María del Mar Contreras

Reversed-phase ultra-high-performance liquid chromatography coupled to electrospray ionization-quadrupole-time-of-flight mass spectrometry as a powerful tool for metabolic profiling of vegetables: Lactuca sativa as an example of its application

Lettuce (Lactuca sativa), a leafy vegetal widely consumed worldwide, fresh cut or minimally processed, constitutes a major dietary source of natural antioxidants and bioactive compounds. In this study, reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) coupled to electrospray ionization-quadrupole-time-of-flight mass spectrometry (ESI-QTOF-MS) was applied for the comprehensive profiling of polar and semi-polar metabolites from three lettuce cultivars (baby, romaine, and iceberg). The UHPLC systems allowed the use of a small-particle-size C18 column (1.8 μm), with very fine resolution for the separation of up to seven isomers, and the QTOF mass analyzer enabled sensitive detection with high mass resolution and accuracy in full scan. Thus, a total of 171 compounds were tentatively identified by matching their accurate mass signals and suggested molecular formula with those previously reported in family Asteraceae. Afterwards, their structures were also corroborated by the MS/MS data provided by the QTOF analyzer. Well-known amino acids, organic acids, sesquiterpene lactones, phenolic acids and flavonoids were characterized, e.g. lactucin, lactucopicrin, caftaric acid, chlorogenic acid, caffeoylmalic acid, chicoric acid, isochlorogenic acid A, luteolin, and quercetin glycosides. For this plant species, this is the first available report of several isomeric forms of the latter polyphenols and other types of components such as nucleosides, peptides, and tryptophan-derived alkaloids. Remarkably, 10 novel structures formed by the conjugation of known amino acids and sesquiterpene lactones were also proposed. Thus, the methodology applied is a useful option to develop an exhaustive metabolic profiling of plants that helps to explain their potential biological activities and folk uses.

UHPLC-ESI-QTOF-MS-based metabolic profiling of Vicia faba L. (Fabaceae) seeds as a key strategy for characterization in foodomics

Vicia faba (Fabaceae) is a popular food in many countries and a good source of nutrients. However, little is known about its phytochemical composition, specially referring to phenolic compounds. In the present work, the dietary metabolites from a hydro‐methanolic extract of V. faba seeds were thoroughly characterized by a nontargeted analytical approach based on reversed‐phase ultra‐HPLC (UHPLC) coupled to QTOF‐MS. A total of 155 primary and secondary metabolites of various structural types were characterized: carbohydrates, amino acids, organic acids, alkaloids, terpenoids, jasmonates, and, mainly, polyphenols. Among the latter group, 73 compounds were characterized for the first time in this legume. In addition, 24 new structures, belonging to jasmonates and glycosylated N‐containing compounds, were also proposed. Thus, this methodology could be implemented in foodomics as a characterization strategy to complement the knowledge of the phytochemical composition of vegetables.

Identification of polyphenols and their metabolites in human urine after cranberry-syrup consumption.

As the beneficial effects of American cranberry (Vaccinium macrocarpon) can be partly attributed to its phenolic composition, the evaluation of the physiological behaviour of this fraction is crucial. A rapid and sensitive method by ultra-performance liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) has been used to identify phenolic metabolites in human urine after a single dose of cranberry syrup. Prior to the analysis, metabolites were extracted using an optimised solid-phase extraction procedure. All possible metabolites were investigated based on retention time, accurate mass data and isotope and fragmentation patterns. Free coumaroyl hexose (isomer 1 and 2), dihydroxybenzoic acid, caffeoyl glucose, dihydroferulic acid 4-O-β-d-glucuronide, methoxyquercetin 3-O-galactoside, scopoletin, myricetin and quercetin, together with other 23 phase-I and phase-II metabolites, including various isomers, could be tentatively identified in the urine. Afterwards, the metabolites were simultaneously screened in the urine of different subjects at 0, 2, 4, and 6h after the ingestion of cranberry syrup by Target Analysis(TM) software.

Profiling of phenolic and other compounds from Egyptian cultivars of chickpea (Cicer arietinum L.) and antioxidant activity: a comparative study

Chickpeas are basic food in many countries with several cultivars distributed all over the world. However, little is known about their secondary metabolites. Thus, this work is focused on the study of the phenolic profiles of seven Egyptian cultivars of chickpea. Selecting the most appropriate extraction method and analytical conditions using reversed-phase high-performance liquid chromatography-diode array detection, with a core–shell column, and coupled with quadrupole-time-of-flight-mass spectrometry (MS), a total of 96 phenolic compounds were characterized based on their retention time, UV spectra, and accurate MS and MS2 data. Among them, the major phenolic subclasses were hydroxybenzoic acids and flavonoids. Moreover, other minor and major metabolites including organic acids, amino acids, nucleosides, peptides and soyasaponins were characterized. Using standards, 22 compounds were unequivocally identified. Remarkably, 88 of these compounds were tentatively reported for the first time in chickpeas. The total phenol content of the cultivars was determined as well as the antioxidant activity by the trolox equivalent antioxidant capacity assay.

New insights into the qualitative phenolic profile of Ficus carica L. fruits and leaves from Tunisia using ultra-high-performance liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry and their antioxidant activity

Ficus carica L. fruits have been consumed from the earliest times, and other parts of the tree have been used for traditional medicinal purposes. Nowadays, the beneficial properties of this and other Ficus species are attributed to the presence of key phytochemicals. To increase our knowledge about this topic, the present study has conducted phenolic profiling of the leaves and whole fruits from two Tunisian cultivars, ‘Temri’ and ‘Tounsi’, using reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) coupled to two detection systems: diode-array detection (DAD) and quadrupole time-of-flight (QTOF) mass spectrometry (MS). UV-Vis absorption was a valuable tool for classifying phenolic compounds into families, while MS using electrospray ionization (ESI) and MS/MS allowed the molecular formula to be established and structural information to be obtained. The total phenol content and the antioxidant activity were also assessed. As a result, in the negative ionization mode 91 phenolic compounds were characterized including hydroxybenzoic acids, hydroxycinnamic acids, hydroxycoumarins and flavanoids (flavonols, flavones, flavanones, flavanonols, flavanols and isoflavones). This work was complemented by the detection of other 18 phenolic compounds in the positive ionization mode, including anthocyanins and furanocoumarins. To the best of our knowledge, this is the first time most of these compounds have been tentatively reported in F. carica. These results indicate the complexity of this family of secondary metabolites in F. carica, as well as the potential of this analytical method for characterization purposes. In conclusion, the qualitative phenolic profile, total phenolic content and antioxidant activity differed especially between leaves and fruits.

Bioactive chemical compounds in Eremurus persicus (Joub. & Spach) Boiss. essential oil and their health implications

The genus Eremurus is native to Eastern Europe and temperate Asia. Particularly, Eremurus persicus (Joub. & Spach) Boiss. is highly valued in traditional foods and medicine. Scientific knowledge about E. persicus chemical composition and bioactivity is required. Therefore, the present study is aimed to determine the volatile composition of E. persicus essential oil (EO) by means of gas chromatography coupled to flame ionization/mass spectrometry detection. Moreover, the antioxidant, antimicrobial, anticancer, and acetylcholinesterase inhibitory activities of the EO were tested. Interestingly, the anti-dermatophyte potency was close to that of the drug griseofulvin, with minimum fungicidal concentration ranging between 0.7 and 4.5% depending on the fungi strain. The EO was also effective against hepatocellular carcinoma (Hep-G2) and breast adenocarcinoma (MCF-7) human cancer cell lines in a concentration (200-1500 ng/mL)-dependent manner, with a decrease of the cell viability up to 65% and 52%, respectively. The E. persicus EO was rich in terpenes and oxygenated terpene derivatives. Individually, limonene (16.25%), geranylgeraniol (15.23%), n-nonanal (9.48%), geranyl acetone (9.12%), benzene acetaldehyde (8.51%), linalool (7.93%), α-pinene (6.89%), and 1,8-cineol (5.22%) were the most abundant volatile compounds and could be chosen as analytical markers of this essential oil. In conclusion, our results suggested that this EO possesses a wide range of bioactive properties that could be useful in nutraceutical, functional foods and cosmeceutical formulations.

Carvacrol and human health: A comprehensive review: Carvacrol and Human Health

Carvacrol (CV) is a phenolic monoterpenoid found in essential oils of oregano (Origanum vulgare), thyme (Thymus vulgaris), pepperwort (Lepidium flavum), wild bergamot (Citrus aurantium bergamia), and other plants. Carvacrol possesses a wide range of bioactivities putatively useful for clinical applications such antimicrobial, antioxidant, and anticancer activities. Carvacrol antimicrobial activity is higher than that of other volatile compounds present in essential oils due to the presence of the free hydroxyl group, hydrophobicity, and the phenol moiety. The present review illustrates the state‐of‐the‐art studies on the antimicrobial, antioxidant, and anticancer properties of CV. It is particularly effective against food‐borne pathogens, including Escherichia coli, Salmonella, and Bacillus cereus. Carvacrol has high antioxidant activity and has been successfully used, mainly associated with thymol, as dietary phytoadditive to improve animal antioxidant status. The anticancer properties of CV have been reported in preclinical models of breast, liver, and lung carcinomas, acting on proapoptotic processes. Besides the interesting properties of CV and the toxicological profile becoming definite, to date, human trials on CV are still lacking, and this largely impedes any conclusions of clinical relevance.

Antihyperlipidemic and Antioxidant Activities of Edible Tunisian Ficus carica L. Fruits in High Fat Diet-Induced Hyperlipidemic Rats

The phenolic constituents of the aqueous-ethanolic extract of Tunisian Ficus carica (F. carica) fruit (FE) and its antihyperlipidemic and antioxidant activities in high-fat diet-induced hyperlipidemic rats (HFD) were evaluated. The obtained results demonstrated that the FE improved the lipid profile by decreasing the total cholesterol, triglyceride, low-density lipoprotein cholesterol and increasing high-density lipoprotein cholesterol levels. It also reduced the content of thiobarbituric acid-reactive substances and increased the antioxidant enzymes in liver, heart and kidney in HFD-fed rats. These antihyperlipidemic effects and in vivo antioxidative effects correlated with the in vitro phenolic content scavenging ability. Thus, the major phenolic compounds were identified using reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) coupled with two detection systems: diode-array detection (DAD) and quadrupole time-of-flight (QTOF) mass spectrometry (MS). Therefore, in the negative ionization mode, 28 phenolic compounds, including hydroxybenzoic acids, hydroxycinnamic acids, flavanoids and hydroxycoumarins were characterized. Dihydroxybenzoic acid di-pentoside, the flavonol quercetin 3-O-rutinoside and the flavone assigned as apigenin 8-C-glucoside were the main representative compounds in ‘Tounsi’ fruits. This work was complemented by the detection of seven other phenolic compounds in the positive ionization mode, including anthocyanins and furanocoumarins. Overall, these results have shown that the FE has a significant hypocholesterolemic effect and antioxidant activity in HFD-fed rats. This beneficial effect may be partly due to these phenolic constituents, especially vitexin, dihydroxybenzoic acid di-pentoside as well as rutin.

Anti-inflammatory activity of hydroalcoholic extracts of Lavandula dentata L. and Lavandula stoechas L.

ETHNOPHARMACOLOGICAL RELEVANCE Plants from genus Lavandula have been used as anti-inflammatory drugs in Mediterranean traditional medicine. Nowadays, there is a growing interest for complementary medicine, including herbal remedies, to treat inflammatory bowel disease (IBD). AIM OF THE STUDY To test the anti-inflammatory properties of Lavandula dentata and Lavandula stoechas extracts in two inflammatory experimental models: TNBS model of rat colitis and the carrageenan-induced paw edema in mice, in order to mimic the intestinal conditions and the extra-intestinal manifestations of human IBD, respectively. MATERIAL AND METHODS The extracts were characterized through the qualitative HPLC analysis. Then, they were assayed in vitro and in vivo. In vitro studies were performed in BMDMs and CMT-93 epithelial cells with different concentrations of the extracts (ranging from 0.1 to 100µg/ml). The extracts were tested in vivo in the TNBS model of rat colitis (10 and 25mg/kg) and in the carrageenan-induced paw edema in mice (10, 25 and 100mg/kg). RESULTS L. dentata and L. stoechas extracts displayed immunomodulatory properties in vitro down-regulating different mediators of inflammation like cytokines and nitric oxide. They also showed anti-inflammatory effects in the TNBS model of colitis as evidenced by reduced myeloperoxidase activity and increased total glutathione content, indicating a decrease of neutrophil infiltration and an improvement of the oxidative state. Besides, both extracts modulated the expression of pro-inflammatory cytokines and chemokines, and ameliorated the altered epithelial barrier function. They also displayed anti-inflammatory effects in the carrageenan-induced paw edema in mice, since a significant reduction of the paw thickness was observed. This was associated with a down-regulation of the expression of different inducible enzymes like MMP-9, iNOS and COX-2 and pro-inflammatory cytokines, all involved in the maintenance of the inflammatory condition. CONCLUSION L. dentata and L. stoechas extracts showed intestinal anti-inflammatory effect, confirming their potential use as herbal remedies in gastrointestinal disorders. In addition, their anti-inflammatory effect was also observed in other locations, thus suggesting a possible use for the treatment of the extra-intestinal symptoms of IBD.

Nano-liquid chromatography coupled to time-of-flight mass spectrometry for phenolic profiling: A case study in cranberry syrups

A new method based on nano-liquid chromatography coupled to time-of-flight mass spectrometry (nano-LC-TOF-MS) using lock-mass calibration was developed to facilitate the accurate and routine characterization and quantification of phenolic compounds. Thus, it was applied to study cranberry syrups, in which, using negative ionization mode, a total of nine phenolic compounds were unequivocally identified using standards and 38 tentatively taking into account their retention time, accurate mass (errors<5 ppm) data and isotope pattern, as well as literature. Among them, 13 compounds, belonging to flavonols and iridoids conjugated with phenolic acids, were reported for first time in cranberry or cranberry based-products. The analytical method was also validated using chlorogenic acid, p-coumaric acid, (+)-catechin, (-)-epicatechin, procyanidin A2, quercetin 3-O-glucoside, quercetin 3-O-rhamnoside, quercetin, and myricetin standards. In this way, the analytical method showed adequate linearity, with R(2) above 0.99, and acceptable values of intra- and inter-day repeatability of the retention time and peak area. The detection limits and quantification were between 1.0-15.6 ng mL(-1) and 2.0-62.5 ng mL(-1), respectively. The method can be extended to characterize phenolic compounds in other food and plant matrices, and as well biological samples.