Thomas Michel

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.

Estrogenic effects of Ficus umbellata Vahl. (Moraceae) extracts and their ability to alleviate some menopausal symptoms induced by ovariectomy in Wistar rats

ETHNOPHARMACOLOGICAL RELEVANCE Ficus umbellata Vahl. (Moraceae) is a medicinal plant used in Cameroon to treat amenorrhea as well as other physiological disorders related to menopause. AIM OF STUDY In order to justify scientifically its traditional use, the estrogen-like properties of the aqueous (AE) and methanol (MeOH) extracts of F. umbellata were investigated. MATERIAL AND METHODS In vitro, the ability of different extracts of F. umbellata to activate estrogen receptors α (ERα) and β (ERβ) in cell-based reporter gene assays using human embryonic kidney (HEK293T) cells transfected with ERs was tested. In vivo, a 3-day uterotrophic assay and the capacity of the extracts to alleviate hot flushes in ovariectomized adult rats were tested. Using a bioassay-guided fractionation the major compound of F. umbellata was isolated and tested in vitro on HEK293T-ERα and ERβ cells. RESULTS AE and MeOH extracts significantly altered ERα as well as ERβ activities. In vivo, both extracts significantly increase the uterine and vaginal epithelium thickness, and uterine total protein levels in a dose dependent manner. Interestingly, both extracts of F. umbellata at the dose of 100 mg/kg BW significantly decreased the total number, average duration as well as frequency of hot flushes in experimental rats compared to age-matched OVX controls. Finally, 7-methylumbelliferone, a coumarin was characterized as the major compound of F. umbellata; however this compound did not transactivate ERα as well ERβ in vitro. CONCLUSION These aforementioned results suggest that F. umbellata extracts as used by the traditional practitioner have estrogen-like effects and may alleviate some menopausal problems such as vaginal dryness and hot flushes.

Crateva adansonii DC, an African ethnomedicinal plant, exerts cytotoxicity in vitro and prevents experimental mammary tumorigenesis in vivo

ETHNOPHARMACOLOGICAL RELEVANCE Crateva adansonii DC is a plant traditionally used in Cameroon to treat constipation, asthma, snakebites, postmenopausal complaints and cancers. AIM The anticancer potential of the dichloromethane/methanol extract of C. adansonii stem barks was investigated using human breast cancer cell and 7,12 dimethylbenz(a)anththracene (DMBA)-induced mammary tumorigenesis model in rats. MATERIAL AND METHODS The cytotoxicity of C. adansonii extract was assessed in vitro towards breast carcinoma (MCF-7 and MDA-MB-231) and non-tumoral cell lines (NIH/3T3 and HUVEC) by Alamar Blue assay. Furthermore, in vivo studies were performed on female Wistar rats treated either with C. adansonii extract at a dose of 75 or 300mg/kg body weight or with tamoxifen (3.3mg/kg body weight), starting 1 week prior DMBA treatment and lasted 12 weeks. The investigation focused on tumour burden, tumour DNA fingerprint, morphological, histological, hematological, and biochemical parameters. RESULTS CC50 values for the in vitro assays were 289µg/mL against MCF-7 cells and >500µg/mL in others cells, leading to a selectivity index ≥1.73. C. adansonii extract significantly (p<0.001) revealed in vivo the reduction of the cumulative tumour yield (87.23%), total tumour burden (88.64%), average tumour weight (71.11%) and tumour volume (78.07%) at the dose of 75mg/kg as compared to DMBA control group. A weak effect was also observed at 300mg/kg. This extract showed a moderate hyperplasia at the dose of 75mg/kg while at 300mg/kg no significant change was noted as compared to DMBA group. It protected rats from the DNA alteration induced by DMBA and increased antioxydant enzymes activities in mammary gland tissue homogenates. In addition, Ultra-High Performance Liquid Chromatography/ESI-QTOF-Mass Spectrometry analysis of C. adansonii extract detected structure-related of many well-known anticancer agents such as flavane gallate, flavonol, phenylpropanoïds, sesquiterpene derivatives, gallotannins and lignans. The LD50 of C. adansonii was estimated to be greater than 5000mg/kg. CONCLUSIONS These aforementioned results suggest that the C. adansonii extract may possess antitumor constituents, which could combat breast cancer and prevent chemically-induced breast cancer in rats.

Characteristics, Phytochemical Analysis and Biological Activities of Extracts from Tunisian Chetoui Olea europaea Variety

This study selected 10 extracts from Tunisian chetoui O. europaea variety for their total phenolics, flavonoids, and phytochemical analyses as well as for their antioxidant and antimicrobial activities determination. The in vitro antioxidant property was investigated using DPPH, ferric reducing antioxidant capacity (FRAP), oxygen reducing antioxidant capacity (ORAC), and β-carotene-linoleic acid bleaching assays while antimicrobial activity was evaluated using macrodilutions method. For all organs of chetoui O. europaea variety, the investigated activities were found to be higher in the polar extracts (ethyl acetate, methanol, and methanol/water). These activities were correlated with the presence of phenolic compounds. Phytochemical analyses revealed that the crude extracts contain triterpenoids, quinones, and flavonoids. High performance liquid chromatography (HPLC) and high performance thin layer chromatography (HPTLC) confirmed the presence of phenolic compounds in the studied extracts.

Increased water salinity applied to tomato plants accelerates the development of the leaf miner Tuta absoluta through bottom-up effects

Variation in resource inputs to plants may trigger bottom-up effects on herbivorous insects. We examined the effects of water input: optimal water vs. limited water; water salinity: with vs. without addition of 100 mM NaCl; and their interactions on tomato plants (Solanum lycopersicum), and consequently, the bottom-up effects on the tomato leaf miner, Tuta absoluta (Meytick) (Lepidoptera: Gelechiidae). Plant growth was significantly impeded by limited water input and NaCl addition. In terms of leaf chemical defense, the production of tomatidine significantly increased with limited water and NaCl addition, and a similar but non-significant trend was observed for the other glycoalkaloids. Tuta absoluta survival did not vary with the water and salinity treatments, but the treatment “optimal water-high salinity” increased the development rate without lowering pupal mass. Our results suggest that caution should be used in the IPM program against T. absoluta when irrigating tomato crops with saline water.

CHAPTER 4:Microwave‐assisted Extraction

Scientific knowledge about the use of natural products with functional properties, consumer demand for high‐quality products, as well as legislative actions to reduce the use of synthetic food additives have led to an increase in the use of products from natural sources. This chapter aims to provide context information about the potential commercial uses of extracts from natural sources obtained by various extraction techniques, illustrating their importance in industry and identifying the scope of the techniques and aspects discussed in the following chapters. The main commercial applications of extracts from natural sources discussed here comprise their use as colorants, functional food, nutraceuticals, flavors and fragrances, preserving agents, and edible/non‐edible oils and fats applied to cosmetic, pharmaceutical and food industries. However, due to their nature, natural products are complex and therefore several aspects must be considered for each application, including importance, chemical characteristics of main compounds present and possible mechanisms of action.

Ficus umbellata Vahl. (Moraceae) Stem Bark Extracts Exert Antitumor Activities In Vitro and In Vivo

A Ficus umbellata is used to treat cancer. The present work was therefore designed to assess antitumor potentials of F. umbellata extracts in nine different cell lines. Cell cycle, apoptosis, cell migration/invasion, levels of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), caspases activities as well as Bcl-2 and Bcl-xL protein content were assessed in MDA-MB-231 cells. The 7,12-dimethylbenz(a)anthracene (DMBA)-induced carcinogenesis in rats were also used to investigate antitumor potential of F. umbellata extracts. The F. umbellata methanol extract exhibited a CC50 of 180 μg/mL in MDA-MB-231 cells after 24 h. It induced apoptosis in MCF-7 and MDA-MB-231 cells, while it did not alter their cell cycle phases. Further, it induced a decrease in MMP, an increase in ROS levels and caspases activities as well as a downregulation in Bcl-2 and Bcl-xL protein contents in MDA-MB-231 cells. In vivo, F. umbellata aqueous (200 mg/kg) and methanol (50 mg/kg) extracts significantly (p < 0.001) reduced ovarian tumor incidence (10%), total tumor burden (58% and 46%, respectively), average tumor weight (57.8% and 45.6%, respectively) as compared to DMBA control group. These results suggest antitumor potential of F. umbellata constituents possibly due to apoptosis induction mediated through ROS-dependent mitochondrial pathway.

New insights in the chemical composition of benzoin balsams.

Benzoin balsam is an anthropic exudate obtained from the bark of several species of Styrax trees that is mainly used as a perfume fixative as well as a flavouring agent. Benzoe tonkinensis Laos (also commercialized under the denomination Siam benzoin balsam) displaying characteristic vanilla notes and already being largely used to flavour all kinds of edible goods, was intended to be proposed by Agroforex Company to the Codex Committee on Food Additives for evaluation as a food additive. For this purpose, the present paper reports the phytochemical characterisation of both the volatile and non-volatile fractions of benzoin balsams and the quantitation of some of the major components by gas and liquid chromatography techniques. Four coniferyl and two morinol derivatives were characterised for the first time in Benzoe tonkinensis Laos. Finally, two liquid chromatographic methods used to easily discriminate Siam from Sumatra balsam (also known as Benzoe sumatranus Indonesia) were developed.

Essential oil composition and biological activities of Petiveria alliacea L. from Martinique

The volatile constituents obtained from the essential oil using hydrodistillation of the aerial parts of Petiveria alliacea L. growing in Martinique (F.W.I.: French West Indies), were examined by a combination of gas chromatography/flame ionization detection (GC/FID) and GC–mass spectrometry (GC–MS) techniques. Fifty-one components representing 89.1–98.1% of the essential oil total GC/FID chromatogram were identified. The most abundant compounds were toluenethiol (2.3–23.0%), phytol (6.4–40.0%) dibenzyldisulfide (13.2–35.3%) and benzaldehyde (0.8–31.3%). In total, forty new components were characterized in P. alliacea oils. Higher content in sulfur compounds were quantified in P. alliacea oils collected during the rainy season than those collected during the dry season. Antimicrobial activity of the essential oil from plants collected in Prêcheur area was also investigated. Antibacterial and antifungal activities were observed against Aspergillus niger, Candida albicans, Staphylococcus aureus, Escherichia coli, Salmonella arizonae and Listeria innocua at 0.5% and against resistant strains of E. coli (J96) and S. aureus (1799) at 1.0%.

UHPLC-HRMS/MS Based Profiling of Algerian Lichens and Their Antimicrobial Activities

Lichens are complex symbiotic organisms able to produce a vast array of compounds. The Algerian lichen diversity has only prompted little interest even given the 1085 species listed. Herein, the chemodiversity of four Algerian lichens including Cladonia rangiformis, Ramalina farinaceae, R. fastigiata, and Roccella phycopsis was investigated. A dereplication strategy, using ultra high performance liquid chromatography‐high resolution‐electrospray ionization‐mass spectrometry (UHPLC‐HRMS/MS), was carried out for a comprehensive characterization of their substances including phenolics, depsides, depsidones, depsones, dibenzofurans, and aliphatic acids. Some known compounds were identified for the first time in some species. Additionally, the lichenic extracts were evaluated for their antifungal and antimicrobial activities on human pathogenic strains (Candida albicans, C. glabrata, Aspergillus fumigatus, Staphylococcus aureus, and Escherichia coli). Cyclohexane extracts were found particularly active against human pathogenic fungi with MIC80 values ranging from 8 to 62.5 μg/mL, without cytotoxicity. This study highlights the therapeutic and prophylactic potential of lichenic extracts as antibacterial and antifungal agents.