, was conjugated to the anti-CPA antibody fragment. The homogeneous single-step immunoassay could detect CPA in just 5 min and enabled a limit of detection (LOD) of 30 pg mL−1 (1.5 μg kg−1) and an IC50 value of 0.36 ng mL−1. No significant cross-reactivity was observed with other co-produced mycotoxins. Finally, we applied the novel method for the detection of CPA in spiked maize samples using high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD) as a reference method.

Chitosan/Gelatin Scaffolds Loaded with Jatropha mollissima Extract as Potential Skin Tissue Engineering Materials.

This work aimed to develop chitosan/gelatin scaffolds loaded with ethanolic extract of Jatropha mollissima (EEJM) to evaluate the influence of its content on the properties of these structures. The scaffolds were prepared by freeze-drying, with different EEJM contents (0-10% (w/w)) and crosslinked with genipin (0.5% (w/w)). The EEJM were characterized through High Performance Liquid Chromatography coupled to a Diode Array Detector (HPLC-DAD), and the determination of three secondary metabolites contents was accomplished. The physical, chemical and biological properties of the scaffolds were investigated. From the HPLC-DAD, six main substances were evidenced, and from the quantification of the total concentration, the condensed tannins were the highest (431.68 ± 33.43 mg·g-1). Spectroscopy showed good mixing between the scaffolds' components. Adding and increasing the EEJM content did not significantly influence the properties of swelling and porosity, but did affect the biodegradation and average pore size. The enzymatic biodegradation test showed a maximum weight loss of 42.89 within 28 days and reinforced the efficiency of genipin in crosslinking chitosan-based materials. The addition of the extract promoted the average pore sizes at a range of 138.44-227.67 µm, which is compatible with those reported for skin regeneration. All of the scaffolds proved to be biocompatible for L929 cells, supporting their potential application as skin tissue engineering materials.

Removal of the pesticide thiamethoxam from sugarcane juice by magnetic nanomodified activated carbon

The removal of the neonicotinoid and systemic pesticide thiamethoxam (TMX) from water and sugarcane juice by magnetic nanomodified activated carbon (AC-NP) is proposed. This adsorbent was synthesized and characterized by FTIR, XRD, and SEM, and TMX was quantified by high-performance liquid chromatography coupled to a diode array detector (HPLC–DAD). The AC-NP was efficiently synthesized using a co-precipitation method and the impregnation of magnetite (NP) in the activated carbon (AC) was assessed by the crystalline planes found in the AC-NP structure shown in the XRD diffractograms. The AC-NP FTIR analysis also indicated predominant bands of Fe–O stretching of the magnetite at 610–570 cm−1. Functional groups in AC and AC-NP were identified by absorption bands at 3550 and 1603 cm−1, characteristic of O–H and C = C, respectively. The TMX adsorption kinetics in sugarcane juice was the pseudo-second-order type with r2 = 0.9999, indicating a chemical adsorption process. The experimental sorption capacity (SCexp) for both TMX (standard) and TMX-I (insecticide) by AC-NP were 13.44 and 42.44 mg/g, respectively. Seven non-linear isotherm models (Langmuir, Freundlich, Dubinin-Radushkevich, Toth, Hill, Sips, and Redlich-Peterson) were fitted to the experimental adsorption data of TMX and TMX-I by AC-NP. Considering the standard error (SE), Freundlich, Langmuir, and Sips were the most appropriate models to describe the TMX adsorption, and Hill’s best adjusted to TMX-I experimental data. The chromatographic method was highly satisfactory due to its high selectivity and recovery (91–103%). The efficiency of AC-NP in the sorption of TMX was confirmed by the excellent values of SCexp and sorption kinetics.

Identification of Dyes in Coptic Textiles from the Museum of Faculty of Archaeology, Cairo University

High Performance Liquid Chromatography coupled to a Diode-Array-Detector (HPLC-DAD) is used to investigate samples which were extracted from ancient Egyptian textiles (4th–5th c. AD) of the Museum of Faculty of Archaeology, Cairo University. Madder is identified in several samples. According to semi-quantitative results, which are obtained from HPLC peak areas measured at 254 nm, madder that is rich in purpurin and poor in alizarin is identified in samples which were treated (i) only with madder and (ii) with madder and either indigo/woad (Indigofera species and other/Isatis tinctoria L.) or weld (Reseda luteola L.). The madder dye used in these samples could have been originated from Rubia peregrina L. However, the possible use of Rubia tinctorum L. (or other plants of the Rubiaceae family) by the Egyptian dyers cannot be ruled out, particularly if methods were developed by the ancient dyers to affect and control the relative composition of madder dye. The HPLC peak area ratio of alizarin versus purpurin is very high (>2.2) for samples which were treated with madder (probably originated from R. tinctorum) and a tannin source. Finally, in some samples, only indigoid dyes (indigo/woad) are identified.

RP-HPLC-DAD determination of the differences in the polyphenol content of Fucus vesiculosus extracts with similar antioxidant activity

Significant quantities of bioactive compounds have been found in the chemical composition of seaweeds. This source of natural antioxidants such as polyphenols appears to attenuate lipid peroxidation caused by oxidative stress, preventing the harmful effects of a number of injuries including ischemia–reperfusion (I/R). Conventional extraction (CE) has been used for years as a traditional method for obtaining bioactive components from seaweeds. However, recent studies highlight ultrasonic-assisted extraction (UAE) as an alternative and more eco-friendly technique. Therefore, the two methods were optimised and compared to obtain a Fucus vesiculosus extract (FVE) with high antioxidant activity and polyphenol content. The highest antioxidant activity was obtained after 1 h at 25 °C for conventional extraction, and after 5 min at 35 °C for ultrasonic-assisted extraction. Higher concentrations of polyphenols were obtained with the optimal conditions in conventional extraction (13.61 mg PGE/g seaweed), but no significant differences were observed between the antioxidant activity obtained with UAE (89.33%) and CE (89.74%). The characterization of the polyphenols present in both optimised extracts was carried out and compared with reverse-phase high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD). The following compounds were identified: phloroglucinol, gallic acid, catechin, vanillic acid, epicatechin, protocatechuic acid, rutin, gentisic acid, chlorogenic acid, caffeic acid, coumaric acid and ferulic acid. RP-HPLC-DAD results also showed higher concentrations of polyphenols in optimised extracts with CE. Consequently, CE was found to be more effective than UAE in providing extracts with higher concentrations of polyphenols, but UAE constitutes an efficient and more eco-friendly methodology for obtaining a FVE with the highest antioxidant activity.

Phytochemical Characterization and Evaluation of Bioactive Properties of Tisanes Prepared from Promising Medicinal and Aromatic Plants

The chemical composition and biological properties correlation in several medicinal and aromatic plants is still underexplored, especially in its most common form of consumption as tisane. The present study aims to characterize the organic acids and vitamin E composition of five tisanes and their extracts by High-Performance Liquid Chromatography coupled to a diode-array detector (HPLC-DAD) and HPLC coupled to a fluorescence detector techniques, respectively, and the phenolic composition by HPLC-DAD-ESI/MS (mass spectrometry by electrospray ionization). It also focuses on their bioactive properties, namely antioxidant, antimicrobial, anti-inflammatory, cytotoxic, anti-tyrosinase, and anti-diabetic activities. A Principal Component Analysis (PCA) was performed in order to understand the correlation between the chemical composition and bioactive properties of the tisanes. The tisane 5 (T5) composed by lemon thyme, tutsan, cloves, and cinnamon, was the most promising mixture, presenting the lowest values for the lipid peroxidation inhibition, anti-inflammatory, and anti-diabetic activity. It also presented the highest concentration of phenolic acids (caffeoylquinic acids derivatives), and flavan-3-ols (catechin derivatives). Only the dry plants presented tocopherols. For the antihemolytic, antimicrobial, and cytotoxic activity, T2 and T4 (with lemon thyme) were highlighted as the best herbal mixtures. The PCA proved to be a valid tool to select the most promising tisane according to the bioactivity. These results suggest that the studied tisanes can be source of high added-value bioactive compounds with health-promoting effects and potential for application in the food and nutraceutical industries, among others.

Polyphenol Profile and Quantitative Assessment of the Flavonoid Kaempferitrin in Wild and Cultivated Brazilian Amazonian Uncaria guianensis (Rubiaceae)

The Amazonian Rubiaceae species Uncaria guianensis (UG) is locally used as antiinflammatory, antitumor, antidiabetic, anti-ulcers, and others. The phenolic content of its leaves is characterized by the great predominance of the flavonoid kaempferol-3,7-O-(α)-L-dirhamnoside (kaempferitrin). The present study quantitatively evaluates the kaempferitrin content in the leaves and branches of cultivated and wild UG specimens collected in different locations of the Brazilian Amazon rainforest by employing high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD). Besides, the understanding of the polyphenol profile performed by electron spray ionization is deepened by tandem mass spectrometry analysis (ESI-MS/MS), using a previously approached leaf UG extract, and the flavonoid quercetin-3,7-O-(α)-L-dirhamnoside was first isolated from UG. All samples showed quite similar qualitative polyphenol profiles. Kaempferitrin in UG ranged from 1.1 to 1.9 mg 100 mg-1 for dry leaves of adult wild plants, 0.3 to 0.7 mg 100 mg-1 for dry leaves of cultivated young plants and 0.00 to 0.04 mg 100 mg-1 for dry branches of adult wild plants. Besides suggesting the distribution of kaempferitrin in the species, these results reinforce this flavonol as a suitable chemical marker for UG leaves and the products derived from them.

Bioactivities of iridoids and flavonoids present in decoctions from aerial parts of Verbascum betonicifolium

IntroductionVerbascum betonicifolium (V. betonicifolium) is a plant used in traditional medicine for several ailments. The objective of this study was to determine the antioxidant activity and acetylcholinesterase inhibitory activity of the aqueous extract together with the metabolites responsible for these activities. This paper presents the first phytochemical characterization and bioactivities of aqueous extracts from aerial parts. MethodsThe compounds present in the aerial part aqueous extract were identified by high performance liquid chromatography, coupled to a diode-array detector (HPLC-DAD) and by high-resolution mass spectrometry (HRMS), using LC–MS/MS analyses. Antioxidant activity was measured as the capacity to scavenge the free radical DPPH and the AChE activity was determined using the Ellman test. The cytotoxicity was determined used HepG2 cell lines. ResultsSeveral types of metabolites were found, primary metabolites (malic, citric and gluconic acids), phenolic compounds (verbascoside, luteolin), terpenoids (iridoid glycoside, unedide), among others. The total phenol content of 28 μg of gallic acid equivalents/mg of extract was determined. The aqueous extract antioxidant activity had and EC50 of 70 μg/mL and the AChE inhibitory activity an IC50 of 750 μg/mL. No cytotoxicity towards HepG2 cells was detected, even using a concentration of 1 mg/mL. ConclusionsThe phenolic compounds present in the extract may be the main contributors to the bioactivities of V. betonicifolium. These results show for the first time the richness of phytochemicals and the strong bioactivities of V. betonicifolium and that the aqueous extract could be used as new natural sources of bioactive molecules.

Identification of dyes in Egyptian textiles of the first millennium ad from the collection Fill-Trevisiol

High-performance liquid chromatography coupled to a diode-array detector (HPLC-DAD) is used to investigate 30 samples which were removed from 27 ancient Egyptian fabrics of the Fill-Trevisiol collection. Attention is focused in this paper on fabrics of the Roman and Byzantine periods, with red and deep violet–blue wool weft threads which are Z-spun. The following dyes are identified in fabrics which date to the Roman period (first–fifth c. ad): mollusc purple, madder, and indigo/woad (Indigofera species and other, Isatis tinctoria L.). The results for the Byzantine (fifth–seventh c. ad) fabrics are richer in terms of identified dyes: apart from the three aforementioned dyes, the use of kermes (Kermes vermilio Planchon) and cochineal is revealed as the two coccid dyes have been detected in six Egyptian–Byzantine fabrics. Moreover, a yellow dye (probably Reseda luteola L.) is identified in one sample. Finally, samples taken from two fabrics, which date to the Islamic period, were dyed using indigo/woad and lac (Kerria lacca, Kerr). Mixing madder and indigo/woad to imitate true purple was a common practice in ancient Egypt and this is confirmed by the HPLC results in several samples. Semi-quantitative results are obtained from the HPLC peak areas and lead to the following conclusions. Madder dyes which were rich or poor in alizarin, compared to purpurin, and could have been therefore obtained from Rubia tinctorum L. and Rubia peregrina L., respectively are detected in Byzantine fabrics. Only alizarin-rich madder dyes are identified in some samples from Roman fabrics, but in some other samples the relative alizarin-to-purpurin ratio cannot be measured with confidence. Cochineal is found in two samples. The biological source of cochineal (Porphyrophora hamelii Brandt) is possible to be chemically identified only in one sample. The HPLC results of the molluscan dye detected in a Roman fabric are compared with the relative compositions of extracts of the three Mediterranean molluscs leading to the speculation that the Roman purple sample was probably dyed using Hexaplex trunculus L. The molluscan dye was furthermore detected in a Byzantine sample where it was combined with madder. Finally, it is reported that the standard (hydrochloric) acid hydrolysis method which is commonly applied to extract dyes from archaeological samples does not have any noticeable effect on the relative composition of the molluscan dye.

Comprehensive characterization of in vitro and in vivo metabolites of 2′,3′,5′‑tri‑O‑acetyl‑N6‑(3‑hydroxyphenyl) adenosine and study of the metabolites distribution in rats by combined methods of HPLC-DAD, off-line cryoNMR, and HPLC-QTOFMS

The compound 2′,3′,5′‑tri‑O‑acetyl‑N6‑(3‑hydroxyphenyl) adenosine (also known as IMM-H007) is a new adenosine analogue that displays anti-hyperlipidaemic activity in many preliminary studies. To clarify its biotransformation process, in vitro and in vivo metabolic patterns of IMM-H007 in rat liver microsomes (RLMs), urine, feces, serum, and various tissues were investigated using high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD), off-line cryogenically cooled probe nuclear magnetic resonance (cryoNMR), and high-performance liquid chromatography quadrupole TOF MS (HPLC-QTOFMS) measurements. A total of 21 metabolites were detected and identified based on accurate mass measurements, diagnostic product ions, and 1D and 2D NMR data. All of the 21 metabolites were detected in vivo besides the 7 ones (LM1–3, LM4a-b, LM5, LM6 (M8)) in vitro. Among them, eight metabolites were phase I metabolites composed of the hydrolysis products LM1–3, LM4a, LM4b, LM5 and M7–8, and hydrolysis and hydroxylation products M6. Others were phase II metabolites including glucuronidation products M2, M4, M9, M11a-c, and M12a-c; and sulfation products M3, M5, and M10. Notably, 14 metabolites (LM1–3, LM4a-b, LM5, M9–10, M11a-c, M12a-c) were unreported before and the distribution of IMM-H007 and its all metabolites was reported for the first time. The results revealed IMM-H007 was metabolized mainly in the small intestine and serum, kidney, stomach, small and large intestines were important samples for metabolites presence. This work improves understanding of the metabolism, distribution, and excretion of IMM-H007, and demonstrates the HPLC/HPLC-MS/off-line cryoNMR approach can be applied for detection and identification of metabolites in complex biological matrices.

Rapid Identification and Simultaneous Quantification of Aristolochic Acids by HPLC-DAD and Confirmations by MS in Aristolochia chilensis Using a Limited Biomass

Six aristolochic acids were identified in the Chilean species Aristolochia chilensis using high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD) and subsequent confirmation with mass spectrometry (MS). The fractions of each signal were collected and injected directly into an Orbitrap mass detector model Q Exactive Focus (Thermo Scientific). The acids extraction was done with 0.10–0.50 g of lyophilized and pulverized sample and concentrated in Soxhlet extraction equipment. The liquid-liquid separations and a subsequent solid phase extraction (SPE) C18 were performed using 100 µL of the extract that contains the aristolochic acids present in the Aristolochia chilensis plant. The HPLC conditions used a single mobile phase acetonitrile : water (1 : 1) acidified with 0.1% acetic acid and an isocratic elution to 1 mL·min−1. The column InertSustain C18 250 × 4.6 mm and 3 µm was used, the injection volume was 20 µL, and the time of run was reduced to 15 min. Calibration curves were constructed with r 2 being 0.9997. The quantification limit for AAI was 0.138 ± 0.010 µg/mL, and for AAII, it was 0.558 ± 0.042 µg/mL.

An HPLC Procedure for the Quantification of Aloin in Latex and Gel from Aloe barbadensis Leaves.

Aloin is an anthraquinone-C-glycoside present in Aloe vera. This compound is extremely variable among different species and highly depends on the growing conditions of the plants. The quantification of aloin in different extraction preparations has been a frequent problem due to the high instability of the compound. The aim of the present study is to develop and validated an analytical method for aloin detection in fresh and dry samples of Aloe barbadensis gel and latex using high performance liquid chromatography coupled to a diode array detector (HPLC-DAD). Phosphate buffered saline (pH 3) was selected as the extraction solvent. The aloin was separated using a Zorbax Eclipse AAA column (4.6 × 150 mm) at 35°C, and water and acetonitrile were used as the mobile phase at a flow rate of 0.9 mL/min. The linearity was satisfactory with a correlation coefficient greater than 0.999. Under these conditions, the method precision (relative standard deviation) was 3.71% for FL, 4.41% for dry latex, 0.81% for fresh gel and 4.42% for dry gel samples. Aloe latex was determined to have a greater amount of aloin than aloe gel. The method validation was satisfactory and exhibited adequate linearity, repeatability and accuracy.

Optimisation and validation of analytical methods for the simultaneous extraction of antioxidants: Application to the analysis of tomato sauces

In the present study, simultaneous extraction of natural antioxidants (phenols and carotenoids) in complex matrices, such as tomato sauces, is presented. The tomato sauce antioxidant compounds studied were the phenolics hydroxytyrosol, from virgin olive oil, quercetin and its derivatives, from onions, and quercetin-rutinoside as well as the carotenoid, lycopene (cis and trans), from tomatoes. These antioxidant compounds were extracted simultaneously with n-hexane/acetone/ethanol (50/25/25, v/v/v). The phenolics were analysed by ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC–MS/MS), and lycopene (cis- and trans-forms) was analysed using high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD). After studying the parameters of these methods, they were applied to the analysis of virgin olive oil, fresh onion, tomato concentrate and tomato powder, and commercial five tomato sauces. Subsequently, the results obtained in our laboratory were compared with those from the Gallina Blanca Star Group laboratory.

Determination of nitration degrees for the birch pollen allergen Bet v 1

Nitration of tyrosine residues in the major birch pollen allergen Bet v 1 may alter the allergenic potential of the protein. The kinetics and mechanism of the nitration reaction, however, have not yet been well characterized. To facilitate further investigations, an efficient method to quantify the nitration degree (ND) of small samples of Bet v 1 is required. Here, we present a suitable method of high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD) that can be photometrically calibrated using the amino acids tyrosine (Tyr) and nitrotyrosine (NTyr) without the need for nitrated protein standards. The new method is efficient and in agreement with alternative methods based on hydrolysis and amino acid analysis of tetranitromethane (TNM)-nitrated Bet v 1 standards as well as samples from nitration experiments with peroxynitrite. The results confirm the applicability of the new method for the investigation of the reaction kinetics and mechanism of protein nitration.

In Vitro Antioxidant Activity and Effect of Parkia biglobosa Bark Extract on Mitochondrial Redox Status

Aqueous-methanolic extract of Parkia biglobosa bark (PBB) was screened for its polyphenolic constituents, in vitro antioxidant activity, and effect on mitochondria redox status. The in vitro antioxidant activity was assessed by using the scavenging abilities and the reducing powers of 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) diammonium salt radical cation against Fe3+. Subsequently, the ability of PBB to inhibit lipid peroxidation induced by FeSO4 (10 μm) and its metal-chelating potential were investigated. The effects of the extract on basal reactive oxygen species (ROS) generation and on the mitochondrial membrane potential (ΔΨm) in isolated mitochondria were determined by using 2′, 7′-dichlorodihydrofluorescin (DCFH) oxidation and safranin fluorescence, respectively. PBB mitigated the Fe(II)-induced lipid peroxidation in rat tissues and showed dose-dependent scavenging of DPPH (IC50: 98.33 ± 10.0 μg/mL) and ABTS. (trolox equivalent antioxidant concentration, TEAC value = 0.05), with considerable ferric-reducing and moderate metal-chelating abilities. PBB caused slight decreases in both the liver and the brain mitochondria potentials and resulted in a significant decrease (p < 0.001) in DCFH oxidation. Screening for polyphenolics using high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD) revealed the presence of caffeic acid, gallic acid, catechin, epigalocatechin, rutin, and quercetin. These results demonstrate for the first time the considerable in vitro antioxidant activity and favorable effect of PBB on mitochondria redox status and provide justification for the use of the plant in ethnomedicine.

New flavanone glycoside from the leaves of Faramea marambaiae (Rubiaceae)

The species Faramea marambaiae M. Gomes (Rubiaceae) is endemic in the city of Rio de Janeiro, Brazil, and there are no reports about its chemical composition or therapeutic potential. From the methanol extract of its leaves were isolated, among other compounds, by liquid-liquid partition followed by reverse phase (C18) column chromatography (CC) and semi-preparative reverse-phase (C18) high performance liquid chromatography coupled to a diode array detector (HPLC-DAD) the novel flavanone glycoside: 5-hydroxy-4'-methoxy-flavanone-7-O-s-D-apiofuranosyl-(1 – 6)-s-D-glucopyranoside. Structural determination was made by nuclear magnetic resonance (NMR) techniques in one and two dimensions (1H NMR, 13C NMR, COSY, HSQC and HMBC), ultraviolet spectroscopy (UV) and optical rotation. Preliminary test for in vitro cytotoxicity of the methanol extract towards a human breast cancer tumor cell line (MCF-7) showed no cytotoxicity. Tests for antioxidant activity with DPPH and phototoxic activity (photosensitizer action to produce singlet oxygen – 1O2) of the flavonoid rich methanol:water 9:1 fraction, obtained by partition of the methanol extract, showed moderate antioxidant activity (IC50= 174.96 µg/mL) and absence of phototoxicity. Acknowledgments: CNPq and FAPERJ (Brazil), Oswaldo Cruz Foundation NMR Lab.

Rapid and Simple Method for the Determination of Emodin in Tartary Buckwheat (Fagopyrum tataricum) by High-Performance Liquid Chromatography Coupled to a Diode Array Detector

A simple and rapid method for determining emodin, an active factor presented in tartary buckwheat (Fagopyrum tataricum), by high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD) has been developed. Emodin was separated from an extract of buckwheat on a Kromasil-ODS C(18) (250 mm × 4.6 mm × 5 μm) column. The separation is achieved within 15 min on the ODS column. Emodin can be quantified using an external standard method detecting at 436 nm. Good linearity is obtained with a correlation coefficient exceeding 0.9992. The limit of detection and the limit of quantification are 5.7 and 19 μg/L, respectively. This method shows good reproducibility for the quantification of the emodin with a relative standard deviation value of 4.3%. Under optimized extraction conditions, the recovery of emodin was calculated as >90%. The validated method is successfully applied to quantify the emodin in tartary buckwheat and its products.