Extracts of the Tiger Milk Mushroom (Lignosus rhinocerus) Enhance Stress Resistance and Extend Lifespan in Caenorhabditis elegans via the DAF-16/FoxO Signaling Pathway.

The tiger milk mushroom, Lignosus rhinocerus (LR), exhibits antioxidant properties, as shown in a few in vitro experiments. The aim of this research was to study whether three LR extracts exhibit antioxidant activities in Caenorhabditis elegans. In wild-type N2 nematodes, we determined the survival rate under oxidative stress caused by increased intracellular ROS concentrations. Transgenic strains, including TJ356, TJ375, CF1553, CL2166, and LD1, were used to detect the expression of DAF-16, HSP-16.2, SOD-3, GST-4, and SKN-1, respectively. Lifespan, lipofuscin, and pharyngeal pumping rates were assessed. Three LR extracts (ethanol, and cold and hot water) protected the worms from oxidative stress and decreased intracellular ROS. The extracts exhibited antioxidant properties through the DAF-16/FOXO pathway, leading to SOD-3 and HSP-16.2 modification. However, the expression of SKN-1 and GST-4 was not changed. All the extracts extended the lifespan. They also reduced lipofuscin (a marker for aging) and influenced the pharyngeal pumping rate (another marker for aging). The extracts did not cause dietary restriction. This novel study provides evidence of the functional antioxidant and anti-aging properties of LR. Further studies must confirm that they are suitable for use as antioxidant supplements.

Antifungal and anti-inflammatory potential of the endangered aromatic plant Thymus albicans.

Thymus albicans is an endemic species of the Iberian Peninsula with a vulnerable conservation status. In an attempt to contribute to the valorization of this species, the present study brings new insights on the antifungal and anti-inflammatory mechanism of action of T. albicans essential oil. The antifungal activity of the oil and its major compounds was assessed for the first time against standard and clinically isolated strains of yeasts and filamentous fungi. The effect on the two major virulence factors of Candida albicans (germ tube formation and biofilm disruption) was considered in more detail. At 0.08 µL/mL, the oil inhibited C. albicans germ tube formation by more than 40% and decreased biofilm biomass at MIC values, thus pointing out its antivirulent potential. The anti-inflammatory activity of the essential oil was investigated on LPS-stimulated mouse macrophages (RAW 264.7) by evaluating the levels of several pro-inflammatory mediators, namely nitric oxide (NO), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). T. albicans oil reduced the production of nitrites, a NO derived sub-product, at non-cytotoxic concentrations of 0.32 and 0.64 µL/mL, by 27 and 41%, respectively. In addition, the iNOS protein levels of essential oil pre-treated cells were reduced by 14%. Overall, the high essential oil yield of T. albicans as well as its bioactive effects at concentrations without cytotoxicity, encourage further studies on the potential pharmacological applications of this species. Furthermore, these results raise awareness for the need to preserve endangered species that may hold relevant medicinal value.

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.

Pro-oxidant and lifespan extension effects of caffeine and related methylxanthines in Caenorhabditis elegans.

Caffeine and related purine alkaloids are common ingredients of many stimulating drinks. Studies have shown that lower concentrations of caffeine have a protective role in aging-related disorders. However, the associated mode of action of caffeine and its related methylxanthines is still not clear. In this study, we demonstrated that caffeine and theophylline promote longevity in Caenorhabditis elegans. Lifespan studies with the wild type, DAF-16 and SKN-1 mutant strains indicated that the methylxanthines-mediated lifespan extension in C. elegans was independent of DAF-16/FOXO and SKN-1. All the tested methylxanthines could protect C. elegans against acute oxidative stress. At early stages of life, an increase of ROS (reactive oxygen species) induced the translocation of DAF-16 and SKN-1, resulting in upregulation of several antioxidant genes, for example, sod-3p::GFP, gst-4p::GFP, gcs-1p::GFP; and downregulation of hsp-16.2p::GFP. RT-PCR corroborates the upregulation of gst-4 and skn-1 genes. The expression of DAF-16 decreased although its nuclear translocation was induced.

Styphnolobium japonicum (L.) Schott Fruits Increase Stress Resistance and Exert Antioxidant Properties in Caenorhabditis elegans and Mouse Models.

Styphnolobium japonicum (L.) Schott is a popular Asian tree widely used in traditional medicine. The current study explored the potential stress resistance and antioxidant activities of its fruits. Phytochemical profiling of the hydroalcoholic fruit extract was done via high performance liquid chromatography-photodiode array-electrospray ionization-mass/mass (HPLC-PDA-ESI-MS/MS). Twenty four phenolic constituents were tentatively identified in the extract. The Caenorhabditis elegans (C. elegans) nematode model in addition to trimethyltin (TMT)-induced neurotoxicity mouse model were used for in vivo evaluation of its antioxidant properties. The ability of the extract to enhance stress resistance was manifested through increasing survival rate by 44.7% and decreasing basal reactive oxygen species (ROS) levels by 72.3% in C. elegans. In addition, the extract increased the levels of the stress response enzyme superoxide dismutase-3 (Sod-3) by 55.5% and decreased the expression of heat shock protein-16.2 (Hsp-16.2) in nematodes, which had been challenged by juglone, by 21%. Using a mouse model, the extract significantly decreased the expression of the oxidative stress marker malondialdehyde (MDA). Furthermore, an elevation in the levels of the antioxidant marker glutathione (GSH), SOD and heme oxygenase-1 (HO-1) enzymes were observed. Our findings imply that Styphnolobium japonicum has the potential to be used in future studies focusing on diseases associated with oxidative stress.

Phenolic Compounds from Populus alba L. and Salix subserrata Willd. (Salicaceae) Counteract Oxidative Stress in Caenorhabditis elegans.

Utilizing bioassay- and TLC-guided column chromatography, fifteen secondary metabolites from Populus alba and eight compounds from Salix subserrata were isolated, including a novel plant metabolite salicyl ether and characterized using ultralviolet light (UV) absorbance, mass spectrometry (MS), 1H-, 13C-NMR (nuclear magnetic resonance), heteronuclear single quantum coherence spectroscopy (HSQC) and heteronuclear multiple bond correlation (HMBC). The extracts, their sub-fractions and the isolated compounds exhibited promising antioxidant activities in vitro in DPPH and FRAP assays. Also, the extracts of P. alba leaf (PL), shoots (PS), and S. subserrata leaf (SL) demonstrated substantial antioxidant activities in vivo in the multicellular model organism Caenorhabditis elegans. For the first time, the isolated secondary metabolites, aromadendrin, tremuloidin, salicin, isorhamnetin-3-O-ß-d-rutinoside, gallocatechin, triandrin, and chrysoeriol-7-O-glucuronide were investigated. They exhibited substantial antioxidant activities in vivo. Salicin, isorhamnetin-3-O-ß-d-rutinoside and gallocatechin, in particular, protected the worms against a lethal dose of the pro-oxidant juglone (80 µM), decreased the endogenous reactive oxygen species (ROS) level to 45.34%, 47.31%, 68.09% and reduced juglone- induced hsp-16.2::GFP (green fluorescence protein) expression to 79.62%, 70.17%, 26.77%, respectively. However, only gallocatechin induced higher levels of sod-3 expression. These findings support the traditional use of Populus alba and Salix subserrata for treating inflammation especially when ROS are involved.

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.

The marine Gram-negative bacterium Novosphingobium sp. PP1Y as a potential source of novel metabolites with antioxidant activity.

OBJECTIVE: The antioxidant activity and protective effect of a methanolic extract obtained from the marine Gram-negative bacterium Novosphingobium sp. PP1Y, isolated from the surface water of a polluted area in the harbour of Pozzuoli (Naples, Italy), was evaluated. RESULTS: The extract was tested in vitro on epithelial colorectal adenocarcinoma cells and in vivo on Caenorhabditis elegans. It showed strong protective activity against oxidative stress, in both experimental systems, by preventing ROS accumulation. In the case of the cells, pre-treatment with methanolic extract was also able to maintain unaltered intracellular GSH levels and phosphorylation levels of mitogen-activated protein kinases p38. Instead, in the case of the worms, the extract was able to modulate the expression levels of stress response genes, by activating the transcription factor skn-1. CONCLUSIONS: From a biotechnological and economical point of view, antioxidants from microorganisms are convenient as they provide a valid alternative to chemical synthesis and respond to the ever-growing market demand for natural antioxidants.

Evaluation of antioxidant and neuroprotective activities of Cassia fistula (L.) using the Caenorhabditis elegans model.

BACKGROUND: Cassia fistula (L.) (Fabaceae) is a medicinal plant from tropical Asia. It is known for its marked antioxidant activity, which is attributed to its high phenolic content. The present study aims at testing both the antioxidant and neuroprotective effects of a hydroalcoholic extract from the aerial parts of Cassia fistula using the Caenorhabditis elegans model, which is widely used in this context. METHODS: Chemical profiling of secondary metabolites that seem to be responsible for both antioxidant and neuroprotective capacities was carried out by HPLC/PDA/ESI-MSn. Antioxidant activity was tested in vitro by CUPRAC and DPPH assays. In vivo antioxidant and neuroprotective activities were investigated using the C. elegans model. RESULTS: The Cassia extract improved the survival rate of the nematodes and protected them against oxidative stress. In addition, a decrease in the accumulation of reactive oxygen species (ROS) was observed. The important role of DAF-16/FOXO pathway was confirmed through an increased nuclear localization of the DAF-16 transcription factor, increased expression of SOD-3 stress response gene and decreased expression of HSP-16.2. Furthermore, the putative involvement of SKN-1/NRF2 pathway was demonstrated by a decrease in GST-4 levels. A neuroprotective activity of the Cassia extract was shown by a decline in polyglutamine (polyQ40) aggregate formation and a delay in paralysis caused by amyloid beta (Aß1-42) accumulation. DISCUSSION: The Cassia extract exhibits substantial antioxidant and neuroprotective activities in vivo, which might provide a rich and novel source of natural antioxidants and neuroprotective compounds to be further studied for the use in various food and cosmetic industrial fields.

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.

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.