This study optimized subcritical water extraction (SWE) to recover bioactive compounds from Mediterranean agro-industrial by-products: citrus peels, artichoke bracts, and date pits. Extraction parameters were optimized using response surface methodology to maximize phenolic content and antioxidant capacity. The resulting extracts were characterized by their total phenolic and flavonoid contents, and phenolic profiles analyzed by HPLC. Despite compositional differences, all SWE extracts exhibited high phenolic contents (>126 mg GAE/g) and strong antioxidant activity as evaluated by DPPH (>132 mg TE/g). The antioxidant potential observed in chemical assays was complemented by the ability of the extracts to significantly reduce ROS and NO production in LPS-stimulated RAW 264.7 macrophages. These results highlight an efficient and integrated strategy to valorize Mediterranean by-products using an optimized green technology SWE to produce extracts with promising antioxidant and anti-inflammatory properties.

Consumption of (+)-catechin and (-)-epicatechin is associated with beneficial effects on human health. However, their limited systemic circulation in native form suggests that their metabolites may contribute to these effects. The aim of this study was to produce and structurally characterize glucuronide metabolites of (+)-catechin and (-)-epicatechin, which are not commercially available, and to assess their presence in plasma from volunteers who consumed a blend of grape and wild blueberry extracts. Firstly, four monoglucuronides of each flavanol were produced using rat liver microsomes and characterized by UHPLC-DAD-MS/MS and NMR. Using these compounds, we were able to confirm the presence in human plasma of three glucuronides: one known (-)-epicatechin glucuronide and two previously unidentified (+)-catechin glucuronides. Further research is needed to understand their biological role.

Background: TOTUM-854 is a patented plant extract blend characterized by its components that have previously been described for their potential health benefits in limiting hypertension onset. However, most of the literature data remain descriptive regarding the mode of action at the cellular level, especially in humans, and further investigations are required for optimized therapeutic strategies. Methods: We first demonstrated in an L-NAME mouse model that TOTUM-854 supports the prevention of hypertension in vitro and in vivo. Then, we designed an ex vivo clinical innovative approach considering the circulating metabolites produced by the digestive tract upon TOTUM-854 ingestion in humans. Human serum was collected in healthy volunteers before and after the acute intake of 3.71 g of TOTUM-854. The bioavailability of circulating metabolites was confirmed and characterized by UPLC-MS. Human serum containing TOTUM-854-derived metabolites was further processed for incubation with human endothelial cells (HUVECs), in the absence or presence of palmitate (200 µM). Results: HUVEC protection against lipotoxicity was characterized by (1) decreased ACE-1 activity (−32% p < 0.0001); (2) the inhibition of oxidative stress with decreased ROS (−12% observed by DCFDA and DHE fluorescent microscopy) and decreased Nox2 gene expression (−6.7 fold change vs. palmitate, p < 0.01); and (3) the inhibition of an inflammatory response, with a decrease in IL-1β release (−37% compared to palmitate, p < 0.001) and decreased MCP-1 and VCAM-1 gene expression (−93% p < 0.001 and −77% p < 0.001, respectively). Conclusions: Overall, this study provides insightful data regarding the protective role of TOTUM-854 in human endothelial cells. Using an innovative clinical ex vivo approach, our data support the role of TOTUM-854 circulating metabolites in vascular protection in humans.

Lipotoxic and inflammatory environment drives metabolic dysfunction-associated steatotic liver disease (MASLD) onset. As most conventional treatments present adverse side effects, alternative options such as preventive nutritional interventions have been developed, though further clinical validation is needed. In this study, we conducted an innovative ex vivo clinical investigation to examine how circulating metabolites generated after oral intake of TOTUM-448 (a plant-based, polyphenol-rich formulation) may influence hepatocyte function. UHPLC-MS/MS analysis confirmed and characterized the bioavailable polyphenol metabolites present in human serum. This metabolite-enriched serum was further used to treat HepG2 hepatocytes, with or without palmitate pretreatment (250 µM). The effects of TOTUM-448–derived metabolites on hepatocytes were evaluated by monitoring cell viability, lipid metabolism, inflammation, oxidative stress, and endoplasmic reticulum (ER) stress, all of which are central features of MASLD. Treated hepatocytes exhibited resistance to palmitate-induced lipotoxic stress, showing reduced intracellular lipid accumulation. TOTUM-448–derived metabolites also prevented the palmitate-induced upregulation of inflammatory gene expression. Additionally, while palmitate strongly upregulated CHOP and XBP1 mRNA expression as well as ATF6 and Caspase-3 activities, the presence of TOTUM-448–derived metabolites restored these ER stress markers to normal levels.

Opuntia stricta var. dillenii extracts exhibit anti-oxidative and anti-inflammatory properties, which are of significant interest for the prevention and management of metabolic dysfunction-associated fatty liver disease (MAFLD). The present study is the first to investigate the potential anti-steatotic effect of Opuntia stricta var. dillenii extracts. The aim is to evaluate the anti-steatotic effect of extracts from various parts of the plant (whole fruit, peel, pulp, and the industrial by-product, bagasse) in an in vitro model using both murine AML12 and human HepG2 hepatocytes. Results have demonstrated that all tested extracts, including those from the whole fruit, peel, pulp, and bagasse, exert an anti-steatotic effect. In murine hepatocytes, the whole fruit extract at 100 μg/mL and the peel extract at 10 μg/mL presented the highest capacity to reduce PA-induced triglyceride accumulation. In fact, the peel was the most potent extract, preventing lipid accumulation at the lowest dose used. In human HepG2 hepatocytes, the peel, pulp, and bagasse extracts at 100 μg/mL demonstrated the greatest triglyceride reduction, suggesting that the human model is less responsive. Regarding the main mechanism of action, the peel and pulp extracts seem to inhibit de novo lipogenesis. Additionally, the downregulation of the fatty acid transporter CD36 appears to contribute to the prevention of triglyceride accumulation in both extracts.

Lipotoxic and inflammatory environment drives metabolic dysfunction-associated steatotic liver disease (MASLD) onset. Since most related treatments evidence side effects, alternatives have emerged, including preventive nutritional strategies, however they require further clinical validation. In this study, we conducted an innovative ex vivo clinical study considering the circulating metabolites produced by the digestive tract following the oral intake of TOTUM-448 (a plant-based, polyphenol-rich ingredient) in humans, to provide insights on whether and how these metabolites may influence hepatocytes behavior. The bioavailability of circulating polyphenol metabolites was confirmed and characterized by UHPLC-MS/MS. Then, human serum enriched with polyphenol metabolites was further incubated with human hepatocytes (HepG2), pretreated or not with palmitate (250µM). Hepatocyte responses were monitored to determine the effects of TOTUM-448’s metabolites on cell viability, lipid metabolism, inflammation, oxidative stress and endoplasmic reticulum (ER) stress which are all key features of MASLD.Treated hepatocytes showed resistance to the induced lipotoxic stress with reduced palmitate-induced intracellular lipid storage. TOTUM-448’s metabolites also inhibited palmitate-induced inflammatory gene expression. Additionally, while palmitate potently induced both CHOP and XBP1 mRNA expression, ATF-6 and Caspase-3 activities, the presence of TOTUM-448’s metabolites normalized these ER stress markers.

Addressing the physiological effects of bioactive compounds in metabolic diseases (i.e., obesity, diabetes, liver steatosis) and establishing their mechanisms of action have been a major interest for the last decades. However, methodologies that can be applied to achieve this can vary greatly, leading to a limited type of information. Thus, the accuracy, robustness, reliability and potential (human) translation are highly reliant on the experimental design and selected methodological models. This review presents an update exploring the main features, advantages and disadvantages of most important pre-clinical models used at the present time to study the effects of bioactive compounds on metabolic diseases. Moreover, future challenges in developing new methods are also depicted. In vitro models (enzyme assays and standard two-dimensional cultures of adipocytes, skeletal muscle cells) are intrinsically well established and constitute the first choice and most widely used methods to study bioactive compounds in metabolic diseases. However, novel models such as three-dimensional cultures (spheroids, organoids) are also starting to emerge and complement traditional culture systems. Models of small organisms (C. elegans, D. melanogaster) and non-mammal vertebrates (D. rerio) represent a scientific advantage and a middle-step before traditional mammalian models (rats and mice). This article provides extensive information and a critical overview of a wide range of methods that represent present and future avenues towards a further understanding of metabolic diseases. Combining and developing new methods will be key for future progression on the effects of bioactive compounds on metabolic diseases, as well as to minimize the use of mammalian models due to ethical reasons.

Resveratrol and its glucoside, piceid, are the primary stilbenes present in wine. These compounds are well known for their pharmaceutical properties. However, these compounds can undergo chemical transformations in wines, such as polymerization in the presence of metallic reagents. This study investigates the oxidative coupling of resveratrol and piceid to form dimers, including δ-viniferin and δ-viniferin-diglucoside. These dimers were synthesized using silver acetate. The formation of these stilbenes was monitored in wine model solutions and red wines. The results indicated that resveratrol and piceid underwent transformation during heat treatment, forming their respective dimers. The polymerization of both compounds is temperature-dependent, with higher conversion rates at elevated temperatures. Notably, piceid was more reactive than resveratrol in wine. Finally, the anti-inflammatory effects of these compounds were evaluated on the RAW 264.7 macrophage cell line.

Opuntia stricta var. dillenii, a member of the Cactaceae family, produces a fruit known as prickly pear. This fruit is rich in bioactive compounds, including betalains and phenolic compounds, which play an important role in health promotion due to their antioxidant and anti-inflammatory properties. This study aims to investigate the impact of prickly pear extracts obtained from the whole fruit, peel, pulp, and an industrial by-product (bagasse) on the differentiation of 3T3-L1 pre-adipocytes. During the differentiation process, 3T3-L1 pre-adipocytes were treated with prickly pear extracts at concentrations ranging from 10 to 100 μg/mL from day 0 to day 8 post-induction. Moreover, the potential mechanisms justifying the observed effects were assessed by RT-PCR. All extracts led to an increase in both triacylglycerol accumulation and cell number. In conclusion, the analysed extracts demonstrated adipogenic effects in 3T3-L1 maturing pre-adipocytes by increasing the expression of the c/ebp-β, srebf-1, and c/ebp-α genes. Additionally, a potential anti-inflammatory effect was observed through the upregulation of adiponectin.

The therapeutic effects of saffron have been reported and described in relation to its major derivatives. Among them, in terms of saffron’s properties, crocin and crocetin absorption and bioavailability have been the most studied. Nevertheless, the metabolism of these major compounds of saffron has not yet been entirely elucidated. Current data indicate that the phase 2 metabolism of crocetins go through conjugation reactions. Crocetins could also be present in isomeric forms such as other carotenoids. Nonetheless, there are still shadow areas in regard to the measurements of the different circulating forms of crocetins after oral saffron extract administration (Safr’Inside™). In using various approaches, we propose the identification of a new cis isomeric form of crocetin, the 6-cis-crocetin. This compound was found in human serum samples after an oral administration of saffron extract. The 6-cis-crocetin represents 19% of the total crocetin measured after 45 min of consumption. These data mark, for the first time, the presence of a cis isomeric form of crocetin in human serum samples. Moreover, this study led to the development of an analytical method that is able to identify and quantify both isomeric forms (trans and cis).

Safe and anti-inflammatory plant-based natural products present an increasing focus in the treatment of chronic inflammatory diseases such as osteoarthritis or inflammatory bowel diseases. Among them, saffron, a spice derived from the stigma of Crocus sativus, could have anti-inflammatory properties and would be therefore a promising therapeutic agent for the treatment of such conditions. However, the anti-inflammatory molecular mechanisms of saffron in humans are still understudied and unclear. In this study, combining human serum metabolites and cell cultures, we evaluated the effect of circulating metabolites from the consumption of a patented saffron extract (Safr’InsideTM) on the chondrocytes and colon epithelial cell responses to inflammatory stress. Parametric or non-parametric Analysis of Variance with post hoc tests was performed. We demonstrated that human serum containing metabolites from saffron intake attenuated IL-1β-stimulated production of PGE2 and MMP-13 in chondrocyte cells and limited the increase in ICAM-1, MCP-1, iNOS, and MMP-3 in human epithelial cells following combined IL-1β and TNF-α inflammatory stimulation. Altogether, these data provide new findings into the mechanisms underlying the beneficial effects of saffron on chondrocytes and enterocyte cells at the cellular level and in the context of chronic inflammatory disorders.

Trans-ε-viniferin, a resveratrol dimer found mainly in grapevine wood, has shown protective capacities against hepatic steatosis in vivo. Nevertheless, this compound is very poorly bioavailable. Thus, the aim of the present study is to determine the potential anti-steatotic properties of 1 and 10 µM of trans-ε-viniferin and its four glucuronide metabolites in AML-12 cells treated with palmitic acid as an in vitro model of hepatic steatosis. The effect of the molecules in cell viability and triglyceride accumulation, and the underlying mechanisms of action by Real-Time PCR and Western Blot were analysed, as well as the quantification of trans-ε-viniferin and the identified bioactive metabolite inside cells and their incubation media. Interestingly, we were able to determine the triglyceride-lowering property of one of the glucuronides (trans-ε-viniferin-2-glucuronide), which acts on de novo lipogenesis, fatty acid uptake and triglyceride assembly. The glucuronides of trans-ε-viniferin would therefore be partly responsible for the in vivo observed anti-steatotic properties of the parent compound.

Colorectal cancer (CRC) is the third most frequent cancer worldwide, accounts for about 10% of the total cancer cases, and ranks as the second cause of death by cancer. CRC is more prevalent in developed countries in close causal relation with occidental diets. Due to anatomy, the diet has a strong impact on CRC. High contents in meat are acknowledged risk factors whereas a diet rich in fruits and vegetables is an established CRC protective factor. Fruits and vegetables contain numerous Bioactive Food Components (BFCs), physiologically active food compounds, beneficial on health. Preventive and therapeutic benefits of BFCs in cancer have increasingly been reported over the past 20 years. BFCs show both chemopreventive and anti-tumor properties in CRC but more interestingly, abundant research describes BFCs as enhancers of conventional cancer treatments. Despite these promising results, their clinical transferability is slowed down by bioavailability interrogations and their poorly understood hormetic effect. In this review, we would like to reposition BFCs as well-fitted for applications in CRC. We provide a synthetic overview of trustworthy BFC applications in CRC, with a special highlight on combinatory approaches and conventional cancer treatment potentiation strategies.

Viniferin is a phenolic compound belonging to the group of stilbenoids. In particular, ε-viniferin is a dimer of resveratrol, found in many plant genders, among which grapes (Vitis vinifera) are a primary source. Due to the fact that ε-viniferin is mainly present in the woody parts of plants, their use as a source of this bioactive compound is a very interesting issue in a circular economy. Both, in vitro studies carried out in pre-adipocytes and mature adipocytes and in vivo studies addressed in mice show that ε-viniferin is able to reduce fat accumulation. Moreover, it prevents the development of some obesity co-morbidities, such as type 2 diabetes, dyslipidemias, hypertension and fatty liver. ε-viniferin can be absorbed orally, but it shows a very low bioavailability. In this scenario, further research on animal models is needed to confirm the effects reported in a great number of studies; to determine which metabolites are involved, including the main one responsible for the biological effects observed and the mechanisms that justify these effects. In a further phase, human studies should be addressed in order to use ε-viniferin as a new tool for obesity management, as a nutraceutical or to be included in functional foods.

The control of oxidative stress with natural active substances could limit the development of numerous pathologies. Our objective was to study the antiradical effects of resveratrol (RSV), ε-viniferin (VNF), and vitisin B (VB) alone or in combination, and those of a standardized stilbene-enriched vine extract (SSVE). In the DPPH-, FRAP-, and NO-scavenging assays, RSV presented the highest activity with an IC50 of 81.92 ± 9.17, 13.36 ± 0.91, and 200.68 ± 15.40 µM, respectively. All binary combinations resulted in additive interactions in the DPPH- and NO-scavenging assays. In the FRAP assay, a synergic interaction for RSV + VNF, an additive for VNF + VB, and an antagonistic for RSV + VB were observed. The ternary combination of RSV + VNF + VB elicited an additive interaction in the DPPH assay and a synergic interaction in the FRAP- and NO-scavenging assays. There was no significant difference between the antioxidant activity of the SSVE and that of the combination of RSV + VNF. In conclusion, RSV presented the highest effects, followed by VNF and VB. The interactions revealed additive or synergistic effects, depending on the combination of the stilbenes and assay.

Abstract Hydrocortisone (HyC), a hydrophobic pharmaceutical active, was encapsulated in multi-lamellar liposomes (MLLs) composed of P100, a mixture of phospholipids, and Tween®80. Three different HyC-loaded formulations were designed to target the stratum corneum, the living epidermis and the hypodermis. The impact of encapsulation on their size, elasticity and zeta potential, the three key factors controlling MLLs skin penetration, was studied. Raman mapping of phospholipids and HyC allowed the localisation of both components inside an artificial skin, Strat-M®, demonstrating the efficiency of the targeting. Percutaneous permeation profiles through excised human skin were performed over 48 h, supporting results on artificial skin. Their modelling revealed that HyC encapsulated in MLLs, designed to target the stratum corneum and living epidermis, exhibited a non-Fickian diffusion process. In contrast, a Fickian diffusion was found for HyC administered in solution, in a pharmaceutical cream and in transdermal MLLs. These results allowed us to propose a mechanism of interaction between HyC-containing MLLs and the skin.

TOTUM-070 is a patented polyphenol-rich blend of five different plant extracts showing separately a latent effect on lipid metabolism and potential synergistic properties. In this study, we investigated the health benefit of such a formula. Using a preclinical model of high fat diet, TOTUM-070 (3 g/kg of body weight) limited the HFD-induced hyperlipemia with a reduction in triglyceride (−32% after 6 weeks; −20.3% after 12 weeks) and non-HDL cholesterol levels (−21% after 6 weeks; −38.4% after 12 weeks). To further investigate such a benefit and its underlying mechanisms in humans, we designed an ex vivo clinical approach to collect the circulating bioactives resulting from TOTUM-070 ingestion and to determine their biological activities on human hepatocytes. Human serum was obtained from healthy subjects before and after intake of TOTUM-070 (4995 mg). The presence of circulating metabolites was assessed by UPLC-MS/MS. Serum containing metabolites was further incubated with hepatocytes cultured in a lipotoxic environment (palmitate, 250 µM). RNA sequencing analyses show that lipid metabolism was one of the most impacted processes. Using histologic, proteomic, and enzymatic assays, the effects of human TOTUM-070 bioactives on hepatocyte metabolism were characterized by (1) the inhibition of lipid storage, including both (2) triglycerides (−41%, p < 0.001) and (3) cholesterol (−50%, p < 0.001) intracellular content, (4) a reduced de novo cholesterol synthesis (HMG-CoA reductase activity −44%, p < 0.001), and (5) a lowered fatty acid synthase protein level (p < 0.001). Altogether, these data support the beneficial impact of TOTUM-070 on lipid metabolism and provide new biochemical insights in human mechanisms occurring in liver cells.

Our previous work showed that the size, elasticity and charge of multi-lamellar liposomes (MLLs) could not be considered separately to predict the fate of MLLs in the skin [1]. Based on this study, we developed several MLLs formulations containing a corticosteroid, betamethasone 17-valerate (B17) to transport the drug into the stratum corneum, living epidermis, dermis or through the skin. MLLs encapsulation efficiency was found to exceed 74 ± 3 % in all cases. In addition, we showed that MLLs protected the corticosteroid from thermal degradation. Comparing the penetration depth of all MLLs within artificial skin measured by Raman imaging, we established an equation for its determination, given the MLLs elasticity and size. This equation was verified experimentally on human explants: quantification of B17 in each skin layer, as well as its transdermal passage by ultra-high performance liquid chromatography, confirmed that B17 was predominantly and significantly transported in the desired layer. Eventually, we showed the benefits in using B17-loaded MLLs instead of a B17-containing pharmaceutical cream in terms of B17 penetration and thermal degradation.

Resveratrol belongs to the Bioactive Food Component (BFC) family. It seems admitted that its cytotoxic action impacts tumor cells and spares healthy cells, but the published proofs remain rare. We hypothesized that cells may differentially metabolize resveratrol and lead to different systemic impacts. For this, resveratrol metabolization was evaluated by ultra-high-performance liquid chromatography (UHPLC) coupled with diode array detection (DAD), and correlated with the expression of Uridyl-diphosphate-Glucuronosyl Transferase 1A (UGT1A) genes. The expression of UGT1A genes in human colorectal tissues was studied with RNAseq databases. Functional validation of UGT1A enzymes implication in resveratrol sensitivity of colorectal cells established by UGT1A expression modulation. As resveratrol impacts the S phase of the cell cycle, nucleotide metabolic balance was assessed. We found that resveratrol was more cytotoxic in cells with downregulation of UGTs, i.e. tumor cells. Conversely, overexpression of the UGT1A10 gene in an initial resveratrol-sensitive tumor cell line restored the metabolization accompanied by cytotoxicity diminution. Resveratrol affected intestinal sensitive tumor cell homeostasis with a cell growth/proliferation decoupling, cell-cycle modulation, and UXP/AXP nucleotide imbalance resulting in a global reduction of transcription and translation. This impact on global cell activity was restricted to tumor cells. This study improves resveratrol's general knowledge and explains how its antitumor action can spare non-tumor cells. It also paves the way to select colorectal tumors eligible for resveratrol treatment potentiation without additional toxicity to healthy digestive tissues.