Piquiá Shells (Caryocar villosum): A Fruit by-Product with Antioxidant and Antiaging Properties in Caenorhabditis elegans.

In a context of rising demand for sustainable antiaging interventions, fruit processing by-products are a promising source of bioactive compounds for the production of antiaging dietary supplements. Piquiá (Caryocar villosum) is a native Amazonian fruit consisting of 65% nonedible shells. In the present study, the phytochemical profile of a hydroalcoholic extract of piquiá shells (CV) was characterized by LC-MS/MS analysis. Its antioxidant and antiaging activities were investigated using the nematode Caenorhabditis elegans as an in vivo model. CV is mainly composed by hydrolysable tannins and triterpenoid saponins. The extract enhanced stress resistance of wild-type and mutant worms by reducing the intracellular levels of reactive oxygen species (ROS) and by increasing their survival against a lethal dose of the prooxidant juglone. These effects involved the upregulation of sod-3 and downregulation of gst-4 and hsp-16.2, studied through the GFP fluorescent reporter intensity and at the transcriptional level by qRT-PCR analysis. CV extended the lifespan of wild-type worms in a DAF-16/FoxO- and SKN-1/Nrf-dependent manner. Taken together, our findings indicate piquiá shells as potential candidates for nutraceutical applications. Further studies are needed to validate the relevance of our findings to antiaging interventions in humans.

Antioxidant Activity of an Aqueous Leaf Extract from Uncaria tomentosa and Its Major Alkaloids Mitraphylline and Isomitraphylline in Caenorhabditis elegans.

Uncaria tomentosa (Rubiaceae) has a recognized therapeutic potential against various diseases associated with oxidative stress. The aim of this research was to evaluate the antioxidant potential of an aqueous leaf extract (ALE) from U. tomentosa, and its major alkaloids mitraphylline and isomitraphylline. The antioxidant activity of ALE was investigated in vitro using standard assays (DPPH, ABTS and FRAP), while the in vivo activity and mode of action were studied using Caenorhabditis elegans as a model organism. The purified alkaloids did not exhibit antioxidant effects in vivo. ALE reduced the accumulation of reactive oxygen species (ROS) in wild-type worms, and was able to rescue the worms from a lethal dose of the pro-oxidant juglone. The ALE treatment led to a decreased expression of the oxidative stress response related genes sod-3, gst-4, and hsp-16.2. The treatment of mutant worms lacking the DAF-16 transcription factor with ALE resulted in a significant reduction of ROS levels. Contrarily, the extract had a pro-oxidant effect in the worms lacking the SKN-1 transcription factor. Our results suggest that the antioxidant activity of ALE in C. elegans is independent of its alkaloid content, and that SKN-1 is required for ALE-mediated stress resistance.

Bark Extract of the Amazonian Tree Endopleura uchi (Humiriaceae) Extends Lifespan and Enhances Stress Resistance in Caenorhabditis elegans.

Endopleura uchi (Huber) Cuatrec (Humiriaceae), known as uxi or uxi-amarelo in Brazil, is an endemic tree of the Amazon forest. In traditional medicine, its stem bark is used to treat a variety of health disorders, including cancer, diabetes, arthritis, uterine inflammation, and gynecological infections. According to HPLC analysis, the main constituent of the bark extract is the polyphenol bergenin. In the current study, we demonstrate by in vitro and in vivo experiments the antioxidant potential of a water extract from the stem bark of E. uchi. When tested in the model organism Caenorhabditis elegans, the extract enhanced stress resistance via the DAF-16/FOXO pathway. Additionally, the extract promoted an increase in the lifespan of the worms independent from caloric restriction. It also attenuated the age-related muscle function decline and formation of polyQ40 plaques, as a model for Huntington's disease. Thus, these data support anti-aging and anti-oxidant properties of E. uchi, which has not yet been described. More studies are needed to assess the real benefits of E. uchi bark for human health and its toxicological profile.

Calycophyllum spruceanum (Benth.), the Amazonian "Tree of Youth" Prolongs Longevity and Enhances Stress Resistance in Caenorhabditis elegans.

The tree popularly known in Brazil as mulateiro or pau-mulato (Calycophyllum spruceanum (Benth.) K. Schum.) is deeply embedded in the herbal medicine of the Amazon region. Different preparations of the bark are claimed to have anti-aging, antioxidant, antimicrobial, emollient, wound healing, hemostatic, contraceptive, stimulant, and anti-diabetic properties. The current study aims to provide the first step towards a science-based evidence of the beneficial effects of C. spruceanum in the promotion of longevity and in the modulation of age-related markers. For this investigation, we used the model system Caenorhabditis elegans to evaluate in vivo antioxidant and anti-aging activity of a water extract from C. spruceanum. To chemically characterize the extract, HPLC MS (High Performance Liquid Chromatography Mass Spectrometry)/MS analyses were performed. Five secondary metabolites were identified in the extract, namely gardenoside, 5-hydroxymorin, cyanidin, taxifolin, and 5-hydroxy-6-methoxycoumarin-7-glucoside. C. spruceanum extract was able to enhance stress resistance and to extend lifespan along with attenuation of aging-associated markers in C. elegans. The demonstrated bioactivities apparently depend on the DAF-16/FOXO pathway. The data might support the popular claims of mulateiro as the "tree of youth", however more studies are needed to clarify its putative benefits to human health.

Anti-Aging and Antioxidant Potential of Paullinia cupana var. sorbilis: Findings in Caenorhabditis elegans Indicate a New Utilization for Roasted Seeds of Guarana.

Background: Roasted seeds of Amazonian guarana (Paullinia cupana var. sorbilis; Sapindaceae) are popular in South America due to their stimulant activity on the central nervous system (CNS). Rich in purine alkaloids, markedly caffeine, the seeds are extensively used in the Brazilian beverage industry for the preparation of soft drinks and as additives in energy drinks. Methods: To investigate the putative anti-aging and antioxidant activity of guarana, we used the model organism Caenorhabditis elegans. Chemical analyses were performed using high-performance liquid chromatography (HPLC) and electrospray ionization-mass spectrometry (ESI-MS/MS). Results: When tested in the model system Caenorhabditis elegans, the water extract from roasted guarana seeds enhanced resistance against oxidative stress, extended lifespan and attenuated aging markers such as muscle function decline and polyQ40 aggregation. Conclusions: In the current study, we demonstrate that guarana extracts can work as a powerful antioxidant in vivo; moreover, guarana extracts exhibit anti-aging properties. Our results suggest that the biological activities of guarana go beyond the extensively reported CNS stimulation.

Cucurbitacins: elucidation of their interactions with the cytoskeleton.

Cucurbitacins, a class of toxic tetracyclic triterpenoids in Cucurbitaceae, modulate many molecular targets. Here we investigated the interactions of cucurbitacin B, E and I with cytoskeletal proteins such as microtubule and actin filaments. The effects of cucurbitacin B, E and I on microtubules and actin filaments were studied in living cells (Hela and U2OS) and in vitro using GFP markers, immunofluorescence staining and in vitro tubulin polymerization assay. Cucurbitacin B, E and I apparently affected microtubule structures in living cells and cucurbitacin E inhibited tubulin polymerization in vitro with IC50 value of 566.91 ± 113.5 µM. Cucurbitacin E did not affect the nucleation but inhibited the growth phase and steady state during microtubule assembly in vitro. In addition, cucurbitacin B, E and I all altered mitotic spindles and induced the cell cycle arrest at G2/M phase. Moreover, they all showed potent effects on actin cytoskeleton by affecting actin filaments through the depolymerization and aggregation. The interactions of cucubitacin B, E and I with microtubules and actin filaments present new insights into their modes of action.

Identification of phenolic secondary metabolites from Schotia brachypetala Sond. (Fabaceae) and demonstration of their antioxidant activities in Caenorhabditis elegans.

BACKGROUND: Schotia brachypetala Sond. (Fabaceae) is an endemic tree of Southern Africa whose phytochemistry and pharmacology were slightly studied. The present work aimed at profiling the major phenolics compounds present in the hydro-alcohol extract from S. brachypetala leaves (SBE) using LC/HRESI/MS/MS and NMR and prove their antioxidant capabilities using novel methods. METHODS: In vitro assays; DPPH, TEAC persulfate decolorizing kinetic and FRAP assays, and in vivo assays: Caenorhabditis elegans strains maintenance, Intracellular ROS in C. elegans, Survival assay, GFP expression and Subcellular DAF-16 localization were employed to evaluate the antioxidant activity. RESULTS: More than forty polyphenols, including flavonoid glycosides, galloylated flavonoid glycosides, isoflavones, dihydrochalcones, procyanidins, anthocyanins, hydroxy benzoic acid derivatives, hydrolysable tannins, and traces of methylated and acetylated flavonoid derivatives were identified. Three compounds were isolated and identified from the genus Schotia for the first time, namely gallic acid, myricetin-3-O-α-L-1C4-rhamnoside and quercetin-3-O-L-1C4-rhamnoside. The total phenolics content of SBE was (376 mg CAE/g), followed by flavonoids (67.87 QE/g). In vitro antioxidant activity of SBE was evidenced by DPPH radical scavenging activity (IC50 of 9 µg/mL), FRAP ferric reducing activity (5,000 mol Fe2+ E/mg) and ABTS peroxide inhibiting activity (1,054 mM Trolox E/mg). The tested extract was able to protect the worms against juglone induced oxidative stress, an increased survival rate (up to 41%) was recorded, when compared with the control group (11%) and attenuate the reactive oxygen species (ROS) accumulation in dose-dependent and reached up to 72% for the highest tested concentration. SBE was also able to attenuate the levels of heat shock protein (HSP) expression in dose-dependent up to 60% in the 150 µg SBE/mL group. In DAF-16 Subcellular localization SBE treated worms showed nuclear localization pattern up to 78%, while it was only 5% in the untreated control group. DISCUSSION: A pronounced antioxidant activity in vivo, which can be attributed to its ability to promote the nuclear translocation of DAF-16/FOXO, the main transcription factor regulating the expression of stress response genes. The remarkable antioxidant activity in vitro and in vivo correlates to SBE rich phenolic profile.

The Interference of Selected Cytotoxic Alkaloids with the Cytoskeleton: An Insight into Their Modes of Action.

Alkaloids, the largest group among the nitrogen-containing secondary metabolites of plants, usually interact with several molecular targets. In this study, we provide evidence that six cytotoxic alkaloids (sanguinarine, chelerythrine, chelidonine, noscapine, protopine, homoharringtonine), which are known to affect neuroreceptors, protein biosynthesis and nucleic acids, also interact with the cellular cytoskeleton, such as microtubules and actin filaments, as well. Sanguinarine, chelerythrine and chelidonine depolymerized the microtubule network in living cancer cells (Hela cells and human osteosarcoma U2OS cells) and inhibited tubulin polymerization in vitro with IC50 values of 48.41 ± 3.73, 206.39 ± 4.20 and 34.51 ± 9.47 µM, respectively. However, sanguinarine and chelerythrine did not arrest the cell cycle while 2.5 µM chelidonine arrested the cell cycle in the G2/M phase with 88.27% ± 0.99% of the cells in this phase. Noscapine and protopine apparently affected microtubule structures in living cells without affecting tubulin polymerization in vitro, which led to cell cycle arrest in the G2/M phase, promoting this cell population to 73.42% ± 8.31% and 54.35% ± 11.26% at a concentration of 80 µM and 250.9 µM, respectively. Homoharringtonine did not show any effects on microtubules and cell cycle, while the known microtubule-stabilizing agent paclitaxel was found to inhibit tubulin polymerization in the presence of MAPs in vitro with an IC50 value of 38.19 ± 3.33 µM. Concerning actin filaments, sanguinarine, chelerythrine and chelidonine exhibited a certain effect on the cellular actin filament network by reducing the mass of actin filaments. The interactions of these cytotoxic alkaloids with microtubules and actin filaments present new insights into their molecular modes of action.

Cytoskeletal interference - A new mode of action for the anticancer drugs camptothecin and topotecan.

The anticancer drugs camptothecin (CPT) and topotecan (TPT) are known DNA topoisomerase I inhibitors which cause DNA damage and lead to cell death. In this study we provide evidence that CPT and TPT also interfere with the elements of cytoskeleton - microtubules and actin filaments which could be partly responsible for their cytotoxic properties. CPT and TPT apparently affected microtubule structures in living cells (Hela and U2OS) and inhibited tubulin polymerization in vitro with IC50 values of 74.57±9.96µM and 121.55±58.68µM, respectively. TPT significantly affected the nucleation and growth phase during the microtubule assembly in vitro, whereas the mode of action of CPT was different in that it specifically affected the 'tread milling' of polymerized microtubules. Cell cycle effects of CPT and TPT varied with their concentrations. CPT and TPT induced G2/M arrest and promoted the population to 76.94±11.20% and 83.91±2.43% at a concentration of 9.4nM and 46.9nM, respectively. As the concentration increased, cells were blocked in S phase with a dose-dependent reduction in G2/M population. In addition, CPT and TPT exhibited a certain effect on actin filaments by reducing the mass of actin filaments. The interactions of CPT and TPT with microtubules and actin filaments present new insights into their modes of action.

Chemical composition and biological activity of the essential oil from Thymus lanceolatus.

Thymus lanceolatus is a rare species, which grows wild in Algeria and Tunis. It is used traditionally as a drink and to flavor and preserve meat and poultry. The composition of the essential oil was determined by GLC/FID and GLC/MS. Forty-nine components were identified and quantified, accounting for 96.75% of the total detected components in the oil. The oxygenated monoterpenes (74.85%) constitute the major class of volatile secondary metabolites in the oil. Thymol was the most abundant constituent (69.61%) followed by γ-terpinene (8.38%). The antioxidant activity was evaluated using both diphenylpicrylhydrazyl (DPPH˙) reduction and 2-deoxyribose (2-DR) degradation prevention methods. The oil showed a very potent antioxidant activity with IC(50) values of 0.20 ± 0.07 and 4.96 ± 0.39 µg/mL for the DPPH˙ and 2-DR methods, respectively. The antimicrobial activity of the oil was assessed using the agar diffusion method, and the in vitro cytotoxicity on five different cancer cells was examined using the MTT assay. The oil revealed promising inhibitory activity against Gram positive bacteria, especially Bacillus subtilis and Streptococcus pyogenes with an MIC value of 62.5 µg/mL. Additionally, the highest cytotoxic activity was observed against the HL-60 cells with an IC(50) of 113.5 µg/mL. These results validate some of their traditional uses in food preservation.

An Anthocyanin-Rich Extract of Acai (Euterpe precatoria Mart.) Increases Stress Resistance and Retards Aging-Related Markers in Caenorhabditis elegans.

Acai fruits (Euterpe precatoria) are rich in antioxidant anthocyanins. Acai consumption is believed to have many health benefits; however, relevant detailed scientific investigations are limited. The current study aimed to investigate an anthocyanin-rich extract from E. precatoria fruits (AE) with regard to its antioxidant and antiaging properties using the model organism Caenorhabditis elegans. AE can protect the worms against oxidative stress and can ameliorate accumulation of reactive oxygen species in vivo. The expression of stress-response genes, such as sod-3::GFP, was upregulated while hsp-16::GFP was down-regulated after AE treatment. Studies with DAF-16/FOXO mutants indicated that some of the antioxidant effects are mediated by this transcription factor. AE can modulate the development of age-related markers, such as pharyngeal pumping. Despite the apparent antioxidant activity, no lifespan-prolonging effect was observed.

Combinations of alkaloids affecting different molecular targets with the saponin digitonin can synergistically enhance trypanocidal activity against Trypanosoma brucei brucei.

The flagellate Trypanosoma brucei causes sleeping sickness in humans and nagana in animals. Only a few drugs are registered to treat trypanosomiasis, but those drugs show severe side effects. Also, because some pathogen strains have become resistant, new strategies are urgently needed to combat this parasitic disease. An underexplored possibility is the application of combinations of several trypanocidal agents, which may potentiate their trypanocidal activity in a synergistic fashion. In this study, the potential synergism of mutual combinations of bioactive alkaloids and alkaloids with a membrane-active steroidal saponin, digitonin, was explored with regard to their effect on T. b. brucei. Alkaloids were selected that affect different molecular targets: berberine and chelerythrine (intercalation of DNA), piperine (induction of apoptosis), vinblastine (inhibition of microtubule assembly), emetine (intercalation of DNA, inhibition of protein biosynthesis), homoharringtonine (inhibition of protein biosynthesis), and digitonin (membrane permeabilization and uptake facilitation of polar compounds). Most combinations resulted in an enhanced trypanocidal effect. The addition of digitonin significantly stimulated the activity of almost all alkaloids against trypanosomes. The strongest effect was measured in a combination of digitonin with vinblastine. The highest dose reduction indexes (DRI) were measured in the two-drug combination of digitonin or piperine with vinblastine, where the dose of vinblastine could be reduced 9.07-fold or 7.05-fold, respectively. The synergistic effects of mutual combinations of alkaloids and of alkaloids with digitonin present a new avenue to treat trypanosomiasis but one which needs to be corroborated in future animal experiments.