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Quantitative Structure-Retention Relationship models were developed to identify phenolic compounds using a typical LC- system, with both UV and MS detection. A new chromatographic method was developed for the separation of fifty-two standard phenolic compounds. Over 5000 descriptors for each standard were calculated using AlvaDesc software and then selected through Genetic Algorithm. The selected descriptors were used as variables for models construction and to obtain a better understanding of the retention behaviour of phenols during reverse-phase separation. Three distinct molecule sets, including fifty-two phenolic compounds (Set 1), 32 flavonoids (Set 2) and 15 mono-substituted flavonoids were divided into training and validation sets to build Partial Least Square, Multiple Linear Regression and Partial Least Square-Artificial Neural Network models. To assess the predictivity of the models, these were tested on a bergamot juice sample. Partial Least Square and Partial Least Square-Artificial Neural Network exhibit the lowest prediction error, and the latter showed the best predictive power in real sample recognition. The building and implementation of such predictive models showed to be a powerful tool to identify phenolic compounds based on retention data and avoiding the use of expensive and sophisticated detectors such as tandem MS., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Cervical cancer is a type of cancer which affects the cervix cells. The conventional treatments for cervical cancer including surgery, chemotherapy, and radiotherapy are only effective in premature stages and less effective in late stages of this tumor. Therefore, the therapeutic strategies based on biologically active substances from plants are needed to develop for the treatment of cervical cancer. The aim of the present study was to assess in vivo toxicity, hematological and biochemical blood parameters in Wistar rats fed Retama sphaerocarpa aqueous leaf extract (RS-AE), as well as to perform in silico molecular docking studies and dynamic simulation of phenolic compounds against HPV16 oncoprotein E6 in order to identify potential inhibitors. RS-AE was found not to induce acute or sub-acute oral toxicity or significant alterations in hematological and biochemical blood parameters in Wistar rats. A total of 11 phenolic compounds were identified in RS-AE, including dihydrodaidzein glucuronide, chrysoperiol pentoside, genistin and vitexin, which turned out to have the highest binding affinity to HPV16 oncoprotein E6. Based on these results, these RS-AE phenolic compounds could be used as natural drugs against the HPV16 E6 oncoprotein., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Within a study focused on Sinapis pubescens subsp. pubescens wild from Sicily (Italy), an edible species still unexplored, our earlier published work has demonstrated good in vitro antioxidant properties for the flower and leaf hydroalcoholic extracts, exhibiting quite different qualitative-quantitative phenolic profiles. Herein, further research was designed to elucidate the role played by phenolic compounds in the different antioxidant mechanisms highlighted for the extracts. To achieve this goal, the crude extracts were subjected to liquid-liquid partitioning with solvents of increasing polarity; then, the fractions were investigated for their antioxidant properties using different in vitro assays. For both flowers and leaves, the ethyl acetate fractions exhibited the best activity in DPPH and reducing power assays, followed by n-butanol. The total phenolic content determination indicated these fractions as the phenolic-rich ones, which were characterized by HPLC-PDA/ESI-MS analysis. Conversely, the phenolic-rich fractions did not show any chelating activity, which was highlighted for the more hydrophobic ones., (© 2024 Wiley-VHCA AG, Zurich, Switzerland.)

Pyrolysis processes are an alternative to minimize the environmental problem associated to agrifood industrial wastes. The main product resulting from these processes is a high-value liquid product, called bio-oil. Recently, the use of comprehensive two-dimensional liquid chromatography (LC × LC) has been demonstrated as a useful tool to improve the characterization of the water-soluble phases of bio-oils, considering their complexity and high water content. However, the precise composition of bio-oils from different agrifood byproducts is still unknown. In the present study, the qualitative and quantitative screening of eight aqueous phases from different biomasses, not yet reported in the literature, using LC × LC is presented. The two-dimensional approach was based on the use of two reverse phase separations. An amide column in the first dimension together with a C18 column in the second dimension were employed. Thanks to the use of diode array and mass spectrometry detection, 28 compounds were identified and quantified in the aqueous phase samples with good figures of merit. Samples showed a distinct quali-quantitative composition and a great predominance of compounds belonging to aldehydes, ketones and phenols, most of them with high polarity., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Olive oil is an important product in the Mediterranean diet, due to its health benefits and sensorial characteristics. Picholine marocaine is the most cultivated variety in Morocco. The present research aims to evaluate the phenolic compounds, vitamin E and fatty acids of commercial Picholine marocaine virgin olive oils (VOOs) from five different North Moroccan provinces (Chefchaouen, Taounate, Errachidia, Beni Mellal and Taza), using HPLC-photodiode array (PDA)/electrospray ionization (ESI)-MS, normal phase (NP)-HPLC/ fluorescence detector (FLD) and GC-flame ionization detector (FID)/MS, respectively. The obtained results showed an average content of 130.0 mg kg -1 of secoiridoids (oleuropein aglycone, 10-hydroxy-oleuropein aglycone and ligstroside aglycone, oleocanthal and oleacein), 108.1 mg kg -1 of phenolic alcohols (tyrosol and hydroxytyrosol), 34.7 mg kg -1 of phenolic acids (caffeic acid, ferulic acid and elenolic acid), and 8.24 mg kg -1 of flavonoids (luteolin, luteolin glucoside, apigenin). With regard to vitamin E, α-tocopherol was the most abundant vitamin E (57.9 mg kg -1 ), followed by α-tocotrienol (2.5 mg kg -1 ), γ-tocopherol (4.5 mg kg -1 ) and β-tocopherol (1.9 mg kg -1 ), while δ-tocopherol was not detected. Moreover, 14 fatty acids were found and, among them, oleic acid (76.1%), linoleic acid (8.1%) palmitic acid (8.7%) and stearic acid (2.5%) were the major fatty acids detected. Finally, heat map and principal component analysis allowed us to classify the studied provinces in terms of VOO chemical composition: Chefchaouen (tyrosol and hydroxytyrosol), Taounate (oleuropein aglycone), Errachidia (ferulic acid, w -3 and w -6), Beni Mellal (oleocanthal) and Taza (luteolin and oleic acid).

A HPLC-DAD/ESI-MS method has been developed and validated for the analysis of the most representative phenolic compounds in extra-virgin olive oil (EVOO) samples using a green extraction approach based on deep eutectic solvents (DESs) at room temperature. We examined ten DESs based on choline chloride and betaine in combination with different hydrogen bond donors comprising six alcohols, two organic acids, and one urea. Five phenolic compounds, belonging to the classes of secoiridoids and phenolic alcohols, were selected for the evaluation of extraction efficiency. A betaine-based DES with glycerol (molar ratio 1:2) was found to be the most effective for extracting phenolic compounds as compared to a conventional solvent. The optimization of the extraction method involved the study of the quantity of water to be added to the DES and evaluation of the sample-to-solvent ratio optimal condition. Thirty percent of water added to DES and sample to solvent ratio 1:1 (w/v) were selected as the best conditions. The chromatographic method was validated by studying LOD, LOQ, intraday and interday retention time precision, and linearity range. Recovery values obtained spiking seed oil sample aliquots with standard compounds at 5 and 100 μg/g concentration were in the range between 75.2% and 98.7%., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The purpose of this study was to conduct a comparative evaluation of some biological properties of methanol and water extracts of leaves of five Juniperus taxa growing in Turkey: J. communis L. var. communis ( Jcc ), J. communis L. var. saxatilis Pall. ( Jcs ), J. drupacea Labill. ( Jd ), J. oxycedrus L. subsp. oxycedrus ( Joo ), J. oxycedrus L. subsp. macrocarpa (Sibth. & Sm.) Ball. ( Jom ). The antioxidant properties were examined in vitro ; both in the DPPH and in the reducing power tests, Joo methanol extract resulted the most active (IC 50  = 0.09 ± 0.01mg/mL and ASE/mL = 2.56 ± 0.06). In the TBA assay, Jcs methanol extract exhibited the highest activity (IC 50  = 4.39 ± 0.47 μg/mL). The extracts displayed bacteriostatic activity against Staphylococcus aureus , and Jd methanol extract resulted the most effective (MIC = 19.53 μg/mL); no effect on the S. aureus biofilm formation was observed. The extracts resulted non-toxic in the Artemia salina lethality bioassay. Finally, the phenolic profile of the methanol extracts was characterized by HPLC-PDA/ESI-MS.

Olea europaea, meaning "European olive," is a small tree belonging to the family Oleaceae, occurring in the Mediterranean Basin. Olive oil is an essential component of a balanced diet because of its nutritional value. Among micronutrients, phenolic compounds did show important beneficial effects for human health. The majority of the research studies on the phenol content are carried out by liquid chromatography combined to photodiode array and/or mass spectrometry detection; however, because of matrix complexity, one-dimensional liquid chromatography cannot be sometimes sufficient to obtain rewarding separations, requiring more advanced analytical techniques. In this work, comprehensive two-dimensional liquid chromatography, incorporating RP-Amide and C18 stationary phases, in the first and second dimension, respectively, both under reversed phase conditions, was investigated for the determination of the phenolic fraction in extra virgin olive oil samples. As far as detection is concerned, triple quadrupole mass spectrometry was employed under multi reaction monitoring mode offering superior selectivity and sensitivity. The reduction of matrix effects, when using comprehensive two-dimensional liquid chromatography with respect to conventional one-dimensional liquid chromatography, was assessed by comparing the slopes of calibration curves built from standard solutions and spiked olive oil samples., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Brassica incana Ten. is an edible plant belonging to the Brassicaceae family. In this work, the phenolic composition and the antioxidant and cytotoxic properties of the hydroalcoholic extracts obtained from the leaves and the flowering tops of B. incana grown wild in Sicily (Italy) were studied for the first time. A total of 17 and 20 polyphenolic compounds were identified in the leaf and in the flowering top extracts, respectively, by HPLC-PDA-ESI-MS analysis. Brassica incana extracts showed in vitro antioxidant properties; the leaf extract displayed greater radical scavenging activity in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) test than the flowering top extract (IC 50 = 1.306 ± 0.049 mg/mL and 2.077 ± 0.011 mg/mL), which in turn had a stronger ferrous ion chelating ability than the other (IC 50 = 0.232 ± 0.002 mg/mL and 1.147 ± 0.016 mg/mL). The cytotoxicity of the extracts against human colorectal adenocarcinoma (CaCo-2) and breast cancer (MCF-7) cell lines was evaluated through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the lactic dehydrogenase (LDH) release determination. The extracts showed cytotoxic efficacy against Caco-2 cells, with the flowering top extract being the most effective (about 90% activity at the highest concentration tested). In the brine shrimp lethality bioassay, the extracts exhibited no toxicity, indicating their potential safety.

This study was designed to establish the most effective solvent/technique for extracting antioxidant phytoconstituents from leaves and flower buds of Inula viscosa (L.) Aiton (Asteraceae) grown wild in Morocco. Maceration and hot extraction with methanol or water and Soxhlet ethanol extraction were utilized. The antioxidant potential was evaluated in vitro by DPPH, reducing power, and ferrous ions chelating activity assays. I. viscosa leaf and flower bud extracts displayed the strongest effect in the DPPH test, being the Soxhlet ethanol the most active ones (IC 50 = 54.24 ± 0.21 μg/mL and 39.77 ± 0.23 μg/mL); thus, they were selected for further investigations. The antimicrobial efficacy of the Soxhlet ethanol extracts against ATCC and food isolates strains was assayed; the leaf extract showed the best activity, and Candida albicans was the most sensitive strain (MIC = 125 µg/mL). The extracts resulted non-toxic against Artemia salina . Among the phenolics characterised by HPLC-PDA-ESI-MS, hispidulin hexoside, patuletin and spinacetin were identified for the first time.

This study aimed to investigate the potential of four sea water microalgae, isolated and cultivated at M'diq Bay in Morocco, as a new source of natural antioxidants. These microalgae belong to different classes, including Phaedactylium tricornitum (Bacillariophyceae), Nannochloropsis gaditana (Eustigmatophyceae), Nannochloris sp (Trebouxiophyceae), and Tetraselmis suecica (Chlorodendrophycea). The antioxidant properties were screened by the use of in vitro assays, namely 2,2-difenil-1-picrylhydrazyl, Ferric reducing antioxidant power, and Ferrous ions chelating activity, and compoundidentification was carried out in methanol and acetone extracts of both dried and fresh microalgae biomass by HPLC-PDA-MS analysis. Among the investigated microalgae, Phaedactylium tricornutum was the richest one regarding its carotenoid (especially all-E-fucoxanthin) and phenolic (especially protocatechuic acid) contents, as well as antioxidant activity (65.5%), followed by Nannochloris sp, Tetraselmis suicica , and Nannochloropsis gaditana, with antioxidant activity of 56.8%, 54.9%, and 51.1%, respectively.

The analysis of phenolic compounds in extra virgin olive oils was carried out by high-performance liquid chromatography utilizing photodiode array and mass spectrometry detectors. The chromatographic profile of thirty samples from four Italian Regions highlighted the presence of secoiridoids, phenolic alcohols, flavonoids, and phenolic acid classes. A similar qualitative profile was observed with some differences in peak area and fifteen compounds were tentatively identified. Quantitative analysis was performed by UV detection considering eight standard phenolic compounds. The chromatographic method, after optimization, was validated studying some parameters, e.g., intra-day and inter-day retention time precision, limit of detection, limit of quantification, and linearity. Recovery of the method was performed achieving good results (10 and 50 g·g -1 with recovery of 72.9⁻92.1% ( w / w ) and 79.1⁻102.8% ( w / w ), respectively). In all samples secoiridoids were the main compounds ranging from 85 to more than 99% ( w / w ) of the total concentration of detected phenolic compounds while phenolic acids accounted for the lowest percentage (0.1⁻0.6%, w / w ). Finally, total concentration of phenolic compounds and antioxidant activity were determined with different chemical assays. A good and significant correlation among total phenolic compound concentration and antioxidant activity was observed. A significant different phenolic compound concentration and antioxidant activity was determined between samples from Puglia and Sicily. This was studied performing statistical analysis by one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test.

The analysis of pomegranate phenolic compounds belonging to different classes in different fruit parts was performed by high-performance liquid chromatography coupled with photodiode array and mass spectrometry detection. Two different separation methods were optimized for the analysis of anthocyanins and hydrolyzable tannins along with phenolic acids and flavonoids. Two C 18 columns, core-shell and fully porous particle stationary phases, were used. The parameters for separation of phenolic compounds were optimized considering chromatographic resolution and analysis time. Thirty-five phenolic compounds were found, and 28 of them were tentatively identified as belonging to four different phenolic compound classes; namely, anthocyanins, phenolic acids, hydrolyzable tannins, and flavonoids. Quantitative analysis was performed with a mixture of nine phenolic compounds belonging to phenolic compound classes representative of pomegranate. The method was then fully validated in terms of retention time precision, expressed as the relative standard deviation, limit of detection, limit of quantification, and linearity range. Phenolic compounds were analyzed directly in pomegranate juice, and after solvent extraction with a mixture of water and methanol with a small percentage of acid in peel and pulp samples. The accuracy of the extraction method was also assessed, and satisfactory values were obtained. Finally, the method was used to study identified analytes in pomegranate juice, peel, and pulp of six different Italian varieties and one international variety. Differences in phenolic compound profiles among the different pomegranate parts were observed. Pomegranate peel samples showed a high concentration of phenolic compounds, ellagitannins being the most abundant ones, with respect to pulp and juice samples for each variety. With the same samples, total phenols and antioxidant activity were evaluated through colorimetric assays, and the results were correlated among them.

Background: Ficus vasta Forssk. (Moraceae) is traditionally used for the treatment of various ailments; nonetheless, this species has been poorly studied to date. This work aimed to characterize the phenolic profile and to evaluate the antioxidant and antimicrobial properties of a hydroalcoholic extract obtained from F. vasta leaves collected in Egypt., Methods: The phenolic profile of the extract was characterized by HPLC-PDA/ESI-MS. The antioxidant properties were examined by different in vitro systems: DPPH test, reducing power and metal chelating activity assays. Moreover, the ability of the extract to protect Escherichia coli growth and survival from H 2 O 2 -induced oxidative stress was evaluated. The potential toxicity was investigated using Artemia salina lethality bioassay. Finally, the antimicrobial properties against a representative set of Gram-positive and Gram-negative bacterial strains and the yeast C. albicans were assayed by standard methods., Results: By HPLC-PDA/ESI-MS analysis 12 compounds belonging to the groups of phenolic acids and flavonoids were identified. The extract exhibited strong radical scavenging activity in DPPH test (IC 50  = 0.0672 ± 0.0038 mg/mL), reducing power (3.65 ± 0.48 ASE/mL) and chelating activity (IC 50  = 0.801 ± 0.007 mg/mL). A total protection against H 2 O 2 -induced damage on E. coli was observed. No toxicity against A. salina was found (LC 50  > 1000 μg/mL). The extract exhibited bacteriostatic activity against almost all the bacteria tested (MICs: 250-62.5 μg/mL)., Conclusions: The obtained results demonstrate the potential of F. vasta leaves as safe sources of natural antioxidant and antimicrobial compounds.

The present work focused on the evaluation of the antioxidant and cytotoxic activities of the phenolic-rich fraction (ItJ-EAF) obtained from cauline leaves collected in January from Isatis tinctoria L. (Brassicaceae) growing wild around Acireale (Sicily, Italy). The total phenolic, flavonoid, and condensed tannin contents of the fraction were determined spectrophotometrically, whereas the phenolic profile was assessed by HPLC-PDA/ESI-MS analysis. A total of 20 compounds were positively identified and twelve out of them were never previously reported in I. tinctoria leaves. The fraction exhibited good radical scavenging activity in DPPH test (IC 50  = 0.6657 ± 0.0024 mg/ml) and reducing power (3.87 ± 0.71 ASE/ml), whereas, it neither showed chelating activity nor was able to counteract H 2 O 2 induced oxidative stress damage in Escherichia coli. The antiproliferative effect was evaluated in vitro on two human anaplastic thyroid carcinoma cell lines (CAL-62 and 8505C) by MTT assay. At the highest tested concentration ItJ-EAF significantly reduced (80%) the growth of CAL-62 cells. No cytotoxicity against Artemia salina was observed. It can be concluded that I. tinctoria cauline leaves represent a source of phenolic compounds which could be potentially used as chemopreventive or adjuvant agents against cancer., (© 2017 Wiley-VHCA AG, Zurich, Switzerland.)

In this study, a simple and rapid methodology to analyze and quantify principal flavanones in citrus fruit juices through the use of a nano-LC/UV-Vis apparatus, employing a 75 μm id capillary column packed with sub-2 μm particles C18 stationary phase for 10 cm, was developed. All compounds were baseline resolved working with a step gradient elution mode in 10 min. The developed analytical method was validated and the resulting RSD% for intra- and interday repeatability, related to retention time and peak area, were <4.7 and 5.5%, respectively. LOD and LOQ values corresponded to 0.40 and 1.56 μg/mL for didymin, hesperitin, and narinegenin, while for the other flavanones were 0.78 and 3 μg/mL, respectively. Good linearity with acceptable determination coefficients R(2) was obtained in the range between LOQ concentration and 200 μg/mL (500 μg/mL naringin and hesperidin). Good recovery values were also obtained. Then, the method was applied to the analysis of selected hand-squeezed and commercial citrus juices. Further, the nano-LC system was coupled to a mass spectrometer to confirm analyte identification. Antioxidant capacity of selected samples was also evaluated measured by Folin-Ciocalteu assay and Trolox equivalent antioxidant capacity assay. Results were compared to determine total flavanones concentrations., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This work aimed to evaluate and compare the phenolic profile and some biological properties of the ripe "berries" methanol extracts of Juniperus oxycedrus L. subsp. oxycedrus (Joo) and Juniperus oxycedrus L. subsp. macrocarpa (Sibth. & Sm.) Ball. (Jom) from Turkey. The total phenolic content resulted about 3-fold higher in Jom (17.89±0.23 mg GAE/g extract) than in Joo (5.14±0.06 mg GAE/g extract). The HPLC-DAD-ESI-MS analysis revealed a similar flavonoid fingerprint in Joo and Jom, whereas a difference in their quantitative content was found (4632 μg/g extract and 12644 μg/g extract). In addition, three phenolic acids were detected in Jom only (5765 μg/g extract), and protocatechuic acid was the most abundant one. The antioxidant capacity of the extracts was evaluated by different in vitro assays: in the DPPH and in the TBA tests a stronger activity in Jom was highlighted, while Joo exhibited higher reducing power and metal chelating activity. Joo and Jom did not affect HepG2 cell viability and both extracts resulted virtually non-toxic against Artemia salina. The extracts were also studied for their antimicrobial potential, displaying efficacy against Gram-positive bacteria., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The present study was designed to define the phenolic profile and the biological potential of berries methanol extract of Juniperus drupacea Labill. from Turkey. The total phenolic content (Folin-Ciocalteau assay) was 48.06±0.99mgGAE/g extract. The HPLC-DAD-ESI-MS analysis allowed the determination of the complete phenolic profile of J. drupacea berries. Phenolic acids represented more than 60% of the total phenolics, and tyrosol was the major one (1324±0.64μg/g extract); within the flavonoids amentoflavone was detected as the main constituent (927±0.35μg/g extract). The extract exhibited good antioxidant properties, as determined by different in vitro models: DPPH test (IC(50) 0.38±0.02mg/mL), reducing power (12.63±0.14ASE/mL), Fe(2+) chelating ability (IC(50) 2.26±0.06mg/mL), and TBA test (IC(50) 2.47±1.13μg/mL). Cytotoxicity against Artemia salina was highlighted (LC(50) 489.47±27.8μg/mL), and a significant decrease (p⩽0.05; p⩽0.01) in HepG2 cells viability was observed at the higher concentrations (5-10μg/mL). The extract displayed good antibacterial activity towards Gram-positive bacteria and in particular Staphylococcus aureus was the most susceptible strain (MIC 78.12μg/mL)., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Infusions or dried leaves from Morus alba (L., Moraceae) have long been used in folk medicine for the prevention or treatment of several diseases. Despite the great interest in determining the role of phytonutrients as potential therapeutic agents, and the rising demand of natural sources with nutraceutic benefits, the antioxidant content of many foodstuffs is unknown, making accurate estimation for human dietary consumption and the correlation to human diseases difficult. The high content in polyphenols mainly accounts for in vitro and in vivo antioxidant activity of extracts obtained from this plant, and it is responsible for their preventive and therapeutic roles in a number of human diseases. In this study, we achieved full characterization and quantitation of flavonoids and other phenolic components extracted from lyophilized mulberry leaves, by maceration with ethanol. To tackle such a task, an RPLC-diode array detector-MS system was optimized, employing a partially porous octadecylsilica column as stationary phase. Identification of mulberry leaves constituents was carried out on the basis of the complementary information obtained from their migration times, diode array spectra, MS ions, and MS/MS fragments. The employment of the hybrid (IT-TOF) mass spectrometer further allowed the structural assignment of a series of structural isomers. To the best of our knowledge, 11 out of 22 identified compounds are being reported in mulberry leaves for the first time, and the relative amounts determined, in the two cultivars analyzed.

A comprehensive two-dimensional liquid chromatography method has been applied for the quantification of polyphenols in red wines and compared to the most commonly employed conventional LC approach. Such methodology comprised the use of a microbore conventional HPLC column packed with totally porous particles in the first dimension and a partially porous column of conventional diameter in the second dimension. Even though a good number of applications in comprehensive LC have been reported, quantification experiments have been rarely described. To this regard, the advantages of comprehensive LC together with the employment of dedicated software capable of detecting and quantifying each peak from the 2D plot, have been taken into account for quantifying the most representative polyphenols in three different commercial Sicilian red wine samples. The optimized method has been validated in terms of linearity, sensitivity, detection and quantification limits. LODs as low as 0.02 ppm were obtained using the one-dimensional HPLC-DAD method, whereas values lower than 0.10 ppm were obtained by comprehensive LC. However, comprehensive LC allowed the quantification of a higher number of compounds with RSD lower than 10% thanks to its improved resolving power. The separation capabilities of comprehensive LC allowed the analysis of complex natural samples without any pre-treatment to effectively reduce the interferences coming from the matrix.

There is growing interest related to rapid screening and full characterization of the constituents of plants with medicinal properties; among these, "Mate" or Yerba Maté is a tea-like beverage widely consumed in South America, obtained from the dried leaves of Ilex paraguariensis. The high content in polyphenols accounts for in vitro and in vivo antioxidant activity of the extracts obtained from this plant; on the other hand, the high complexity of the samples extracted, depending on the method employed, may preclude complete resolution by conventional HPLC techniques. For this purpose, a comprehensive two-dimensional liquid chromatography (LC x LC) system, comprised of an RP-Amide first dimension and a partially porous octadecylsilica column in the second dimension, has been compared with a one-dimensional system. The latter was operated using a partially porous octadecylsilica column, with diode array (DAD) and electrospray/ion trap-time of flight (ESI/IT-TOF) detection for the most complex extracts. The employment of the hybrid mass spectrometer allowed unequivocal identification of several compounds in the mate extracts. Using LC x LC-MS(3), it was possible to discriminate between congeners of chlorogenic acids, along with monoacyl- and diacylchlorogenic acid esters.

In the present work, a comprehensive LC system using a microbore HPLC column in the first dimension and a partially porous column in the second dimension was developed and applied to the separation of polyphenolic components in a red wine sample. The performance of the partially porous short column (3.0 cm) was compared to that of a monolithic column, of comparable dimensions. The results obtained demonstrated the possibility to use partially porous columns to obtain fast analyses, using high flow rates, under repetitive gradient conditions and with very brief reconditioning times. A conventional HPLC system was used since the backpressure generated by the shell-packed column, even at very high flow rates, was well within the operational limits. The use of an increased column temperature (60 degrees C) allowed a further pressure-drop decrease, with no stationary phase degradation, or loss in column performance.

Various combinations of PEG-silica, phenyl-silica and C18 columns in a single-column or serial (tandem) arrangement in the first dimension and a monolithic Chromolith column in the second dimension were tested for comprehensive two-dimensional (2D) LCxLC separation of phenolic and flavone natural antioxidants. The combinations of different stationary phase chemistries provided low selectivity correlations between the first-dimension and the second-dimension separation systems. Improvement in system orthogonality, bandwidths suppression, more regular band distribution over the whole 2D retention plane and increased peak capacity in different 2D setups was achieved by using gradients with matching profiles running in parallel in the two dimensions over the whole 2D separation time range. Instead of two sampling loops, two alternating trapping XTerra columns were used for sample fraction transfer from the first-dimension column to the second dimension. Stronger retention on the XTerra columns in comparison to PEG-silica or phenyl-silica columns in the first dimension allowed using focusing of sample fractions in narrow zones on the top of a trapping column and back-flushing into the second dimension in a very low volume of the mobile phase. This fraction transfer modulation provided significant bandwidth suppression in the second dimension. 2D systems with optimized stationary phase selectivity, parallel gradients and fraction transfer modulation using two trapping columns were applied for the analysis of natural antioxidants in beer and wine samples.

The effects of temperature and mobile phase on LC chromatographic separation of phenolic antioxidants on zirconia-based columns were investigated. Unlike silica-based materials, zirconia columns show excellent thermal stability over a wide range of temperatures and enable high-temperature separations. Enthalpic and entropic contributions to the retention of phenolic compounds on ZR-Carbon and ZR-Carbon C18 columns were determined from retention versus temperature plots in order to elucidate the retention mechanism of sample compounds over the temperature range up to 14 degrees C. High-temperature liquid chromatography on ZR-Carbon columns was used for comprehensive LC x LC two-dimensional separation systems based on the different selectivity of a Zorbax SB micro-column used in the first dimension and a ZR-Carbon column used in the second dimension. Two-dimensional LC x LC systems employing a setup with two alternately operated parallel ZR-Carbon columns in the second dimension were used for the analysis of phenolic antioxidants in beer and wine samples.

The separation selectivity of octadecyl-silica (C18) and of bonded pentafluorophenylpropyl-silica (F5) and PEG-silica columns was compared for natural phenolic antioxidants. The separation selectivities for phenolic antioxidants on C18 and F5 columns are strongly correlated, but low selectivity correlation indicating strong differences in the retention mechanism was observed between the C18 and PEG columns. Hence, the combination of a C18 and a PEG column is useful for separation of phenolic antioxidants that are not fully separated on single columns. Two-dimensional comprehensive liquid chromatography using a short PEG-silica column in the first dimension and a conventional C18-silica in the second dimension has the advantage of on-column focusing of the fractions transferred onto the C18 column in the second dimension, as a weaker mobile phase is used in the first dimension than in the second dimension. However, a stop-flow set-up in the first dimension system is necessary after the transfer of each fraction to the second dimension. Peak capacity is considerably larger but the separation time is much longer than with serially coupled PEG and C18 columns, which were employed for separation of beer and hop extract samples in connection with coulometric detection.

This study was designed to define and compare the antioxidant and cytotoxic properties of polar extracts obtained from basal leaves (It-BL), cauline leaves (It-CL) and flowers (It-F) of Isatis tinctoria L. growing wild around Acireale (Sicily, Italy). The phenolic profile was characterized by HPLC-PDA-ESI-MS analysis and the correlation between phenolic content and the observed biological effects was established. Further, LC/MS analysis showed that the extracts contain glucosinolates at very low concentrations. The antioxidant activity of the extracts was tested in vitro; It-F was the most effective in the DPPH test (IC50 = 0.437 ± 0.003 mg/mL), whilst It-CL showed the best reducing power (1.546 ± 0.006 ASE/mL) and ferrous ions chelating activity (IC50 = 0.564 ± 0.011 mg/mL). The extracts exhibited anti-proliferative effects against three different human thyroid carcinoma cell lines, and It-BL displayed the strongest activity; particularly, it markedly inhibited the growth of CAL-62 cells, causing nearly 85% reduction of viability at the highest tested dose. No cytotoxicity against Artemia salina was observed. The results of our investigations indicate that the polar extracts obtained from I. tinctoria are a potential source of antioxidant and anticancer compounds, which could be suitable for nutraceutical and therapeutic applications. [ABSTRACT FROM AUTHOR]

Thirty-one red wines of "Protected Geographical Indication", including most of the wine cultivars grown in Sicily, were assayed for their phenolic content. Individual phenolic compounds were identified and quantified by HPLC with a photo-diode array (PDA) coupled on-line with a MS system. The total phenolic content was evaluated by the Folin-Ciocalteau method and the antioxidant capacity was measured by the DMPD (N,N-dimethyl-p-phenylendiamine) method. The results provide new insight into the polyphenolic composition of commercial Sicilian wines, and highlight some interesting points about the different wine typologies. In particular, the Merlot wines produced in Sicily, similar to those produced by other allochthonous grapes, generally have a higher polyphenolic content. Based on the data given, Merlot wines appear to be an important source of dietary polyphenolic antioxidants. It may be supposed that wines produced from this grape typology in Sicily are endowed with properties that have potential health benefits. [ABSTRACT FROM AUTHOR]