Contribution of Phenolic Compounds to the Antioxidant Activity of Leaf and Flower Extracts of Sinapis pubescens L. subsp. pubescens (Brassicaceae).

Within a study focused on Sinapis pubescens subsp. pubescens wild from Sicily (Italy), an edible species still unexplored, our earlier published work has demonstrated good in vitro antioxidant properties for the flower and leaf hydroalcoholic extracts, exhibiting quite different qualitative-quantitative phenolic profiles. Herein, further research was designed to elucidate the role played by phenolic compounds in the different antioxidant mechanisms highlighted for the extracts. To achieve this goal, the crude extracts were subjected to liquid-liquid partitioning with solvents of increasing polarity; then, the fractions were investigated for their antioxidant properties using different in vitro assays. For both flowers and leaves, the ethyl acetate fractions exhibited the best activity in DPPH and reducing power assays, followed by n-butanol. The total phenolic content determination indicated these fractions as the phenolic-rich ones, which were characterized by HPLC-PDA/ESI-MS analysis. Conversely, the phenolic-rich fractions did not show any chelating activity, which was highlighted for the more hydrophobic ones.

Comparative Study on Phenolic Profile and Biological Activities of the Aerial Parts of Sinapis pubescens L. subsp. pubescens (Brassicaceae) Wild from Sicily (Italy).

This work aimed to investigate Sinapis pubescens subsp. pubescens spontaneously grown in Sicily (Italy) as new potential source of active metabolites; specifically, a comparative study on leaf, flower, and stem hydroalcoholic extracts was performed. Polyphenols were quantitatively determined by spectrophotometric methods and characterized by HPLC-PDA/ESI-MS; a total of 55 polyphenolic compounds were identified, highlighting considerably different qualitative-quantitative profiles. The extracts showed antioxidant activity, evaluated by in vitro assays; particularly, the leaf extract displayed the best radical scavenging activity (DPPH test) and reducing power, while the flower extract showed the greatest chelating activity. The antimicrobial properties of the extracts were investigated against bacteria and yeasts by standard methods; no antimicrobial activity was found against the strains tested. The extracts resulted to be non-toxic after preliminary toxicity evaluation by the Artemia salina lethality bioassay. The aerial parts of S. pubescens subsp. pubescens proved to be valuable sources of antioxidants for pharmaceutical and nutraceutical applications.

Improvement in the Biosynthesis of Antioxidant-Active Metabolites in In Vitro Cultures of Isatis tinctoria (Brassicaceae) by Biotechnological Methods/Elicitation and Precursor Feeding.

This study aimed to establish the in vitro shoot culture of Isatis tinctoria L. and its ability to produce antioxidant bioactive compounds. The Murashige and Skoog (MS) medium variants, containing different concentrations (0.1-2.0 mg/L) of benzylaminopurine (BAP) and 1-naphthaleneacetic acid (NAA) were tested. Their influence on the growth of biomass, accumulation of phenolic compounds, and antioxidant potential was evaluated. To improve the phenolic content, agitated cultures (MS 1.0/1.0 mg/L BAP/NAA) were treated with different elicitors, including the following: Methyl Jasmonate, CaCl2, AgNO3, and yeast, as well as with L-Phenylalanine and L-Tyrosine-precursors of phenolic metabolites. The total phenolic content (TPC) of hydroalcoholic extracts (MeOH 70%) obtained from the biomass grown in vitro was determined spectrophotometrically; phenolic acids and flavonoids were quantified by RP-HPLC. Moreover, the antioxidant potential of extracts was examined through the DPPH test, the reducing power, and the Fe2+ chelating assays. The biomass extracts obtained after 72 h of supplementation with Tyr (2 g/L), as well as after 120 and 168 h with Tyr (1 g/L), were found to be the richest in TPC (49.37 ± 0.93, 58.65 ± 0.91, and 60.36 ± 4.97 mg GAE/g extract, respectively). Whereas among the elicitors, the highest TPC achieved was with CaCl2 (20 and 50 mM 24 h), followed by MeJa (50 and 100 µM, 120 h). The HPLC of the extracts led to the identification of six flavonoids and nine phenolic acids, with vicenin-2, isovitexin, syringic, and caffeic acids being the most abundant compounds. Notably, the amount of all flavonoids and phenolic acids detected in the elicited/precursor feeding biomass was higher than that of the leaves of the parental plant. The best chelating activity was found with the extract of biomass fed with Tyrosine 2 g/L, 72 h (IC50 0.27 ± 0.01 mg/mL), the strongest radical scavenging (DPPH test) for the extract obtained from biomass elicited with CaCl2 50 mM, after 24 h of incubation (25.14 ± 0.35 mg Trolox equivalents (TE)/g extract). In conclusion, the in vitro shoot culture of I. tinctoria supplemented with Tyrosine, as well as MeJa and/or CaCl2, could represent a biotechnological source of compounds with antioxidant properties.

Studies on the Accumulation of Secondary Metabolites and Evaluation of Biological Activity of In Vitro Cultures of Ruta montana L. in Temporary Immersion Bioreactors.

The present work focuses on in vitro cultures of Ruta montana L. in temporary immersion PlantformTM bioreactors. The main aim of the study was to evaluate the effects of cultivation time (5 and 6 weeks) and different concentrations (0.1-1.0 mg/L) of plant growth and development regulators (NAA and BAP) on the increase in biomass and the accumulation of secondary metabolites. Consequently, the antioxidant, antibacterial, and antibiofilm potentials of methanol extracts obtained from the in vitro-cultured biomass of R. montana were evaluated. High-performance liquid chromatography analysis was performed to characterize furanocoumarins, furoquinoline alkaloids, phenolic acids, and catechins. The major secondary metabolites in R. montana cultures were coumarins (maximum total content of 1824.3 mg/100 g DM), and the dominant compounds among them were xanthotoxin and bergapten. The maximum content of alkaloids was 561.7 mg/100 g DM. Concerning the antioxidant activity, the extract obtained from the biomass grown on the 0.1/0.1 LS medium variant, with an IC50 0.90 ± 0.03 mg/mL, showed the best chelating ability among the extracts, while the 0.1/0.1 and 0.5/1.0 LS media variants showed the best antibacterial (MIC range 125-500 µg/mL) and antibiofilm activity against resistant Staphylococcus aureus strains.

Wild Artichoke (Cynara cardunculus subsp. sylvestris, Asteraceae) Leaf Extract: Phenolic Profile and Oxidative Stress Inhibitory Effects on HepG2 Cells.

Cynara cardunculus subsp. sylvestris (wild artichoke) is widespread in Sicily, where it has been used for food and medicinal purposes since ancient times; decoctions of the aerial parts of this plant have been traditionally employed as a remedy for different hepatic diseases. In this study, the phenolic profile and cell-free antioxidant properties of the leaf aqueous extract of wild artichokes grown in Sicily (Italy) were investigated. The crude extract was also tested in cells for its antioxidant characteristics and potential oxidative stress inhibitory effects. To resemble the features of the early stage of mild steatosis in humans, human HepG2 cells treated with free fatty acids at the concentration of 1.5 mM were used. HPLC-DAD analysis revealed the presence of several phenolic acids (caffeoylquinic acids) and flavonoids (luteolin and apigenin derivatives). At the same time, DPPH assay showed a promising antioxidant power (IC50 = 20.04 ± 2.52 µg/mL). Biological investigations showed the safety of the crude extract and its capacity to counteract the injury induced by FFA exposure by restoring cell viability and counteracting oxidative stress through inhibiting reactive oxygen species and lipid peroxidation and increasing thiol-group levels. In addition, the extract increased mRNA expression of some proteins implicated in the antioxidant defense (Nrf2, Gpx, and SOD1) and decreased mRNA levels of inflammatory cytokines (IL-6, TNF-α, and IL-1ß), which were modified by FFA treatment. Results suggest that the total phytocomplex contained in wild artichoke leaves effectively modulates FFA-induced hepatic oxidative stress.

Chemical Profile, Antioxidant and Cytotoxic Activity of a Phenolic-Rich Fraction from the Leaves of Brassica fruticulosa subsp. fruticulosa (Brassicaceae) Growing Wild in Sicily (Italy).

Recently, our research team has started a study on Brassica fruticulosa subsp. fruticulosa, an edible plant traditionally used to treat various ailments, little investigated to date. Good in vitro antioxidant properties were highlighted for the leaf hydroalcoholic extract, with the secondary higher than the primary ones. In continuation of the ongoing research, this work was designed to elucidate the antioxidant properties of the phenolic compounds contained in the extract. For this purpose, a phenolic-rich ethyl acetate fraction (Bff-EAF) was obtained from the crude extract by liquid-liquid extraction. The phenolic composition was characterized by HPLC-PDA/ESI-MS analysis and the antioxidant potential was investigated by different in vitro methods. Furthermore, the cytotoxic properties were evaluated by MTT, LDH and ROS determinations on human colorectal epithelial adenocarcinoma cells (CaCo-2) and human normal fibroblasts (HFF-1). Twenty phenolic compounds (flavonoid and phenolic acid derivatives) were identified in Bff-EAF. The fraction exhibited good radical scavenging activity in the DPPH test (IC50 = 0.81 ± 0.02 mg/mL), and moderate reducing power (ASE/mL = 13.10 ± 0.94) and chelating properties (IC50 = 2.27 ± 0.18 mg/mL), contrary to what previously observed for the crude extract. Bff-EAF reduced in a dose-dependent manner CaCo-2 cell proliferation after 72 h of treatment. This effect was accompanied by the destabilization of the cellular redox state due to the antioxidant and pro-oxidant activities displayed by the fraction at lower and higher concentrations. No cytotoxic effect was observed on HFF-1 fibroblasts, used as control cell line.

Different Types of Hypericum perforatum cvs. (Elixir, Helos, Topas) In Vitro Cultures: A Rich Source of Bioactive Metabolites and Biological Activities of Biomass Extracts.

Microshoot agitated and bioreactor cultures (PlantForm bioreactors) of three Hypericum perforatum cultivars (Elixir, Helos, Topas) were maintained in four variants of Murashige and Skoog medium (MS) supplemented with 6-benzylaminopurine (BAP) and 1-naphthaleneacetic acid (NAA) (in the range of 0.1-3.0 mg/L). In both types of in vitro cultures, the accumulation dynamics of phenolic acids, flavonoids, and catechins were investigated during 5- and 4-week growth cycles, respectively. The contents of metabolites in methanolic extracts from biomasses collected in 1-week intervals were estimated by HPLC. The highest total contents of phenolic acids, flavonoids, and catechins were 505, 2386, and 712 mg/100 g DW, respectively (agitated cultures of cv. Helos). The extracts from biomass grown under the best in vitro culture conditions were examined for antioxidant and antimicrobial activities. The extracts showed high or moderate antioxidant activity (DPPH, reducing power, and chelating activity assays), high activity against Gram-positive bacteria, and strong antifungal activity. Additionally, experiments with phenylalanine feeding (1 g/L) in agitated cultures were performed reaching the highest enhancement of the total contents of flavonoids, phenolic acids, and catechins on day 7 after the addition of the biogenetic precursor (2.33-, 1.73- and 1.33-fold, respectively). After feeding, the highest accumulation of polyphenols was detected in the agitated culture of cv. Elixir (4.48 g/100 g DW). The high contents of metabolites and the promising biological properties of the biomass extracts are interesting from a practical point of view.

Ferula communis Root Extract: In Vitro Evaluation of the Potential Additive Effect with Chemotherapy Tamoxifen in Breast Cancer (MCF-7) Cells Part II.

Ferula L., belonging to the Apiaceae family, is represented by about 170 species predominantly present in areas with a mild-warm-arid climate, including the Mediterranean region, North Africa and Central Asia. Numerous beneficial activities have been reported for this plant in traditional medicine, including antidiabetic, antimicrobial, antiproliferative, anti-dysentery, stomachache with diarrhea and cramps remedies. FER-E was obtained from the plant F. communis, and precisely from the root, collected in Sardinia, Italy. A total of 25 g of root was mixed with 125 g of acetone (ratio 1:5, room temperature). The solution was filtered, and the liquid fraction was subjected to high pressure liquid chromatographic separation (HPLC). In particular, 10 mg of dry root extract powder, from F. communis, was dissolved in 10.0 mL of methanol, filtered with a 0.2 µm PTFE filter and subjected to HPLC analysis. The net dry powder yield obtained was 2.2 g. In addition, to reduce the toxicity of FER-E, the component ferulenol was removed. High concentrations of FER-E have demonstrated a toxic effect against breast cancer, with a mechanism independent of the oxidative potential, which is absent in this extract. In fact, some in vitro tests were used and showed little or no oxidizing activity by the extract. In addition, we appreciated less damage on the respective healthy cell lines (breast), assuming that this extract could be used for its potential role against uncontrolled cancer growth. The results of this research have also shown that F. communis extract could be used together with tamoxifen, increasing its effectiveness, and reducing side effects. However, further confirmatory experiments should be carried out.

In Vitro Cultures of Scutellaria brevibracteata subsp. subvelutina as a Source of Bioactive Phenolic Metabolites.

Some of the more than 350 Scutellaria species, such as S. baicalensis and S. lateriflora, have been used in traditional medicine and today play an important role in official phytotherapy. Other species have been less investigated, and their therapeutic potential is unknown. This is one of the few studies on Scutellaria brevibracteata subsp. subvelutina, and the first research of this species' in vitro cultures. The aim of this study was to establish an in vitro culture and analyse its phytochemical profile and biological activity. In the methanolic extracts from biomass cultured on six solid Murashige and Skoog (MS) medium variants supplemented with different combinations of 6-benzylaminopurine (BAP) and 1-naphthaleneacetic acid (NAA) in the range 0.5-3 mg/L analysed by HPLC, the presence of specific flavonoids (baicalein, baicalin, wogonin, wogonoside, scutellarin, chrysin), phenylpropanoid glycosides (verbascoside, isoverbascoside), and phenolic acids (p-hydroxybenzoic, caffeic, ferulic, m-coumaric acids) was confirmed. The dominant metabolites were wogonoside and verbascoside with the highest content of 346 and 457 mg/100 g DW, respectively. Thus, the extract with the highest content of bioactive metabolites was selected for further research and subjected to evaluation of antioxidant and antimicrobial potential. The extract exhibited good free radical scavenging activity (IC50 = 0.92 ± 0.01 mg/mL) and moderate reducing power and chelating activity. The brine shrimp lethality bioassay proved its lack of biotoxicity. Antimicrobial activity was tested against sixteen strains of Gram-positive and Gram-negative bacteria and fungi. The strongest growth inhibitory activity was observed against Trichophyton tonsurans.

Chemical, Antioxidant and Biological Studies of Brassica incana subsp. raimondoi (Brassicaceae) Leaf Extract.

Brassica incana subsp. raimondoi is an endemic taxon present in a restricted area located on steep limestone cliffs at an altitude of about 500 m a.s.l. in eastern Sicily. In this research, for the first time, studies on the phytochemical profile, the antioxidant properties in cell-free and cell-based systems, the cytotoxicity on normal and cancer cells by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay, and on Artemia salina Leach, were performed. The total phenolic, flavonoid, and condensed tannin contents of the leaf hydroalcoholic extract were spectrophotometrically determined. Ultra-performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) analysis highlighted the presence of several phenolic acids, flavonoids, and carotenoids, while High-Performance Liquid Chromatography with Diode-Array Detection (HPLC-DAD) identified various kaempferol and isorhamnetin derivatives. The extract exhibited different antioxidant properties according to the five in vitro methods used. Cytotoxicity by MTT assay evidenced no impact on normal human fibroblasts (HFF-1) and prostate cancer cells (DU145), and cytotoxicity accompanied by necrotic cell death for colon cancer cells (CaCo-2) and hepatoma cells (HepG2), starting from 100 µg/mL and 500 µg/mL, respectively. No cytotoxic effects were detected by the A. salina lethality bioassay. In the H2O2-induced oxidative stress cell model, the extract counteracted cellular reactive oxygen species (ROS) production and preserved non-protein thiol groups (RSH) affected by H2O2 exposure in HepG2 cells. Results suggest the potential of B. incana subsp. raimondoi as a source of bioactive molecules.

Determination of the polyphenolic content of berry juices using focusing-modulated comprehensive two-dimensional liquid chromatography coupled to mass spectrometry detection.

In this work, a comprehensive two-dimensional liquid chromatography system, comprised of a ZIC-HILIC and C18 columns in the first and second dimensions, respectively, was tuned and employed for attaining high resolution profiles of the polyphenolic pattern in seven commercial berry juices. The developed HILIC × RP-LC method was validated in terms of linearity range, correlation coefficients, limit of detection, limit of quantification, precision (intra- and inter-day), and recovery. A total of 104 polyphenolic compounds belonging to different chemical classes (hydroxybenzoic and cinnamic acid derivatives, flavone glycosides, flavonols, flavonol glycosides, dihydroflavonols, and anthocyanin glycosides) have been characterized and quantified in the juices investigated. Despite the constituents being similar, a notable quantitative variation among the analyzed berry species was observed. Elderberry contained the highest amount of polyphenols (918 ± 1.10 mg 100 mL-1), followed by chokeberry (516 ± 0.08 mg 100 mL-1). On the other hand, raspberry contained the lowest amount (104 ± 1.21 mg 100 mL-1). Further, total phenolic, flavonoid, and anthocyanin contents were determined spectrophotometrically, yielding consistent results. The free-radical scavenging activity (DPPH test) and reducing power of the juices, expressed as IC50 (µL mL-1) and mg ASE mL-1, varied from 2.79 ± 0.03 (honeyberry) to 31.66 ± 0.02 (blueberry) and from 1.71 ± 0.01 (blueberry) to 8.89 ± 0.12 (chokeberry), respectively. Such a ZIC-HILIC × C18 platform based on focusing modulation, never employed so far for berry juices, showed a remarkable separation capability with high values of corrected peak capacity (up to 1372) and orthogonality (Ao up to 0.80), thus providing a great applicability to be advantageously employed for other complex food samples.

Volatile composition, antidiabetic, and anti-obesity potential of Brassica incana leaf and flowering top extracts.

CONTEXT: Brassica incana Ten. (Brassicaceae) is an edible plant with very limited available information. Previous studies have demonstrated the polyphenolic profile and the antioxidant and cytotoxic properties of the leaf and flowering top hydroalcoholic extracts. OBJECTIVE: The volatile composition and the antidiabetic and anti-obesity potential of B. incana leaf and flowering top extracts have been investigated. MATERIAL AND METHODS: The volatile characterization of the extracts was attained by HS-SPME-GC/MS analysis. The antidiabetic and anti-obesity potential was investigated spectrophotometrically in vitro by the ability to modulate pancreatic lipase and α-glucosidase at different concentrations using orlistat and acarbose as reference drugs. The inhibition of advanced glycation end-products (AGEs) was measured with aminoguanidine as reference and the antioxidant activity with the xanthine/xanthine oxidase system and Trolox for comparative purposes. RESULTS: Several volatiles belonging to different chemical classes were identified, being sulphur compounds the most abundant in both leaf and flowering top extracts (56.33% and 64.40% of all volatiles). Although the leaf extract showed lower IC50 values in most of the assays (0.968 and 1.921 mg/mL for α-glucosidase; 0.192 and 0.262 mg/mL for AGEs; 0.022 and 0.038 mg/mL for superoxide scavenging), there were no statistically significant differences between both samples. These extracts showed a similar behaviour to Trolox in the xanthine oxidase assay (IC50 values of 0.022 mg/mL for leaf extract; 0.038 mg/mL for flowering top and 0.028 for Trolox). CONCLUSIONS: Leaves and flowering tops from B. incana can be used as sources of functional compounds that could act as antidiabetic and anti-obesogenic agents.

Determination of the Phenolic Profile by Liquid Chromatography, Evaluation of Antioxidant Activity and Toxicity of Moroccan Erica multiflora, Erica scoparia, and Calluna vulgaris (Ericaceae).

This study aimed to investigate the phenolic profile and selected biological activities of the leaf and aerial extracts of three Ericaceae species, namely Erica multiflora, Erica scoparia, and Calluna vulgaris, collected from three different places in the north of Morocco. The phenolic composition of all extracts was determined by LC coupled with photodiode array and mass spectrometry detection. Among the investigated extracts, that of E. scoparia aerial parts was the richest one, with a total amount of polyphenols of 9528.93 mg/kg. Up to 59 phenolic compounds were detected: 52 were positively identified and 49 quantified-11 in C. vulgaris, 14 in E. multiflora, and 24 in E. scoparia. In terms of chemical classes, nine were phenolic acids and 43 were flavonoids, and among them, the majority belonged to the class of flavonols. The antioxidant activity of all extracts was investigated by three different in vitro methods, namely DPPH, reducing power, and Fe2+ chelating assays; E. scoparia aerial part extract was the most active, with an IC50 of 0.142 ± 0.014 mg/mL (DPPH test) and 1.898 ± 0.056 ASE/mL (reducing power assay). Further, all extracts were non-toxic against Artemia salina, thus indicating their potential safety. The findings attained in this work for such Moroccan Ericaceae species, never investigated so far, bring novelty to the field and show them to be valuable sources of phenolic compounds with interesting primary antioxidant properties.

Phenolic and Volatile Composition and Antioxidant Properties of the Leaf Extract of Brassica fruticulosa subsp. fruticulosa (Brassicaceae) Growing Wild in Sicily (Italy).

In continuation of research conducted on species of the spontaneous flora of Sicily (Italy) belonging to the Brassicaceae family, Brassica fruticulosa subsp. fruticulosa was selected. It is an edible species utilized in Sicilian traditional medicine. In this study, for the first time, the phenolic and the volatile compounds and the antioxidant properties of the hydroalcoholic extract obtained from the leaves of B. fruticulosa subsp. fruticulosa were characterized. Through HPLC-PDA/ESI-MS analysis, a total of 22 polyphenolic compounds (20 flavonoids and 2 phenolic acids) were identified, with 3-hydroxiferuloylsophoroside-7-O-glucoside (1.30 mg/g ± 0.01) and kaempferol-3-O-feruloylsophoroside-7-O-glucoside (1.28 mg/g ± 0.01) as the most abundant compounds. Through SPME-GC/MS several volatiles belonging to different chemical classes were characterized, with nitriles and aldehydes accounting for more than 54% of the whole volatile fraction. The extract of B. fruticulosa subsp. fruticulosa showed moderate activity in the DPPH assay (IC50 = 1.65 ± 0.08 mg/mL), weak reducing power (17.47 ± 0.65 ASE/mL), and good chelating properties (IC50 = 0.38 ± 0.02 mg/mL), reaching approximately 90% activity at the highest tested concentration. Lastly, the extract was non-toxic against Artemia salina, indicating its potential safety. According to the findings, it can be stated that B. fruticulosa subsp. fruticulosa represents a new valuable source of bioactive compounds.

In Vitro Anthelmintic Activity of Isatis tinctoria Extracts against Ewes' Gastrointestinal Nematodes (GINs), a Possible Application for Animal Welfare.

Sheep gastrointestinal nematode (GIN) infestation represents a limiting factor for sheep farming and milk production in Italy. The development of anthelmintic resistance to conventionally used drugs suggests the path towards the use of natural remedies as a possible alternative. The purpose of this study is to evaluate the in vitro anthelmintic efficacy of the hydroalcoholic extracts of basal leaves (It-BL), cauline leaves (It-CL) and flowers (It-F) of Isatis tinctoria (Brassicaceae), a spontaneous Sicilian species renowned as an important source of bioactive compounds. The dry extracts of the different parts of the plant were tested using the egg hatch test (EHT) in vitro to verify the efficacy against ovine GIN at different concentrations (1.00, 0.5, 0.25, 0.125 mg/mL). Thiabendazole and deionized water were used as positive and negative controls, respectively. The results obtained from EHT indicated that all the I. tinctoria extracts were highly effective (p < 0.0001) in inhibiting egg hatching within 48 h of exposure. The in vitro inhibitory effect was never less than 84% in all doses tested, and it was only slightly lower than the standard drug thiabendazole (95.6%). The current study documents the anthelmintic activity of I. tinctoria against sheep's GIN, suggesting its application as alternative natural method to limit the use of antiparasitic drugs.

Phenolic Compounds, Antioxidant and Antibacterial Activities of Extracts from Aerial Parts of Thymus zygis subsp. gracilis, Mentha suaveolens and Sideritis incana from Morocco.

Thymus zygis subsp. gracilis, Mentha suaveolens and Sideritis incana (Lamiaceae family) are well recognized for their medicinal, pharmaceutical and aromatic properties. The present study aimed to investigate for the first time the phenolic composition, the antioxidant and antibacterial properties of the extracts obtained from the aerial parts of these species collected from the Ifrane region of Morocco. The phenolic compounds were determined using high-performance liquid chromatography coupled to photodiode array and electrospray ionization mass spectrometry (HPLC-PDA-ESI/MS). The antioxidant activity was investigated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, reducing power and ferrous ion chelating assays. The antibacterial activity was evaluated against three Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium) and three Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis and Listeria monocytogenes). A total of thirty-three, sixteen and thirteen phenolic compounds were positively identified and characterized in T. zygis subsp. gracilis, M. suaveolens and S. incana extracts, respectively. Among the extracts, M. suaveolens exhibited the highest antioxidant activity, followed by S. incana and T. zygis subsp. gracilis in both DPPH and reducing power assays; all extracts showed the lowest activity in the chelating assay. In addition, all extracts demonstrated a bactericidal effect against Gram-positive bacteria and bacteriostatic effect against Gram-negative bacteria. Therefore, the aerial parts of Moroccan T. zygis subsp. gracilis, M. suaveolens and S. incana might be considered as a valuable source of natural antioxidant and antibacterial agents with potential application in food and pharmaceutical industries.

Anti-adipogenic and anti-oxidant effects of a standardised extract of Moro blood oranges (Citrus sinensis (L.) Osbeck) during adipocyte differentiation of 3T3-L1 preadipocytes.

Particular features in the pulp of blood oranges are marked levels of anthocyanin, a class of polyphenolic compounds well known to exert numerous health-promoting actions on human wellbeing including anti-obesity effects. In this study, we investigated in vitro, the antioxidant and anti-adipogenic activities of Morosil®, a standardised extract of Moro blood oranges. During adipocyte differentiation, 3T3-L1 cells were treated with concentrations of extract containing 2.5, 5, 10, 25 µM of anthocyanins. After seven days of treatment and differentiation, we measured reactive oxygen species production, non-proteic thiol groups content, adipokine secretion and triglyceride accumulation together with mRNA expression of adipogenic transcription factors such as PPARγ, C/EBPα, SREBP-1c. Furthermore, both ACCα, FAS protein expression and citrate synthase activity were measured. Results show that Morosil® exerts antioxidant and anti-adipogenic activities during adipocyte differentiation of 3T3-L1 pre-adipocytes.

The Inhibitory Effect of Sulforaphane on Bladder Cancer Cell Depends on GSH Depletion-Induced by Nrf2 Translocation.

Sulforaphane (SFN), an isothiocyanate (ITCs) derived from glucosinolate that is found in cruciferous vegetables, has been reported to exert a promising anticancer effect in a substantial amount of scientific research. However, epidemical studies showed inconsistencies between cruciferous vegetable intake and bladder cancer risk. In this study, human bladder cancer T24 cells were used as in vitro model for revealing the inhibitory effect and its potential mechanism of SFN on cell growth. Here, a low dose of SFN (2.5 µM) was shown to promote cell proliferation (5.18-11.84%) and migration in T24 cells, whilst high doses of SFN (>10 µM) inhibited cell growth significantly. The induction effect of SFN on nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression at both low (2.5 µM) and high dose (10 µM) was characterized by a bell-shaped curve. Nrf2 and glutathione (GSH) might be the underlying mechanism in the effect of SFN on T24 cell growth since Nrf2 siRNA and GSH-depleting agent L-Buthionine-sulfoximine abolished the effect of SFN on cell proliferation. In summary, the inhibitory effect of SFN on bladder cancer cell growth and migration is highly dependent on Nrf2-mediated GSH depletion and following production. These findings suggested that a higher dose of SFN is required for the prevention and treatment of bladder cancer.

Phenylalanine Increases the Production of Antioxidant Phenolic Acids in Ginkgo biloba Cell Cultures.

The aims of this study were to evaluate the antioxidant properties, to investigate the content of major secondary metabolites in Ginkgo biloba cell cultures, and to determine the change in the production of phenolic acids by adding phenylalanine to the culture medium. Three in vitro methods, which depend on different mechanisms, were used for assessing the antioxidant activity of the extract: 1,1-diphenyl-2-picrylhydrazil (DPPH), reducing power and Fe2+ chelating activity assays. The extract showed moderate activity both in the DPPH and in the reducing power assays (IC50 = 1.966 ± 0.058 mg/mL; ASE/mL = 16.31 ± 1.20); instead, it was found to possess good chelating properties reaching approximately 70% activity at the highest tested dose. The total phenolic, total flavonoid, and condensed tannin content of G. biloba cell culture extract was spectrophotometrically determined. The phenolic acid content was investigated by RP-HPLC, and the major metabolites-protocatechuic and p-hydroxybenzoic acids-were isolated and investigated by 1H NMR. The results showed that phenylalanine added to G. biloba cell cultures at concentrations of 100, 150, and 200 mg/150 mL increased the production of phenolic acids. Cultures that were grown for 3 weeks and collected after 4 days of phenylalanine supplementation at high concentration showed maximal content of phenolic acids (73.76 mg/100 g DW).

Phytochemical Constituents, Antioxidant Activity, and Toxicity Assessment of the Aerial Part Extracts from the Infraspecific Taxa of Matthiola fruticulosa (Brassicaceae) Endemic to Sicily.

In a project designed to investigate the specific and infraspecific taxa of Matthiola endemic to Sicily (Italy) as new potential sources of bioactive compounds in this work, the infraspecific taxa of Matthiola fruticulosa were studied, namely, subsp. fruticulosa and subsp. coronopifolia. HPLC-PDA/ESI-MS and SPME-GC/MS analyses of hydroalcoholic extracts obtained from the aerial parts of the two subspecies led to the detection of 51 phenolics and 61 volatile components, highlighting a quite different qualitative-quantitative profile. The antioxidant properties of the extracts were explored through in vitro methods: 1,1-diphenyl-2-picrylhydrazyl (DPPH), reducing power and Fe2+ chelating activity assays. The results of the antioxidant tests showed that the extracts possess a different antioxidant ability: particularly, the extract of M. fruticulosa subsp. fruticulosa exhibited higher radical scavenging activity than that of subsp. coronopifolia (IC50 = 1.25 ± 0.02 mg/mL and 2.86 ± 0.05 mg/mL), which in turn displayed better chelating properties (IC50 = 1.49 ± 0.01 mg/mL and 0.63 ± 0.01 mg/mL). Lastly, Artemia salina lethality bioassay was performed for toxicity assessment. The results of the bioassay showed lack of toxicity against brine shrimp larvae for both extracts. The data presented indicate the infraspecific taxa of M. fruticulosa as new and safe sources of antioxidant compounds.