Integrated Supercritical Fluid Extraction and Pre-Formulation Process of Punica granatum L. Pericarp Polar Compounds.

Pomegranate (Punica granatum L.) is a widely used fruit in the dietary supplement industry due to its richness in bioactive compounds. In this study, an experimental design was applied to optimize supercritical fluid extraction (SFE) of polar compounds of interest (ellagic acid and punicalagins), known for antioxidant and skin care properties from pomegranate's pericarp. The effects of temperature, modifier percentage, and water additive percentage added in the modifier were explored through a Box-Behnken design, followed by a study of the extraction kinetics. The results indicated that 40 °C, 20% EtOH:H2O 80:20 v:v, with an extraction duration of 60 min allowed for the highest recovery of the above-mentioned molecules (19.59 mg/g). Due to solubilization issues encountered by the extract, a screening of cosmetic solvents was carried out to solubilize SFE pomegranate extracts and a composition of Gly:H2O 80:20 v:v was selected. Furthermore, an integrated SFE pre-formulation process of pomegranate pericarp extract (PPE) was elaborated. This allowed for the recovery of the extracts in cosmetic solvent, avoiding a full evaporation. Finally, the stability of the pre-formulated extracts was evaluated and showed high stability for over 3 months at 5 °C.

Exploring the Sequential-Selective Supercritical Fluid Extraction (S3FE) of Flavonoids and Esterified Triterpenoids from Calendula officinalis L. Flowers.

One of the many advantages of supercritical fluid extraction (SFE) is the possibility of using it in sequential and selective approaches. This is due to the use of a dynamic extraction mode in addition to the possibility of altering the composition of the modifier during the extraction process. In this study, the optimization of Calendula officinalis L. extraction of non-polar and polar compounds was achieved using three-level Box-Behnken designs (BBD). For non-polar compounds, the factors were pressure, temperature, and EtOH percentage. As for the polar compounds, the three variables were temperature, the total modifier percentage, and H2O added in the modifier as an additive. The recovery of selectively rich extracts in triterpendiol esters and narcissin was possible using a sequential two-step SFE. The first step was performed at 80 °C and 15% EtOH, and the second at 40 °C and 30% EtOH:H2O 80:20 v:v with a total of 60 min of extraction. Additionally, the SFE extraction of non-polar compounds was scaled up on a pilot-scale extractor, demonstrating similar results. Finally, the SFE results were compared to ultrasound-assisted extraction (UAE).

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 UHPLC-Q-TOF-HRMS based determination of discrimating compounds for oak species Quercus robur L. and Quercus petraea Liebl. identification.

INTRODUCTION: Two species of oak are dominant in French forests: pedunculate oak (Quercus robur L.) and sessile oak (Quercus petraea Liebl.). Differentiating oak species is difficult, since features such as morphological characters, geographical origin and grain are not always relevant. Even if the former is generally richer in tannin compounds while the latter is often richer in aromatic compounds, the intra-species variability is high. The characterisation of the oak species remains a suitable indicator of the molecular composition and quality of the wood. OBJECTIVES: The aim of this study was to determine differentiating molecules allowing oak species identification in order to assist in a suitable wood selection for a better oak tree valorisation since the selection of the oak wood to be used in the production of barrels plays an essential role in wine ageing. MATERIALS AND METHODS: Oak wood samples were collected both in forests and in cooperage timber yards. An untargeted metabolomic approach using ultra-high-pressure liquid chromatography qualitative time-of-flight high-resolution mass spectrometry (UHPLC-Q-TOF-HRMS) associated to multivariate statistical analyses (hierarchical ascendant clustering and partial least squares discriminant analysis) was implemented to determine molecular markers of oak species. RESULTS: Heartwood was identified as the suitable wood part to distinguish oak species. Discriminating molecules did not depend on the sample set. The pedunculate species showed overexpression of bartogenic derivatives while sessile oak presented a higher content in oak lactone precursors and in quercotriterpenosids. CONCLUSION: The developed method allowed the identification of relevant compounds for oak species identification to a better wood valorisation and selection.

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.

Evaluation of the extraction and stability of chlorophyll-rich extracts by supercritical fluid chromatography.

In this study, a rapid (less than 10 min) analytical method by reverse-phase supercritical fluid chromatography was developed with an isocratic mobile phase, enabling the separation of 11 compounds, chlorophyll a and b, pheophytin a and numerous allomers or epimers. This method was used to examine the stability of chlorophyll pigments of plant extracts obtained with various extraction methods including microwave-assisted extraction (MAE), supercritical fluid extraction (SFE), pressurized liquid extraction (PLE) and ultrasound-assisted extraction (UAE), with ethanol as solvent or modifier. The effect of storage was studied for both liquid and dried extracts. Irrespective of the extraction method, the evaporation of the extracts induced partial degradation of the chlorophyll pigments. It was found that liquid extracts could be stored at 4 °C for 3 weeks without a dramatic change in allomer forms of chlorophylls. However, during this storage period, epimerization appears to be important, leading to a significant decrease in the chlorophyll b native form. Graphical abstract.

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.

Different Ways to On-Line Hyphenate Centrifugal Partition Chromatography and Mass Spectrometry: Application to Prenylated Xanthones from Garcinia mangostana.

Centrifugal partition chromatography is a liquid-liquid separation method well adapted for the fractionation or purification of natural compounds from plant extracts. However, following the preparative isolation, the fractions collected must be analysed by high-performance thin-layer chromatography or high-performance liquid chromatography to evaluate their composition and/or their purity. These additional steps are time-consuming and increase the risk of compound degradation. In order to get an instantaneous analysis of fraction content with structural information on the phytochemicals eluted, it is possible to hyphenate on-line centrifugal partition chromatography with mass spectrometry. Depending on the complexity of the extract, two different kinds of centrifugal partition chromatography-mass spectrometry coupling can be performed: centrifugal partition chromatography-mass spectrometry or centrifugal partition chromatography-high-performance liquid chromatography-mass spectrometry coupling. In the first case, one part of the centrifugal partition chromatography effluent is directly introduced in the mass spectrometry ionisation source to identify the eluted compounds, while in the second case, it is directed to a high-performance liquid chromatography-mass spectrometry system where compounds are first separated thanks to high-performance liquid chromatography and then identified using mass spectrometry.

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.

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.

Intensification of the SFE Using Ethanol as a Cosolvent and Integration of the SFE Process with sc-CO2 Followed by PLE Using Pressurized Ethanol of Black Soldier Fly (Hermetia illucens L.) Larvae Meal-Extract Yields and Characterization.

The objective of this research was to investigate and compare the results obtained in the intensification and integration of (sc-CO2) under different pressure conditions (25 and 30 MPa) at 60 °C. When intensifying the process, ethanol (10%) was used as a co-solvent (sc-CO2 + EtOH). In the process integration, black soldier fly larvae flour, defatted via supercritical extraction (SFE), was the raw material for pressurized liquid extraction (PLE) using ethanol as solvent. The extract yields, fatty acid profile, free fatty acids, triacylglycerols (TAGs), oxidative stability, and nutritional quality of the oil obtained using sc-CO2 + EtOH were evaluated. The composition of bioactive compounds (carotenoids, acidity, antioxidant compounds, tocopherols, and phospholipids) was determined in both extracts. The yields of the extracts were different by 32.5 to 53.9%. In the extracts obtained with sc-CO2 + EtOH (10%), the predominant fatty acids were oleic, palmitic, and linoleic, with considerable levels of desirable fatty acids (DFA), tocopherols, and phospholipids. The nutritional indices showed good values for polyunsaturated and saturated fatty acids (PUFAs/SFAs), above 0.45%. Extracts from larvae meal defatted with SFE showed carotenoids, phenolic compounds, and antioxidant activity. HPTLC and HPLC analyses indicated the presence of amino acids, sugars, phenolics, and organic acids in their composition. This study revealed that the supercritical fluid extraction (SFE) process, or its conditions, can modify the fatty acid composition and the presence of minor bioactive compounds in the obtained extracts.

Oil/Water Biphasic Solvent System for the Eco-Extraction and Cosmetic Formulation of Bixa orellana L.

Annatto, obtained from the seeds of achiote (Bixa orellana L.), is a widely used orange pigment rich in bixin and other apocarotenoids. This work reports the optimisation of a green extraction method of pigments and antioxidant compounds from achiote as well as its integration in a one-step green extraction-cosmetic formulation process. A biphasic solvent system of water and oil was used to recover simultaneously polar polyphenols, and less polar compounds, such as δ-tocotrienol and bixin. The optimisation of the ultrasound assisted extraction is presented, as well as a comparison of different vegetable oils used as extraction solvents. The composition, physicochemical properties and antioxidant activity of the oils were studied and their extraction performance was compared. Refined sunflower oil proved to be a better solvent than virgin olive, jojoba, coconut and grapeseed oils. Both aqueous and oil phases displayed an interesting antioxidant capacity. The oil phase contained 0.9% of bixin, as well as minor apocarotenoids and δ-tocotrienol. Twelve compounds, mainly phenolics, were identified by UHPLC-DAD-HRMS/MS in the aqueous phase. Twenty-one volatile compounds were identified in the volatile fraction by SPME-GC-MS. Lastly, a one-step green process is proposed to combine the extraction and the cosmetic formulation of the bioactive compounds.

Characterization of Corn Silk Extract Using HPLC/HRMS/MS Analyses and Bioinformatic Data Processing.

In addition to having different biological activities of interest, corn silks play a role in the defense of plants. While benzoxamines and flavonoids have already been identified as molecules of plant defense and growth mechanisms, knowledge on the phytochemical composition of corn silk is lacking. Such knowledge would make it possible to better select the most effective varieties to improve resistance or bioactive properties. In this article, an approach was implemented to map a corn silk extract in two complementary ways. The first one involved working with UHPLC/HRMS data and Kendrick and van Krevelen plots to highlight a homologous series of compounds, such as lipids from 17 to 23 carbons, monoglycosylated flavonoids from 21 to 24 carbons, diglycosylated flavonoids of 26 to 28 carbons and organic acids of 14 to 19 carbons. The second way was to analyze the sample in UHPLC/HRMS2 and to plot mass spectral similarity networks with the GNPS platform and Cytoscape software to refine identification. By combining the information obtained, we were able to propose an identification for 104 detected molecules, including 7 nitrogenous, 28 lipidic and 67 phenolic compounds, leading to the first detailed phytochemical analysis of corn silk extract.

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.

New "hyphenated" CPC-HPLC-DAD-MS strategy for simultaneous isolation, analysis and identification of phytochemicals: application to xanthones from Garcinia mangostana.

Centrifugal partition chromatography (CPC) coupled online with high-performance liquid chromatography (HPLC) with diode-array detection (DAD) and mass spectrometry (MS) is presented in this work. This strategy offers the possibility to obtain simultaneously CPC fractionation of natural extracts, the HPLC fingerprint of separated fractions and structural information on molecules contained in each fraction. This new approach was applied to the fractionation and purification of xanthones from Garcinia mangostana (Clusiaceae) pericarp. A biphasic solvent system of heptane/ethyl acetate/methanol/water (2:1:2:1, v/v) was used for the CPC separation of 175 mg crude ethanolic extract. The HPLC analysis was conducted with a reversed-phase monolithic column allowing fast and repeatable separation. This combined CPC-HPLC-DAD-MS method led to isolation of 33 mg α-mangostin and 6 mg γ-mangostin at 98% and 98.5% purity, respectively, in 140 min. Furthermore, in the same time a total of 16 other xanthones were detected in the extract, and ten of them were identified on the basis of their UV and MS spectra.

Screening and Evaluation of Dermo-Cosmetic Activities of the Invasive Plant Species Polygonum cuspidatum.

Polygonum cuspidatum (P. cuspidatum) is among the world's most problematic invasive plant species with negative ecological, socio-economic and security consequences. Management operations in areas invaded systematically generate a large quantity of plant waste, most often without outlets. Using this plant material could constitute a new alternative treatment for sustainable management. P. cuspidatum is well known to have numerous biological properties, containing notably stilbenes, quinones, flavonoids and phenolic acids. The present work proposes a reliable strategy using powerful techniques for the screening and the evaluation of the dermo-cosmetic potential of its aerial parts (AP) and root parts (RP). To the best of our knowledge, only antioxidant and anti-tyrosinase activities were previously evaluated on P. cuspidatum among the targets studied (superoxide dismutase, hyaluronidase, elastase, collagenase and tyrosinase). The results revealed strong antioxidant and anti-collagenase activities, moderate anti-hyaluronidase activity, while weak anti-elastase and anti-tyrosinase activities were observed for ethanolic extracts. Different standards selected and screened on the same targets made it possible to correlate the observed residual activities of produced extracts of P. cuspidatum from Savoie Mont Blanc and their chemical compositions. A structure-activity study was thus conducted on main molecular families, widely represented in the genus Polygonum.

Oak Species Quercus robur L. and Quercus petraea Liebl. Identification Based on UHPLC-HRMS/MS Molecular Networks.

Two species of oak are dominant in French forests: pedunculate oak (Quercus robur L.) and sessile oak (Quercus petraea Liebl.). Their differentiation is not straightforward but is essential to better understand their respective molecular content in order to better valorize them. Thus, to improve oak species identification, an untargeted UHPLC-HRMS/MS method associated with a two-step data treatment was developed to analyze a wide range of specialized metabolites enabling the comparison of both species of oak extracts. Pooled extracts from sessile and pedunculate oaks, composed of extracts from several trees of pure species from various origins, were compared using first the Venn diagram, as a quick way to get an initial idea of how close the extracts are, and then using a molecular network to visualize, on the one hand, the ions shared between the two species and, on the other hand, the compounds specific to one species. The molecular network showed that the two species shared common clusters mainly representative of tannins derivatives and that each species has specific molecules with similar fragmentation patterns, associated in specific clusters. This methodology was then applied to compare these two pooled extracts to unknown individuals in order to determine the species. The Venn diagram allowed for the quick presumption of the species of the individual and then the species could be assigned more precisely with the molecular network, at the level of specific clusters. This method, developed for the first time, has several interests. First, it makes it possible to discriminate the species and to correctly assign the species of unknown samples. Moreover, it gave an overview of the metabolite composition of each sample to better target oak tree utilization and valorization.

Selective extraction of bioactive compounds from plants using recent extraction techniques: A review.

Plant extraction has existed for a long time and is still of interest. Due to technological improvements, it is now possible to obtain extracts with higher yields. While global yield is a major parameter because it assesses the extraction performance, it can be of interest to focus on the extraction of particular compounds (specific metabolites) to enrich the sample and to avoid the extraction of unwanted ones, for instance the primary metabolites (carbohydrates, triacylglycerols). The objective then is to improve extraction selectivity is then considered. In solid-liquid extraction, which is often called maceration, the solvent has a major impact on selectivity. Its polarity has a direct influence on the solutes extracted, related to the chemical structure of the compounds, and modelling compound/solvent interactions by using various polarity or interaction scales is a great challenge to favor the choice of the appropriate extracting liquid. Technical advances have allowed the development of recent, and sometimes green, extraction techniques, such as Microwave-Assisted Extraction (MAE), Ultrasound-Assisted Extraction (UAE), Pressurized Liquid Extraction (PLE) and Supercritical Fluid Extraction (SFE). This review focuses on the specificity of these recent techniques and the influence of their physical parameters (i.e. pressure, intensity, etc.). In addition to the solvent selection, which is of prime interest, the physical parameters applied by the different techniques influence the extraction results in different ways. Besides, SFE is a versatile and green technique suitable to achieve selectivity for some compounds. Due to its properties, SC-CO2 allows tailoring conditions to improve the selectivity.

Sequential extraction of carnosic acid, rosmarinic acid and pigments (carotenoids and chlorophylls) from Rosemary by online supercritical fluid extraction-supercritical fluid chromatography.

A high degree of selectivity is required during the plant extraction process in order to obtain extracts enriched in specific compounds or to avoid the extraction of unwanted ones. Rosemary is well known for its antioxidant compounds (carnosic acid, carnosol and rosmarinic acid). The plant also contains pigments (i.e. carotenoids, chlorophylls) which may cause a colour problem during the use of the extract in cosmetic formulations, for example. Supercritical fluid extraction is considered as a selective technique for plant extraction. Due to the physico-chemical properties of supercritical fluids, related to pressure, temperature and modifier addition, it is possible to carry out sequential extraction with successive conditions to collect different fractions that are rich either in pigments or in bioactive compounds. The aim of this study was to selectively extract bioactive compounds (i.e. carnosic acid and rosmarinic acid) and pigments (carotenoids and chlorophylls) from rosemary using supercritical fluid extraction. The optimisation of the extraction method was carried out using supercritical fluid extraction online coupled with a supercritical fluid chromatography (SFE-SFC) system. Two columns of different polarities were coupled to achieve the separation of the targeted compounds every five minutes during the extraction. Four fractions were obtained: a first one rich in carotenoids obtained with pure CO2 (25°C and 20 MPa), a second rich in carnosic acid obtained with 3% polar modifier (EtOH:water 50/50 v/v), a third fraction rich in rosmarinic acid using 10% of the same modifier and a fourth fraction rich in chlorophylls with 30% of ethanol as modifier. These four samples were then analysed by UHPLC-DAD-ESI-QTOF-HRMS in order to identify other extracted compounds and to study how the selected conditions impacted their extraction.