Characterization and Biological Activities of In Vitro Digested Olive Pomace Polyphenols Evaluated on Ex Vivo Human Immune Blood Cells.

Olive pomace (OP) represents one of the main by-products of olive oil production, which still contains high quantities of health-promoting bioactive compounds. In the present study, three batches of sun-dried OP were characterized for their profile in phenolic compounds (by HPLC-DAD) and in vitro antioxidant properties (ABTS, FRAP and DPPH assays) before (methanolic extracts) and after (aqueous extracts) their simulated in vitro digestion and dialysis. Phenolic profiles, and, accordingly, the antioxidant activities, showed significant differences among the three OP batches, and most compounds showed good bioaccessibility after simulated digestion. Based on these preliminary screenings, the best OP aqueous extract (OP-W) was further characterized for its peptide composition and subdivided into seven fractions (OP-F). The most promising OP-F (characterized for its metabolome) and OP-W samples were then assessed for their potential anti-inflammatory properties in ex vivo human peripheral mononuclear cells (PBMCs) triggered or not with lipopolysaccharide (LPS). The levels of 16 pro-and anti-inflammatory cytokines were measured in PBMC culture media by multiplex ELISA assay, whereas the gene expressions of interleukin-6 (IL-6), IL-10 and TNF-α were measured by real time RT-qPCR. Interestingly, OP-W and PO-F samples had a similar effect in reducing the expressions of IL-6 and TNF-α, but only OP-W was able to reduce the release of these inflammatory mediators, suggesting that the anti-inflammatory activity of OP-W is different from that of OP-F.

Tart cherry (Prunus cerasus L.) dietary supplement modulates visceral adipose tissue CB1 mRNA levels along with other adipogenesis-related genes in rat models of diet-induced obesity.

PURPOSE: There is increasing evidence for the involvement of dietary bioactive compounds in the cross-talk modulation of endocannabinoid system and some of the key regulators of transcriptional control for adipogenesis. METHODS: We aimed to characterize the expression of cannabinoid CB1/CB2 receptors and fatty acid amide hydrolase (FAAH) along with selected adipogenesis-related genes (PPARγ, SREBP-1c and PREF-1), adipocyte-secreted factors (leptin and adiponectin), mitochondrial bioenergetic modulators (PGC-1A and UCP-2), and transient receptor potential vanilloid subtype 1 (TRPV1) and 2 (TRPV2) channels in visceral adipose tissue of rats fed with a high-fat diet (HFD) containing either tart cherry seeds alone or tart cherry seeds and juice for 17 weeks. The visceral adipose tissue was weighed and checked the expression of different markers by qRT-PCR, Western blot and immunohistochemistry. RESULTS: Tart cherry supplements were able to downregulate the HFD-induced mRNA expression of CB1 receptor, SREBP-1c, PPARγ, leptin, TRPV1 and TRPV2 resulting in potential anti-adipogenic effects. CONCLUSION: The present study points out that the intake of bioactive constituents of tart cherry may attenuate the effect of adipogenesis by acting directly on the adipose tissue and modulating the interplay between CB1, PPARγ and TRPV channel gene transcription.

Antioxidant and Antisteatotic Activities of Fucoidan Fractions from Marine and Terrestrial Sources.

Fucoidan is a fucose-rich sulfated polysaccharide typically found in the cell wall of marine algae but also recently isolated from terrestrial sources. Due to a variety of biological activities, including antioxidant properties, fucoidan exhibits an attractive therapeutic potential against a wide array of metabolic diseases associated with oxidative stress. We used FTIR, 1H NMR and 13C NMR spectroscopy to investigate the structural features of a fucoidan fraction extracted from the brown alga Cystoseira compressa (CYS). The antioxidant potential of CYS was measured by DPPH, ABTS and FRAP assays, which revealed a radical scavenging capacity that was confirmed in in vitro cellular models of hepatic and endothelial cells. The same antioxidant effects were observed for another fucoidan fraction previously identified in the terrestrial tree Eucalyptus globulus (EUC). Moreover, in hepatic cells, CYS and EUC exhibited a significant antisteatotic action, being able to reduce intracellular triglyceride content through the regulation of key genes of hepatic lipid metabolism. EUC exerted stronger antioxidant and antisteatotic effects as compared to CYS, suggesting that both marine and terrestrial sources should be considered for fucoidan extraction and therapeutic applications.

Antioxidant and Antisteatotic Activities of a New Fucoidan Extracted from Ferula hermonis Roots Harvested on Lebanese Mountains.

Fucoidan is a fucose-rich sulfated polysaccharide with attractive therapeutic potential due to a variety of biological activities, including antioxidant action. Fucoidan is typically found in the cell wall of marine brown algae, but extra-algal sources have also been discovered. In the present work, for the first time we extracted a water soluble fucoidan fraction from the roots of the terrestrial shrub Ferula hermonis. This fucoidan fraction was termed FUFe, and contained fucose, glucose, sulfate, smaller amounts of monosaccharides such as galactose and mannose, and a minor quantity of proteins. FUFe structural features were investigated by FTIR, 1H NMR and 13C NMR spectroscopy. The antioxidant property of FUFe was measured by DPPH, ABTS and FRAP assays, which revealed a high radical scavenging capacity that was confirmed in in vitro cellular models. In hepatic and endothelial cells, 50 µg/mL FUFe could reduce ROS production induced by intracellular lipid accumulation. Moreover, in hepatic cells FUFe exhibited a significant antisteatotic action, being able to reduce intracellular triglyceride content and to regulate the expression of key genes of hepatic lipid metabolism. Altogether, our results candidate FUFe as a possible bioactive compound against fatty liver disease and related vascular damage.

Tart Cherry Juice and Seeds Affect Pro-Inflammatory Markers in Visceral Adipose Tissue of High-Fat Diet Obese Rats.

BACKGROUND: Tart cherries (Prunus cerasus L.) are a rich source of anthocyanins. They are phytochemical flavonoids found in red and blue fruits, and vegetables that can reduce hyperlipidemia. Visceral Adipose Tissue (VAT) has emerged as a major player in driving obesity-related inflammatory response. METHODS: This study has investigated the potential positive effects of tart cherries on rats with Diet-Induced Obesity (DIO). In particular, the inflammatory status in retroperitoneal (RPW) and perigonadal (PGW) adipose tissue were studied. Rats were fed ad libitum for 17 weeks with a hypercaloric diet with the supplementation of tart cherries seeds powder (DS) and seeds powder plus tart cherries juice containing 1mg of anthocyanins (DJS). In RPW and PGW, expression of CRP, IL-1 ß, TNF-α, CCL2 and CD36, were measured by qRT-PCR, Western blot and immunohistochemistry techniques. RESULTS: No differences in the weight of RPW and PGW animals were found between DS and DJS groups compared to DIO rats. However, an increase of inflammatory markers was observed in DIO group in comparison with control lean rats. A modulation of these markers was evident upon tart cherry supplementation. CONCLUSION: Study results suggest that tart cherry enriched-diet did not modify the accumulation of visceral fat, but it decreased inflammatory markers in both tissues. Therefore, this supplementation could be useful, in combination with healthy lifestyles, to modify adipose tissue cell metabolism limiting-obesity related organ damage.

Synthesis, Characterization and Assessment of the Antioxidant Activity of Cu(II), Zn(II) and Cd(II) Complexes Derived from Scorpionate Ligands.

Seeking to enrich the yet less explored field of scorpionate complexes bearing antioxidant properties, we, here, report on the synthesis, characterization and assessment of the antioxidant activity of new complexes derived from three scorpionate ligands. The interaction between the scorpionate ligands thallium(I) hydrotris(5-methyl-indazolyl)borate (TlTp4Bo,5Me), thallium(I) hydrotris(4,5-dihydro-2H-benzo[g]indazolyl)borate (TlTpa) and potassium hydrotris(3-tert-butyl- pyrazolyl)borate (KTptBu), and metal(II) chlorides, in dichloromethane at room temperature, produced a new family of complexes having the stoichiometric formula [M(Tp4Bo,5Me)2] (M = Cu, 1; Zn, 4; Cd, 7), [M(Tpa)2] (M = Cu, 2; Zn, 5; Cd, 8), [Cu(HpztBu)3Cl2] (3), [Zn(TptBu)Cl] (6) and [Cd(BptBu)(HpztBu)Cl] (9). The obtained metal complexes were characterized by Fourier transform infrared spectroscopy, proton nuclear magnetic resonance and elemental analysis, highlighting the total and partial hydrolysis of the scorpionate ligand TptBu during the synthesis of the Cu(II) complex 3 and the Cd(II) complex 9, respectively. An assessment of the antioxidant activity of the obtained metal complexes was performed through both enzymatic and non-enzymatic assays against 1,1-diphenyl-2-picryl- hydrazyl (DPPH·), 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+·), hydroxyl (HO·), nitric oxide (NO·), superoxide (O2-) and peroxide (OOH·) radicals. In particular, the complex [Cu(Tpa)2]⋅0.5H2O (2) exhibited significant antioxidant activity, as good and specific activity against superoxide (O2-·), (IC50 values equal to 5.6 ± 0.2 µM) and might be identified as auspicious SOD-mimics (SOD = superoxide dismutase).

Hairy Garlic (Allium subhirsutum) from Sicily (Italy): LC-DAD-MSn Analysis of Secondary Metabolites and In Vitro Biological Properties.

Allium subhirsutum, known as hairy garlic, is a bulbous plant widespread in the Mediterranean area and locally used as a food and spice. In the present study, the chemical profile of the ethanolic extracts from bulbs (BE) and aerial parts (APE) were analyzed by HPLC-ESI-MSn, and antioxidant properties were evaluated by DPPH, ABTS and TEAC assays. The traditional use in the diet, and the well documented biological activity of Allium species suggest a potential as a new nutraceutical. For this reason, the potential usefulness of this food can be considered in the treatment and prevention of degenerative Alzheimer disease. For this reason, acetylcholinesterase inhibitory property was investigated. Furthermore, due to the observed presence of sulfur-containing and phenolic constituents, the cytotoxicity on tumor cells line was investigated. Results revealed significant AChE inhibitory activity for BE and APE. Both extracts exhibited also moderate antioxidant properties in the in vitro assays. Finally, limited cytotoxic activity was observed towards Human colon carcinoma and adenocarcinoma cell line, with differences between the individual parts tested. HPLC-ESI-MSn analysis showed that hairy garlic is a good source of sulphur compounds, flavonoids and phenylpropanoids derivatives, thus being a valid alternative to the common garlic (A. sativum). This work opens new opportunities for the application of A. subhirsutum as a health-promoting food.

The Nonvolatile and Volatile Metabolites of Prangos ferulacea and Their Biological Properties.

Prangos ferulacea (L.) Lindl. (Fam. Apiaceae), an orophilous species of eastern Mediterranean and western Asia, possesses a number of biological properties that are worthy of exploitation in different fields. Phytochemical investigations revealed the presence of coumarins, prenyl-coumarins, and furano-coumarins as the main constituents of this species, as well as several flavonoids. Among prenyl-coumarins, osthol is a promising apoptotic agent quite selective toward cancer cells. In addition, the essential oils have been extensively investigated, and several chemotypes have been identified. This work reviews the literature on this species published between 1965 and 2018, describes its volatile and nonvolatile metabolites, and outlines its pharmacological effects.

Exploring the Insecticidal Potential of Boldo (Peumus boldus) Essential Oil: Toxicity to Pests and Vectors and Non-target Impact on the Microcrustacean Daphnia magna.

Every year Chile exports about 2000 tons of boldo folium (Peumus boldus), which is used around the world as a traditional herbal medicinal product (THMP), mostly to relieve gastrointestinal disorders. This biomass may be a resource for the agrochemical industry to manufacture botanical insecticides. In this regard, the insecticidal potential of boldo has been poorly investigated. In the present work, hydrodistillation of a commercial boldo folium gave 1.5% (w/w) of a yellowish essential oil (boldo essential oil, BEO) containing 1,8-cineole (20.7%), p-cymene (18.5%), limonene (9.1%), ascaridole (9.1%) and ß-phellandrene (6.4%) as the main constituents, as determined by gas chromatography-mass spectrometry (GC-MS). NMR analysis allowed us to determine that ascaridole was mainly represented by the cis-isomer. BEO was toxic to larvae of the filariasis vector Culex quinquefasciatus and adults of the housefly Musca domestica, showing LC50/LD50 values of 67.9 mg·L-1 and 98.5 µg·adult-1, respectively. On the other hand, lower insecticidal activity was observed against larvae of the moth pest Spodoptera littoralis (LD50 of 268.9 µg·larva-1). It is worth noting that, when tested at LC90 concentration, BEO was significantly less toxic to aquatic microcrustacean Daphnia magna than the conventional insecticide α-cypermethrin. Finally, in the attempt to explore the BEO mode of action, we tested it for acetylcholinesterase (AChE) inhibitory properties using the Ellman method, obtaining negligible effects (IC50 = 0.45 mg·mL-1). Taken together, these results gave new insights into the potential of BEO as a future ingredient of botanical insecticides.

Bioactive Constituents of Juniperus turbinata Guss. from La Maddalena Archipelago.

A comprehensive phytochemical study of Juniperus turbinata (Cupressaceae) collected from La Maddalena Archipelago (Sardinia, Italy) is reported. Both the essential oil and the ethanolic extract obtained from the aerial parts were analyzed. The essential oil appears to belong to a new chemotype compared to other Mediterranean juniper accessions, as it was favored by geographic isolation of the isles. It showed a low content of monoterpene hydrocarbons and α-terpineol, ent-manoyl oxide, 1,10-di-epi-cubenol as the major constituents. The ethanolic fraction contained mainly diterpenoids. Among these, 15-formyloxyimbricatolic acid (7) is a new natural product since it has hitherto been obtained only by synthetic route. The phenolic fraction contained biflavonoids: cupressuflavone (9), followed by minor amounts of amentoflavone (10) and hinokiflavone (11). The essential oil and six purified compounds (1 - 4, 8 and 9) were assessed for biological activities, namely antioxidant (assessed by DPPH· , ABTS·+ and FRAP methods) and cytotoxic effects towards selected human tumor cell lines (MDA-MB 231, A375 and HCT116 cells). Compound 3 exhibited higher radical scavenging activity against ABTS·+ radical than the reference Trolox. Noteworthy, compound 8 showed powerful effects towards tumor cell lines, with IC50 values in the range of 0.060 - 0.201 µm, which make it a promising anticancer drug candidate.

Efficacy of the Volatile Oil from Water Celery (Helosciadium nodiflorum, Apiaceae) against the Filariasis Vector Culex quinquefasciatus, the Housefly Musca domestica, and the African Cotton Leafworm Spodoptera littoralis.

Water celery (Helosciadium nodiflorum, Apiaceae) is a common and invasive hydrophyte occurring in rivers and slow waters of Europe. In the past, it was consumed as a vegetable and spices, but currently it has mostly been abandoned. As a result, this plant produces an unexploited biomass occluding rivers and slow waters. With the aim to valorise water celery as an important biomass source for the development of botanical insecticides, in this work, we investigated the inhibitory effects of the essential oil hydrodistilled from its aerial parts against larvae of the filariasis vector Culex quinquefasciatus, adults of the housefly Musca domestica, and larvae of the African cotton leafworm Spodoptera littoralis. The essential oil, which was characterized by the insecticidal limonene (39.8%) and myristicin (35.3%), exerted important toxic effects on 3rd instar larvae of C. quinquefasciatus and S. littoralis and on adults of M. domestica, with LD50 of 50.4 µl l-1 , 116.7 µg larva-1 , and 45.1 (male) and 70.1 (female) µg adult-1 , respectively. The oil did not show any noticeable inhibitory activity on the acetylcholinesterase enzyme. Our findings add scientific evidences highlighting water celery as an important biomass source to be used for the control of insect pests and vectors of high economic importance.

Cytotoxic Essential Oils from Eryngium campestre and Eryngium amethystinum (Apiaceae) Growing in Central Italy.

Eryngium campestre and E. amethystinum are thorny herbs belonging to the Apiaceae family and spontaneously growing in stony pastures and dry meadows, preferentially on calcareous substrates. In the Mediterranean countries, these plants have been used as a food or traditional remedies to treat various ailments. In the present work, we have analyzed the chemical composition of the essential oils distilled from the aerial parts by GC-FID and GC/MS, and evaluated their cytotoxic effects on a panel of human cancer cells, namely A375 (human malignant melanoma), MDA-MB 231 cells (human breast adenocarcinoma), and HCT116 cells (human colon carcinoma), by the MTT assay. Furthermore, the Eryngium essential oils were evaluated for antioxidant and acetylcholinesterase (AChE) activities. The two essential oils were rich in sesquiterpene hydrocarbons, with germacrene D as the major compound, accompanied by allo-aromadendrene, ß-elemene, spathulenol, and ledol. They turned out to be highly cytotoxic on the tumor cells, with IC50 values (1.65 - 5.32 and 1.57 - 2.99 µg/ml for E. amethystinum and E. campestre, respectively) comparable or close to those of the anticancer drug cisplatin. The E. amethystinum essential oil exhibited a moderate antioxidant activity, whereas that of E. campestre a weak AChE inhibition.

Essential Oil of Thymus munbyanus subsp. coloratus from Algeria: Chemotypification and in vitro Biological Activities.

Thymus munbyanus subsp. coloratus (Lamiaceae) is a small shrub endemic to Algeria and Morocco where is found in lawns, rockeries and mountainous regions. From a phytochemical point of view this taxon has never been characterized. In this work we have analysed the chemical compositions of the essential oils obtained from inflorescences and vegetative parts by GC/MS. A new chemotype, i.e. borneol-chemotype, was characterized for the first time in the species. Furthermore, we assessed the biological activities of essential oils, namely the antioxidant, antimicrobial and cytotoxicity on tumor cells that were evaluated by the DPPH, ABTS, and FRAP, disc diffusion, and MTT methods, respectively. Biological assays highlighted a moderate inhibitory effect on Staphylococcus aureus, Escherichia coli and Candida albicans (inhibition zone diameter in the range 9 - 10 mm), and noteworthy cytotoxicity on A375 human melanoma cells (IC50 of 46.95 µg/ml).

The water soluble ruthenium(II) organometallic compound [Ru(p-cymene)(bis(3,5 dimethylpyrazol-1-yl)methane)Cl]Cl suppresses triple negative breast cancer growth by inhibiting tumor infiltration of regulatory T cells.

Ruthenium compounds have become promising alternatives to platinum drugs by displaying specific activities against different cancers and favorable toxicity and clearance properties. Here, we show that the ruthenium(II) complex [Ru(p-cymene)(bis(3,5-dimethylpyrazol-1-yl)methane)Cl]Cl (UNICAM-1) exhibits potent in vivo antitumor effects. When administered as four-dose course, by repeating a single dose (52.4mgkg-1) every three days, UNICAM-1 significantly reduces the growth of A17 triple negative breast cancer cells transplanted into FVB syngeneic mice. Pharmacokinetic studies indicate that UNICAM-1 is rapidly eliminated from kidney, liver and bloodstream thanks to its high hydrosolubility, exerting excellent therapeutic activity with minimal side effects. Immunohistological analysis revealed that the efficacy of UNICAM-1, mainly relies on its capacity to reverse tumor-associated immune suppression by significantly reducing the number of tumor-infiltrating regulatory T cells. Therefore, UNICAM-1 appears very promising for the treatment of TNBC.

Secondary Metabolites, Glandular Trichomes and Biological Activity of Sideritis montana L. subsp. montana from Central Italy.

Sideritis montana subsp. montana is a small annual herb occurring in countries bordering the Mediterranean and Balkan regions. The secondary metabolism of this plant has not been fully explored so far. The aim of the present study was to understand the complex mixture of secondary metabolites and the type of secretory structures. The polar constituents were isolated by column chromatography from the ethanolic extract, and their structure was elucidated by NMR and MS. The essential oil was isolated by hydrodistillation and analysed by GC/MS. The plant indumentum was studied by light and scanning electron microscopy. To complete the work, the essential oil antioxidant activity and cytotoxicity on tumor cells were evaluated by DPPH, ABTS, FRAP, and MTT methods. Four different classes of secondary metabolites were isolated, namely flavonoids, caffeoylquinic derivatives, glycosidic hydroquinones and iridoids. The essential oil was mainly characterized by sesquiterpenene hydrocarbons. Peltate and long-capitate hairs were the main sites where terpenes and polar constituents are produced. The secondary metabolites found in S. montana subsp. montana are of chemotaxonomic interest, some of them being typical of the genus Sideritis. The trichomes types observed partially differ from those described in other members of the genus Sideritis. The essential oil showed noteworthy inhibition on tumor cells.

Bioactive Secondary Metabolites from Schizogyne sericea (Asteraceae) Endemic to Canary Islands.

Schizogyne sericea (Asteraceae) is a halophytic shrub endemic to the Canary Islands and traditionally employed as analgesic, astringent, anti-inflammatory, and vulnerary. A comprehensive phytochemical investigation was conducted on the flowering aerial parts by analyzing both essential oil constituents and polar compounds. The essential oil was dominated by p-cymene, with the noteworthy occurrence of ß-pinene and thymol esters. From the EtOH extract, eight compounds were isolated and structurally elucidated. Essential oil, polar fractions, and isolates (2), (4), and (5) were separately in vitro assayed for antiproliferative activity on human tumor cell lines (A375, MDA-MB 231, and HCT116) by MTT assay, for antioxidant potential by DPPH, ABTS, and FRAP assays, and for antimicrobial activity by the agar disk diffusion method. Results revealed that essential oil and compounds 1 and 2 exert a strong inhibition on tumor cells, and in some cases, higher than that of cisplatin. Fractions containing thymol derivatives (1 and 2) and caffeoylquinic acid derivatives 4 and 5 displayed antioxidant activity comparable to that of Trolox, making S. sericea extract an interesting natural product with potential applications as preservative or in the treatment of diseases in which oxidative stress plays an important role.

Biological Activities of the Essential Oil from Erigeron floribundus.

Erigeron floribundus (Asteraceae) is an herbaceous plant widely used in Cameroonian traditional medicine to treat various diseases of microbial and non-microbial origin. In the present study, we evaluated the in vitro biological activities displayed by the essential oil obtained from the aerial parts of E. floribundus, namely the antioxidant, antimicrobial and antiproliferative activities. Moreover, we investigated the inhibitory effects of E. floribundus essential oil on nicotinate mononucleotide adenylyltransferase (NadD), a promising new target for developing novel antibiotics, and Trypanosoma brucei, the protozoan parasite responsible for Human African trypanosomiasis. The essential oil composition was dominated by spathulenol (12.2%), caryophyllene oxide (12.4%) and limonene (8.8%). The E. floribundus oil showed a good activity against Staphylococcus aureus (inhibition zone diameter, IZD of 14 mm, minimum inhibitory concentration, MIC of 512 µg/mL). Interestingly, it inhibited the NadD enzyme from S. aureus (IC50 of 98 µg/mL), with no effects on mammalian orthologue enzymes. In addition, T. brucei proliferation was inhibited with IC50 values of 33.5 µg/mL with the essential oil and 5.6 µg/mL with the active component limonene. The essential oil exhibited strong cytotoxicity on HCT 116 colon carcinoma cells with an IC50 value of 14.89 µg/mL, and remarkable ferric reducing antioxidant power (tocopherol-equivalent antioxidant capacity, TEAC = 411.9 µmol·TE/g).

Syntheses, structures, and antimicrobial activity of new remarkably light-stable and water-soluble tris(pyrazolyl)methanesulfonate silver(I) derivatives of N-methyl-1,3,5-triaza-7-phosphaadamantane salt - [mPTA]BF4.

Two new silver(I) complexes of formula [Ag(mPTA)4](Tpms)4(BF4) (1) and [Ag(Tpms)(mPTA)](BF4) (2) (mPTA = N-methyl-1,3,5-triaza-7-phosphaadamantane cation, Tpms = tris(pyrazol-1-yl)methanesulfonate anion) have been synthesized and fully characterized by elemental analyses, (1)H and (31)P{(1)H} NMR, ESI-MS, and IR spectroscopic techniques. The single-crystal X-ray diffraction study of 1 discloses a noncoordinated nature of the Tpms species, existing as counterions around the highly charged metal center [Ag(mPTA)](5+), 1 being the first reported coordination compound bearing a κ(0)-Tpms. 1 features high solubility and stability in water (S25 °C ≈ 30 mg·mL(-1)). The two complexes interact with calf thymus DNA via intercalation mode, binding to the BSA with decrease of its tryptophan fluorescence with a static quenching mechanism. The two new silver complexes exhibit significant antibacterial and antifungal activities screened in vitro against the standard strains of Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans.

Engineering color variants of green fluorescent protein (GFP) for thermostability, pH-sensitivity, and improved folding kinetics.

A number of studies have been conducted to improve chromophore maturation, folding kinetics, thermostability, and other traits of green fluorescent protein (GFP). However, no specific work aimed at improving the thermostability of the yellow fluorescent protein (YFP) and of the pH-sensitive, yet thermostable color variants of GFP has so far been done. The protein variants reported in this study were improved through rational multiple site-directed mutagenesis of GFP (ASV) by introducing up to ten point mutations including the mutations near and at the chromophore region. Therefore, we report the development and characterization of fast folder and thermo-tolerant green variant (FF-GFP), and a fast folder thermostable yellow fluorescent protein (FFTS-YFP) endowed with remarkably improved thermostability and folding kinetics. We demonstrate that the fluorescence intensity of this yellow variant is not affected by heating at 75 °C. Moreover, we have developed a pH-unresponsive cyan variant AcS-CFP, which has potential use as part of in vivo imaging irrespective of intracellular pH. The combined improved properties make these fluorescent variants ideal tools to study protein expression and function under different pH environments, in mesophiles and thermophiles. Furthermore, coupling of the FFTS-YFP and AcS-CFP could potentially serve as an ideal tool to perform functional analysis of live cells by multicolor labeling.

Phytochemical Analysis, Biological Activity, and Secretory Structures of Stachys annua (L.) L. subsp. annua (Lamiaceae) from Central Italy.

Stachys annua subsp. annua, well-known in central Italy as 'stregona annuale', is an annual, small, slightly-scented herb, commonly found in fields and uncultivated areas in almost all regions of Italy. In folk medicine, its aerial parts were used as anti-catarrhal, febrifuge, tonic, and vulnerary. In the present work, the chemical composition of the flowering aerial parts was studied. The hydrodistilled volatile oil, analysed by GC/MS, showed sesquiterpenoids as the major fraction (42.5%); phytol (9.8%), germacrene D (9.2%), and spathulenol (8.5%) were the most abundant constituents. The volatile oil was assayed for antioxidant and cytotoxic activity by DPPH, ABTS, FRAP, and MTT methods. The cytotoxicity results against HCT116, A375, and MDA-MB 231 human tumor cell lines were significant, with IC50 values of 23.5, 37.2, and 41.5 µg/ml, respectively, whereas the antioxidant power was negligible. The EtOH extract was composed mainly of three glycosidic flavonoids, namely 7-{[2-O-(6-O-acetyl-ß-D-allopyranosyl)-ß-D-glucopyranosyl]oxy}-5,8-dihydroxy-2-(4-methoxyphenyl)-4H-1-benzopyran-4-one (1), 7-{[6-O-acetyl-2-O-(6-O-acetyl-ß-D-allopyranosyl)-ß-D-glucopyranosyl]oxy}-2-(3,4-dihydroxyphenyl)-5,8-dihydroxy-4H-1-benzopyran-4-one (2), and 7-{[6-O-acetyl-2-O-(ß-D-allopyranosyl)-ß-D-glucopyranosyl]oxy}-2-(3-hydroxy-4-methoxyphenyl)-5,8-dihydroxy-4H-1-benzopyran-4-one (3). On the contrary, iridoids, considered chemotaxonomic markers of the genus Stachys, were absent in this species. Finally, the morphological and histochemical survey showed that glandular trichomes were composed of two main types, i.e. peltate type A and capitate types B and C giving positive response for both lipids and polyphenols.