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This work presents the volatile compounds and phenolic profile investigation of the leaves of Brassica oleracea L. growing in Tunisia, together with antioxidant and antibacterial properties. Volatile constituents were determined by HS-SPME coupled to GC/MS, and the results showed that α-pinene (31.6%) and limonene (16.9%) were the main volatiles. The phenolic profile was determined by HPLC analysis, the methanol extract revealed the presence of four hydroxycinnamic acids (chlorogenic, ferulic, p-coumaric and sinapic acids), two hydroxybenzoic acids (syringic and gallic acids), and four flavonoids (catechol, catechin hydrate, epigallocatechin and epicatechin 3-O-gallate). The methanol extract showed the best significantly antiradical activity by DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS ((2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) antioxidant assays, with EC50 of 0.32 and 0.45 mg/mL, respectively. For antibacterial activity, the methanol extract inhibits all the tested strains. It can be concluded that kohlrabi leaves are rich in bioactive compounds and are a potential source of natural antioxidants and antibacterials.

Aim: Despite its therapeutic value almost nothing is known about potential adverse health effects of Olea europea L. We therefore investigated the in vitro toxicity and genotoxicity of leaf extracts of this plant. Material and Methods: Extracts from olive tree leaves were obtained from four different regions in Tunisia. We investigated the in vitro toxicity, genotoxicity and antigenotoxicity of their aqueous extracts using the neutral red (NR) uptake, Vitotox and alkaline comet assays. Results: None of the extracts were found to be toxic and none of them were genotoxic, although some doubt exists for the extract obtained at Meski (North of Tunisia). On the basis of the Vitotox test only, none of the extracts appeared to have antigenotoxic (or cogenotoxic) properties. Discussion: The negative genotoxicity underline the safe use of the leaves, for example, as hypoglycemic and antidiabetic preparations. Lack of antigenotoxicity may indicate that the previously reported anticancer effects do not result from protection against genotoxicity. HIGHLIGHTS We investigated the in vitro toxicity and genotoxicity of aqueous extracts of olivesThe neutral Red Uptake test, Vitotox and alkaline comet assay were usedLeaf extracts from 4 different origins were investigatedNone of them showed in vitro toxicity or genotoxicityThe extracts also didn’t have antigenotoxic properties Abbreviation list: BaP: benzo(α)pyrene, EMS: ethyl methane sulfonate, LMP: low melting point, NI50: 50% inhibition of NRU, NR: neutral red, NRU: neutral red uptake, OD: optical density, PBS: phosphate buffer saline, SDS: sodium dodecyl sulphate, S/N: signal to noise ratio, 4NQO: 4-nitroquinoline oxide

The aim of this study is to evaluate the polyphenolic and flavonoid contents in the leaves extracts of Ruscus hypophyllum. Antioxidant activity was estimated by α,α-diphenyl-β-picrylhydrazyl (DPPH) and 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) assays. The anticoagulant activity of Ruscus extracts was evaluated in vitro, using the prothrombin time (PT) and a PTT-activated partial thromboplastin time tests. The antibacterial activity was tested against large number of important medically and resistant bacteria by the broth dilution method. In this study, ethyl acetate and chloroform extracts displayed the highest total phenols contents (74.76 mg EAG/g and 73.89 mg EAG/g, respectively) and flavonoid content 40 and 32.43 mg EC/g, respectively. The GC-MS analysis of ethyl acetate extract confirmed the presence of oxygenated sesquiterpenes and hydrocarbon diterpenes with percentages of 16.41% and 10.72%, respectively, but chloroform extract was rich with, oxygenated monoterpenes, and oxygenated diterpenes, with percentages of 6.19 and 3.27%, respectively. Among tested extracts, ethyl acetate exhibited the best antioxidant and anticoagulant activities. Furthermore, ethyl acetate and chloroform extracts showed important antibacterial activity against resistant bacteria methicillin-resistant Staphylococcus aureus (SARM), Acinetobacter imipenem-resistant (IMP/R), P. aeruginosa imipenem-resistant (IMP/R) and extended-spectrum beta-lactamase producing E. cloacae (BLSE) with minimal inhibitory concentration (MIC) values varying between 0.125 and 0.5 mg/mL.

Objective: To examine the potential antioxidant and anti-α-glucosidase inhibitory activities of Tunisian Euphorbia paralias L. leaves and stems extracts and their composition of total polyphenol and flavonoids. Methods: The different samples were tested for their antiradical activities by using 2, 2’- azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays. In α-glucosidase activity, α-glucosidase (0.3 IU/mL) and substrate, 2500 µmol/ L p-nitrophenyl α-D-glucopyranoside were used; absorbance was registered at 405 nm. Results: The leaves acetonic extract exhibited the strongest α-glucosidase inhibition [IC50 = (0.0035 ± 0.001) µg/mL], which was 20-fold more active than the standard product (acarbose) [IC50 = (0.07 ± 0.01) µg/mL]. Acetonic extract of the leaves exhibited the highest quantity of total phenolic [(95.54 ± 0.04) µg gallic acid equivalent/mg] and flavonoid [(55.16 ± 0.25) µg quercetin equivalent/mg]. The obtained findings presented also that this extract was detected with best antioxidant capacity [IC50 = (0.015 ± 0.01) µg/mL] against DPPH and a value of IC50 equal to (0.02 ± 0.01) µg/mL against ABTS. Positive relationship between polyphenolic content of the tested Euphorbia paralias L. leaves and stems extracts and its antioxidant activity (DPPH and ABTS) was detected. Elevated positive linear correlation was got between ABTS and total phenolic (R2 = 0.751). Conclusions: The findings clearly demonstrate that the use of a polar solvent enables extraction of significant quantities of phenol compounds and flavonoids.

Consumers are well-informed about food additives and it is likely that they prefer natural additives over their synthetic analogues. Antioxidants represent a major class of food preservatives, among which tocopherols stand out as one of the most important examples. Interestingly, these compounds are present in relevant amounts in the mycelia of in vitro cultured mushrooms. Accordingly, the mycelia from Ganoderma lucidum, Pleurotus ostreatus and Pleurotus eryngii were used as alternative sources of tocopherols. These extracts were incorporated into different yogurt formulations, which were further compared among each other and with yogurts containing commercial α-tocopherol (E307), regarding their nutritional parameters, fatty acid profile and antioxidant activity. The proposed approach was validated as an effective functionalization strategy, particularly in the case of the G. lucidum mycelium, which showed the highest antioxidant potential, most likely as a result of its tocopherol profile. Furthermore, yogurts prepared with each mycelium extract allowed maintaining the nutritional properties observed in the “blank” yogurt formulation. The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2013), L. Barros and J. Barreira contracts. This work is funded by the European Regional Development Fund (ERDF) through the Regional Operational Program North 2020, within the scope of Project NORTE-01-0145-FEDER-023289: DeCodE and Project Mobilizador Norte-01-0247-FEDER-024479: ValorNatural®. info:eu-repo/semantics/publishedVersion

Ruscus hypophyllum L. is a rare Mediterranean plant which is used in the traditional medicine. We studied its phenolic content and in vitro toxicity and genotoxicity using the neutral red uptake (NRU) test, the bacterial Vitotox test, and the comet assay in human C3A hepatic cells. Aqueous leaf and fruit extracts were investigated. Antigenotoxicity against 4-nitroquinoline-oxide (4NQO, 0.4 mu g/mL) and Benzo(alpha) pyrene (BaP, 800 mu g/mL) was also investigated with the Vitotox test. The extracts appeared to be genotoxic only at high exposure levels in the comet assay. There was no indication of a genotoxic activity in the Vitotox test and also no indication of antigenotoxicity. The moderate polyphenol content may provide an explanation for the absence of antigenotoxicity.

A phytochemical investigation of the Ferula lutea (Poir.) Maire flowers has led to the isolation of a new compound, (E)-5-ethylidenefuran-2(5H)-one-5-O-β-D-glucopyranoside (1), designated ferunide, 4-hydroxy-3-methylbut-2-enoic acid (2), reported for the first time as a natural product, together with nine known compounds, verbenone-5-O-β-D-glucopyranoside (3), 5-O-caffeoylquinic acid (4), methyl caffeate (5), methyl 3,5-O-dicaffeoylquinate (6), 3,5-O-dicaffeoylquinic acid (7), isorhamnetin-3-O-α-L-rhamnopyranosyl(1→6)-β-D-glucopyranoside, narcissin (8), (−)-marmesin (9), isoimperatorin (10) and 2,3,6-trimethylbenzaldehyde (11). Compounds 3–10 were identified for the first time in Ferula genus. Their structures were elucidated by spectroscopic methods, including 1D and 2D NMR experiments, mass spectroscopy and X-ray diffraction analysis (compound 2), as well as by comparison with literature data. The antioxidant, anti-inflammatory and cytotoxic activities of isolated compounds were evaluated. Results showed that compound 7 exhibited the highest antioxidant activity with IC50 values of 18 ± 0.5 µmol/L and 19.7 ± 0.7 µmol/L by DPPH radical and ABTS radical cation, respectively. The compound 6 exhibited the highest anti-inflammatory activity with an IC50 value of 5.3 ± 0.1 µmol/L against 5-lipoxygenase. In addition, compound 5 was found to be the most cytotoxic, with IC50 values of 22.5 ± 2.4 µmol/L, 17.8 ± 1.1 µmol/L and 25 ± 1.1 µmol/L against the HCT-116, IGROV-1 and OVCAR-3 cell lines, respectively.

Genetically modified organisms (GMO) invade more and more the agricultural production in the world. Although there are no legislations on GM labeling and cultivation of GM crops in Tunisia, the present study aims to check the status of GMO in Tunisian market using qualitative and quantitative real time-PCR (QRT-PCR). Three-hundred-sixty five samples were collected and different DNA extraction methods were adapted and optimized. Specific primers targeting 35S promoter from Cauliflower mosaic virus (CaMV) and nopaline synthase terminator from Agrobacterium tumefaciens (At) were used for the detection of the GMO insert and Taxon specific primers for the detection of plant species. Validated Taqman® probes (EU-RL) targeting event specific regions of the maize events MON810, Bt11, and the soybean event RRS were used for the quantification studies. Seven food and feed products showed different amounts of RRS (1.9%), MON810 (2.1%), and Bt11 (1.6%). The results demonstrate for the first time the presence of GMO in Tunisian markets reinforcing the need for the development of accurate quantitative methods in routine analyses.

Objective: To investigate the antioxidant and α-glucosidase properties and phytochemical constituents of roots, stems, leaves and flowers extracts and aerial parts oil of Chrysanthoglossum trifurcatum (Desf.) (C. trifurcatum). Methods: For extraction from roots, stems, leaves and flowers of C. trifurcatum, methanol, ethyl acetate and petroleum ether solvents were used. Phenols, flavonoids, flavonols and tannins contents were evaluated. More, C. trifurcatum aerial parts oil composition was determined using chromatography/mass spectrometry. The antioxidant effect was estimated by DPPH, ABTS and reducing power test systems. The α-glucosidase inhibition was determined by colorimetric assay using the enzyme from Aspergillus niger and the p-nitrophenyl glucopyranoside (pNPG) as substrate. Results: The highest amounts of polyphenols, flavonoids, flavonols and tannins were shown by the methanolic extract of leaves. The main components of the aerial parts oil were limonene (29.21%), γ -terpinene (12.96%), 4-terpenyl acetate (12.18%) and α -pinene (5.76%). The activity evaluated by DPPH, ABTS and reducing power tests was important for stems (IC50=0.68 mg/mL) and flowers (IC50=0.67 mg/mL) methanolic extracts and essential oil (IC50=0.72 mg/mL). Findings of α-glucosidase activity revealed that petroleum ether extracts of leaves and roots together with aerial parts oil showed a highest activity with IC50 of 0.044, 0.045 and 0.049 mg/mL, respectively. Conclusions: Observed antioxidant and α-glucosidase activities of oil and extracts are attributed to the presence of the active phytochemicals in C. trifurcatum organs. Thus, the C. trifurcatum can be used as a source of antioxidant compounds and dietary supplement to treat patients with type 2 diabetes.

The essential oils from the leaves, stems and roots of Chrysanthemum trifurcatum (Desf.) Batt. and Trab. var. macrocephalum (viv.) were obtained by hydrodistillation and their chemical compositions were analysed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS), in order to get insight into similarities and differences as to their active composition. A total of fifty compounds were identified, constituting 97.84%, 99.02% and 98.20% of total oil composition of the leaves, stems and roots, respectively. Monoterpene hydrocarbons were shown to be the main group of constituents of the leaves and stems parts in the ratio of 67.88% and 51.29%, respectively. But, the major group in the roots oil was found to be sesquiterpene hydrocarbons (70.30%). The main compounds in leaves oil were limonene (26.83%), γ-terpinene (19.68%), α-pinene (9.7%) and α-terpenyl acetate (7.16%). The stems oil, contains mainly limonene (32.91%), 4-terpenyl acetate (16.33%) and γ-terpinene (5.93%), whereas the main compounds in roots oil were α-calacorene (25.98%), α-cedrene (16.55%), β-bourbobene (14.91%), elemol (7.45%) and 2-hexenal (6.88%). The crude organic extracts of leaves, stems and roots, obtained by maceration with solvents of increasing polarity: petroleum ether, ethyl acetate and methanol, contained tannins, flavonoids and alkaloids. Meanwhile, essential oils and organic extracts were tested for antibacterial activities against eight Gram-positive and Gram-negative strains, using a microdilution method. The oil and methanolic extact from C. trifurcatum leaves showed a great potential of antibacterial effect against Bacillus subtilis and Staphylococcus epidermidis, with an IC50 range of 31.25-62.5 µg/ml.

KEY MESSAGE : Here, we describe a new developed quantitative real-time PCR method for the detection and quantification of a new specific endogenous reference gene used in GMO analysis. The key requirement of this study was the identification of a new reference gene used for the differentiation of the four genomic sections of the sugar beet (Beta vulgaris L.) (Beta, Corrollinae, Nanae and Procumbentes) suitable for quantification of genetically modified sugar beet. A specific qualitative polymerase chain reaction (PCR) assay was designed to detect the sugar beet amplifying a region of the adenylate transporter (ant) gene only from the species of the genomic section I of the genus Beta (cultivated and wild relatives) and showing negative PCR results for 7 species of the 3 other sections, 8 related species and 20 non-sugar beet plants. The sensitivity of the assay was 15 haploid genome copies (HGC). A quantitative real-time polymerase chain reaction (QRT-PCR) assay was also performed, having high linearity (R (2) 0.994) over sugar beet standard concentrations ranging from 20,000 to 10 HGC of the sugar beet DNA per PCR. The QRT-PCR assay described in this study was specific and more sensitive for sugar beet quantification compared to the validated test previously reported in the European Reference Laboratory. This assay is suitable for GMO quantification in routine analysis from a wide variety of matrices.