Evaluation and Comparison of Dermo-Cosmetic Activities of Three Oak Species by Targeting Antioxidant Metabolites and Skin Enzyme Inhibitors.

The two main species, sessile oak (Quercus petraea Liebl.) and pedunculate oak (Quercus robur L.), predominant in French forests, are mainly used for aging wines and spirits; however, the potential of oak wood extract as a source of natural antioxidants, due to its high polyphenol content, could be more widely exploited. This study focuses on three oak species, the two that are well-known, namely, sessile and pedunculate oak, and a third that has seldom been described and valorized, namely, pubescent oak (Quercus pubescens). Water extracts of these three species were fractionated by semi-preparative HPLC. The antioxidant activities of crude extracts and fractions were measured by colorimetric and enzymatic tests. The anti-elastase and anti-collagenase activities of the extracts and their fractions were also evaluated. In parallel, samples were analyzed by UHPLC-HRMS to correlate the activity with the molecular composition using molecular networks. The results obtained for the total extract of the three species were compared to determine if the activity depended on the species. The results within the same species were also compared to highlight which fraction and, therefore, which molecular family was involved in the activity of the total extract. The various antioxidant tests showed good activity of the total extract for the three species of oak and a very good anti-collagenase activity. The antioxidant activity of oak extract has already been proven in the literature and this is correlated with its richness in polyphenols. This study shows that each molecular family of the extract contributes to the activities of the total extract. Oak extract can be used to neutralize the ROS produced during oxidative stress and to prevent the degradation of collagen and elastase during skin aging. Its complementary properties make oak extract a valuable ingredient to act against skin aging.

Supercritical Fluid Extraction (SFE) of Polar Compounds from Camellia sinensis Leaves: Use of Ethanol/Water as a Green Polarity Modifier.

The use of bioactive plant extracts in cosmetic products is a common practice. Most of these extracts are obtained by maceration in organic solvents, and depending on which solvents are used, the polarity and the structure of the target molecules will vary. Polyphenols are polar compounds that often display antioxidant and/or antibacterial activities. To extract them, ethanol/water mixtures are usually selected as green solvents. This solid-liquid extraction (assisted or not) requires the use of high volumes of solvents and many additional steps like mixing, agitation, filtration, and evaporation. Alternatively, supercritical carbon dioxide (SC-CO2) offers many benefits for plant extraction: economical, non-toxic, and naturally concentrated extracts. However, its low polarity is not suitable to solubilize polar compounds. In this study, an experimental design was used to optimize supercritical fluid extraction (SFE) of caffeine and catechins from Camellia sinensis. Catechins are recognized for skin care use (antioxidant) and caffeine is also used for its skin care properties and to prevent excess storage of fat in cells. The temperature, modifier content, and water additive percentage were used as independent variables. The results showed that while the temperature was an insignificant parameter, a higher percentage of water (up to 20% in ethanol) and modifier favored the extraction of the polar target molecules. Additionally, the SFE results were compared with ultrasound-assisted extraction (UAE). Finally, a sequential selective extraction of caffeine from catechins is also presented.

Untargeted metabolomic and molecular network approaches to reveal tomato root secondary metabolites.

INTRODUCTION: The tomato plant, Solanum lycopersicum L. (Solanaceae), is one of the most widely consumed vegetables in the world and plays an important role in human diet. Tomato cultivars are hosts for diverse types of pests, implying diverse chemical defence strategies. Glycoalkaloids are the main specialised metabolites produced by tomato leaves and fruits to protect against pests. However, the roots have received little attention, leading to limited knowledge about their phytochemical content. OBJECTIVE: The main goal of the current study was the development of an untargeted ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS) based metabolomic approach to study phytochemical variations in tomato roots at two different development stages (i.e. 34th and 62nd day after sowing). METHODS: UHPLC-HRMS was used to establish the fingerprint of 24 batches of tomato roots. Statistical analyses were performed to highlight the compounds that discriminated between young and mature tomato roots. A dereplication strategy using molecular networking and HRMS/MS data was set up to identify the metabolites regulated during early root development. KEY FINDINGS: The main biomarkers were guanidine and adenosine derivatives associated with tryptophan. Secondary metabolites such as glycoalkaloids and steroidal alkaloids were also characterised. Most of the metabolites were up-regulated in young tomato roots (34 days old) while tryptophan was up-regulated in the older roots (62 days old). CONCLUSION: The metabolic changes observed in this work contribute to a deeper understanding of early-stage root development and may help our understanding of the complex processes involved in the tomato root defence arsenal.

TLC-MALDI-TOF-MS-based identification of flavonoid compounds using an inorganic matrix.

INTRODUCTION: Thin layer chromatography (TLC) is frequently used to obtain the fingerprint of a plant extract. Although the retardation factor and the response to visualisation give primary information about compound identification, the direct TLC-mass spectrometry (MS) coupling allows a more detailed characterisation of samples. OBJECTIVES: To demonstrate the potential for the flavonoid dereplication using an inorganic matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) method with and without TLC separation. MATERIAL AND METHODS: Samples derived from wine, apple or rose were deposited on an aluminium-backed silica gel TLC sheet compatible with the MS adapter. Unlike the wine sample, for apple and rose samples compound derivatisation was necessary. These two samples were deposited twice and the plate was cut in two parts. One half was oversprayed with Neu-Peg reagent to visualise flavonoids while the inorganic matrix was deposited on each flavonoid zone on the second half for MS ionisation. RESULTS: Mass spectra obtained for samples without plate development showed numerous ions corresponding to glycosylated flavonoids. The lower m/z observed could be due either to aglycone flavonoids or to in-source fragment ions. After plate development, a separation of many spots was observed and each spot was analysed separately leading to a deeper identification of the present flavonoids. Moreover, isobaric flavonoids with different hRf values could be differentiated. CONCLUSION: TLC-MALDI-TOF-MS using an inorganic matrix enabled the analysis of anthocyanins in positive mode and of flavonols, flavanols, dihydrochalcones and phenolic acids in negative mode, reducing adduct, aggregate forms giving thus simple and reliable spectra for the dereplication approach of flavonoids in complex samples.

Molecular Fingerprint Comparison of Closely Related Rose Varieties based on UHPLC-HRMS Analysis and Chemometrics.

INTRODUCTION: The "Jardin de Granville" modern rose variety not only combines the morphological properties of its two parental cultivars, but also possesses better agronomic characteristics (abundant blooms, strong growth and vitality, high resistance to common rose diseases). In addition, it shows remarkable biological properties such as a high ability to decrease inflammatory and oxidative stress on skin cells. That is why Parfums Christian Dior selected this rose variety to be an active ingredient in luxury cosmetics. OBJECTIVES: To identify the characteristic molecular signature of "Jardin de Granville" compared with its parents "Annapurna" and "John Clare", by the mean of a non-targeted metabolomic comparison. MATERIAL AND METHODS: Wood, flower and leaf hydro-alcoholic extracts were analysed by UHPLC-ESI-HRMS. The fingerprints were then submitted to unsupervised multivariate analyses involving principal component analysis (PCA) and hierarchical ascendant classification (HAC). Analysis of variance (ANOVA) was finally performed to highlight the significant differences in each group of organs. RESULTS: The extracts were composed of phenolic compounds such as hydrolysable and condensed tannins and flavonol derivatives. Three groups of extracts were clustered as a function of the variety. The compounds overexpressed in "Jardin de Granville" variety were highlighted thanks to ANOVA test. Flower was the most discriminative organ with 15 overexpressed molecules. Auto MS/MS analyses led to their tentative identifications. CONCLUSION: The non-targeted metabolomic approach revealed the importance of tannins to discriminate close rose varieties. The overexpressed hydrolysable tannins characteristic of "Jardin de Granville" can be responsible for the antioxidant and anti-inflammatory properties of the rose cosmetic ingredients. Copyright © 2016 John Wiley & Sons, Ltd.

Non-targeted molecular characterisation of a rose flower ethyl acetate extract using Ultra-HPLC with atmospheric pressure photoionisation and quadrupole time-of-flight MS/MS.

INTRODUCTION: A non-targeted approach to characterise the phytochemical composition of the flower organ of an original rose cultivar 'Jardin de Granville'® was developed. Particular attention was paid to the less documented molecular families of intermediate polarity, compared with the polyphenol family (anthocyanins, flavonoids, tannins) and volatile compounds. OBJECTIVE: To develop a molecular fingerprinting method for the rapid qualitative phytochemical characterisation of the rose flower ethyl acetate extract. MATERIAL AND METHODS: An ultra-HPLC with atmospheric pressure photoionisation (APPI) and quadrupole time-of-flight (QTOF) MS/MS combined with microwave-assisted extraction was carried out for ethyl acetate extracts as an intermediate polarity extraction solvent in order to obtain the most exhaustive extract containing a large range of molecular families. An optimised methodology based on the coupling of the UHPLC and APPI source with a QTOF analyser was developed to characterise the extracted molecules. RESULTS: Sixty-one compounds were identified in the extract, covering eight molecular families of intermediate polarity ranging from polyphenols to triglycerides. The presence of flavonoids with anti-oxidant properties and of triterpenoids with potential anti-inflammatory activity was evidenced and cell-wall constituents such as fatty acids, glycolipids, sphingolipids and acylated sterol glycosides were characterised. Some chlorophyll derivatives were also detected. CONCLUSION: The method developed is appropriate for fast phytochemical evaluation of rose ethyl acetate extract. It produced accurate mass and MS/MS spectra, which permitted identification of a wide range of compounds of intermediate polarity.

Phytochemical analysis of Rosa hybrida cv. 'Jardin de Granville' by HPTLC, HPLC-DAD and HPLC-ESI-HRMS: polyphenolic fingerprints of six plant organs.

The Rosa hybrida cultivar 'Jardin de Granville', a delicate clear pink flower, is here investigated through a progressive analytical strategy using complementary phytochemical screening methods in order to characterize the polyphenol content of several parts of the plant. The microwave hydro-ethanolic extract analysis of six different parts of the plant, carried out by High Performance Thin Layer Chromatography (HPTLC) and High Performance Liquid Chromatography coupled with a Diode Array Detector (HPLC-DAD) enabled initial identification of the polar molecular families present in each organ, namely tannins and flavonoids (quercetin and kaempferol derivatives). The HPLC fingerprints displayed different profiles for each organ, attesting to the original composition and potential valuation of the different plant parts. More detailed analyses of the extracts were then carried out by High Performance Liquid Chromatography coupled with electrospray ionization (ESI) mass spectrometry with a Q-TOF analyzer (ESI-HR-Q-TOF). Around 60 compounds were identified, mainly gallo-tannins, ellagi-tannins, catechin derivatives and glycoside derivatives of quercetin and kaempferol. Some compounds such as hyperoside or ellagic acid appeared to be ubiquitous and were found in abundance in each plant part. Woods were the richest organ in catechin and proanthocyanidin derivatives while kaempferol derivatives were more numerous and abundant in bud and flower parts.

New advances in countercurrent chromatography and centrifugal partition chromatography: focus on coupling strategy.

Countercurrent chromatography (CCC) is an attractive separation method because the analytes are partitioned between two immiscible liquid phases avoiding problems related to solid stationary phase. In recent years, this technique has made great progress in separation power and detection potential. This review describes coupling strategies involving high speed CCC (HSCCC) or centrifugal partition chromatography (CPC). It includes on-line extraction-isolation, hyphenation with mass spectrometry (MS) and nuclear magnetic resonance (NMR) detectors, multidimensional CCC (MDCCC), two-dimensional CCC (2D-CCC), on-line coupling with liquid chromatography (LC), and biological tests, and innovative off-line developments. The basic principles of each method are presented and applications are summarized.

Capillary electrophoresis as a novel technique for screening natural flavonoids as kinase inhibitors.

Capillary electrophoresis (CE) was used for the first time to evaluate the inhibition activity of aglycone flavonoids (such as quercetin and isorhamnetin) and some of their glycosylated derivatives toward human kinases. The cyclin-dependant kinase 5 (CDK5/p25) and the glycogen synthase kinase 3ß (GSK3ß) were chosen since they are very promising biological targets for developing treatments against neurodegenerative diseases and cancer. In a previous work, we developed an in-capillary kinase CE assay where the capillary was used as an enzymatic nanoreactor in which the kinase, its substrate, adenosine 5'-triphosphate (ATP) and its potential inhibitor were mixed by using transverse diffusion of laminar flow profiles (TDLFP). The product adenosine 5'-diphosphate (ADP) was then detected at 254nm and quantified. In this work, this assay was improved to reduce, for the first time, the dilution effect commonly observed with the TDLFP approach. Under the new conditions established herein, IC50 values for quercetin, kaempferol and flavopiridol were successfully obtained and were in the same order of magnitude of those reported in the literature using the conventional assay using radioactive (33)P-ATP. It was shown that aglycone flavonoids have an inhibition activity more important than their glycosylated derivatives. CE was also proved to be very efficient for evaluating inhibition activity of complex samples such as crude extracts of sea buckthorn (SBT) berries obtained by solvent-free microwave extraction (SFME). This novel approach to combine SFME technique to a CE-based enzymatic assay is very interesting for evaluating the biological activity of natural material in a fast, simple, economic (no use of neither fluorescent nor radiometric labels) and green (no organic solvents) manner.