Bioavailable Human Metabolites from TOTUM- 448 (Plant-Based, Polyphenol-Rich Ingredient) Maintain Liver Cell Functionality in a Lipotoxic Context that Drives MASLD Onset

Bioavailable Human Metabolites from TOTUM- 448 (Plant-Based, Polyphenol-Rich Ingredient) Maintain Liver Cell Functionality in a Lipotoxic Context that Drives MASLD Onset | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Bioavailable Human Metabolites from TOTUM- 448 (Plant-Based, Polyphenol-Rich Ingredient) Maintain Liver Cell Functionality in a Lipotoxic Context that Drives MASLD Onset Fabien Wauquier, Vivien Chavanelle, Annie Bouchard-Mercier, Line Boutin-Wittrant, and 15 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7460979/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Dec, 2025 Read the published version in Scientific Reports → Version 1 posted 15 You are reading this latest preprint version Abstract 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. Biological sciences/Biochemistry Biological sciences/Cell biology clinical trial ex vivo hypertension plant extract oxidative stress lipotoxic stress human metabolites human endothelial cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Chronic liver disease is a social and economic burden 1 . Among the most common liver disorders is metabolic dysfunction–associated steatotic liver disease (MASLD), redefined in 2023 and previously described as non-alcoholic fatty liver disease (NAFLD) 2 . Its prevalence is estimated at about 30% of the adult population worldwide 3 – 5 while it can reach up to 90% in obese individuals and 60% in people with diabetes 16 . Up to 20% of the normal-weight people are also concerned. MASLD is characterized by the association of excessive fat accumulation in liver and at least one metabolic risk factor including overweight/obesity, type 2 diabetes, metabolic syndrome, hypertriglyceridemia andhypercholesterolemia. 2 . This steatosis results from the accumulation of lipids in liver parenchymal cells and triggers hepatic inflammation and fibrosis. Over time, it leads to cirrhosis and its associated complications 7 . Thus, MASLD is correlated with higher risks of cancer and cardiovascular disease mortality 8 , 9 and considered as the second most common reason for liver transplantation in the United States and Europe 10 , 11 . In light of this, strategies promoting hepatic lipid clearance are of particular interest. Different pharmacological approaches are acknowledged including statins, SGLT-2 inhibitors, GLP-1 agonists, pioglitazone, vitamin E and resmetirom 3 , 12 , 13 . Resmetirom, a liver-directed thyroid hormone receptor beta-selective agonist, has recently been approved by FDA for MASLD treatment 14 – 16 . However, these treatments designed to cure rather than to prevent evidence side effects (e.g., muscle related issues for statins; nausea and diarrhea for resmetirom) 13 and may be quite expensive for patients and healthcare systems 17 . This highlights the need for alternative, safer, and preventive options. In this context, lifestyle interventions including dietary changes and exercise represent the initial step in the treatment as well as nutritional or nutraceutical strategies which have become a major field for therapeutic innovation 1 . Among natural bioactives of interest, polyphenols are plant compounds known for their antioxidant and anti-inflammatory properties. Regarding their role in MASLD, the last five years have accounted for more than half of the total publications related to this theme, demonstrating the growing interest in nutritional approaches in the field. Yet, only about 7% of these investigations have been conducted at the clinical level. Certain plant extracts are already known for their hepato-protective properties. For example, artichoke leaf extract (Cynara scolymus) is widely described in the literature for its choleretic, hepatoprotective 18 and lipid metabolism regulation properties 19 – 22 . Nevertheless, these strategies lack of relevant clinical data and optimization of the expected benefits, particularly through nutritional synergies, require further clinical validation. TOTUM-448 is a patented polyphenol-rich blend of 5 different plant extracts (olive leaf (Olea europaea), bilberry (Vaccinium myrtillus), artichoke leaf (Cynara scolymus), chrysanthellum (Chrysanthellum indicum subsp. afroamericanum B.L. Turner), black pepper (Piper nigrum)) plus choline with potential synergistic properties on liver function. The aim of this work was to investigate whether and how this combination of different polyphenols can maintain, preserve or even improve hepatocyte metabolism in a lipotoxic context, supporting preventive strategies in the early stages of MASLD. To address this working hypothesis, we collected human serum containing circulating bioactive metabolites resulting from TOTUM-448 ingestion and according to the Clinic’n’Cell patented methodology 22 – 29 , we evaluated their ex vivo influence on the behavior and function of human hepatocytes in a lipotoxic environment. Results Kinetic of apparition of circulating metabolites resulting from TOTUM-448 ingestion in humans TOTUM-448 was ingested and digested by fasted healthy volunteers and the appearance of the circulating metabolites in the bloodstream was observed through a kinetic approach to determine the absorption profile. We found 12 detectable circulating human metabolites, including 2 oleuropein glucuronide isomers, 3 luteolin glucuronide isomers, 1 ferulic acid sulfate, 3 hydroxytyrosol sulfate isomers, 1 hydroxytyrosol glucuronide, 1 tyrosol glucuronide, and 1 homovanilic acid sulfate (Fig. 1 A). The Tmax ranged from 40 min to 160 min post-absorption depending on the targeted metabolite and the volunteer. In order to get a representative viewpoint of the diversity of the metabolites detected, we plotted, for each time point, the cumulative absorption for each metabolite family. The resulting curve rapidly reached a peak between 80 and 140 min (Fig. 1 B). Thus, to ensure the collection of serum fractions enriched with the highest diversity of metabolites, the 100 min time point (post-ingestion) was chosen for blood sampling during the second clinical phase. Impact on cell viability following incubation of HepG2 hepatocytes with either naïve or metabolites enriched human serum The final objective of this clinical ex vivo approach was to investigate whether and how these bioavailable human metabolites may preserve human hepatocyte function (in comparison with naïve serum) in a lipotoxic context mimicking MASLD. Therefore, blood was sampled before the ingestion of TOTUM-448 (naïve fraction) and at 100 min post ingestion (enriched fraction) for ex vivo investigations. Cell culture procedures were set as presented in Fig. 2 A. Hepatocytes were pre-incubated in the presence of either naïve serum (NHS) or TOTUM-448 enriched serum (EHS), 24h prior to a lipotoxic stress induction by palmitate (250µM) that lasted for 48 additional hours (total serum incubation: 72 hours). To ensure the biological soundness of this ex vivo approach, we first checked that human serum (either naïve or enriched) had no adverse impact on cell growth and viability (Fig. 2 B). As expected, the absence of fetal calf serum stopped the cell proliferation, while its presence allowed normal cell behavior. The presence of human serum slightly limited cell growth but there was no significant difference between the naïve or the enriched fraction (Fig. 2 B). The presence of palmitate had no impact on this parameter (Fig. 2 C). However, when looking more precisely into the pro-apoptotic mechanisms, we found that palmitate markedly induced caspase-3 activity (Fig. 2 D). This induction was significantly limited in the presence of the enriched serum fraction (-31%, p ≤ 0.01, Fig. 2 D). TOTUM-448 human metabolites limit fat accumulation in human hepatocytes Hepatocytes were stressed by the incubation with 250µM of palmitate to mimic a MASLD context. Stress was performed in the presence of either the naïve or the enriched serum fractions to test whether metabolites were able to contribute to maintain hepatocyte function in such a lipotoxic environment. According to red oil staining, the presence of palmitate dramatically promoted lipid storage in hepatocytes (Fig. 3 A and B). Neither the naïve nor the enriched human serum fraction had any impact on such lipid accumulation alone. In contrast, the impact of palmitate on the staining was significantly reduced when the cells were incubated with TOTUM-448 metabolites. To get more in-depth, we characterized the type of lipids involved in such a lipid accumulation. We found that red oil staining observation remarkably parallels with triglycerides and cholesterol contents (Fig. 3 C and D). Palmitate significantly stimulated both triglycerides and cholesterol accumulation in human hepatocytes, while the presence of TOTUM-448 metabolites significantly prevented it (triglycerides (TG) -27%%, p ≤ 0.001; cholesterol − 52%, p ≤ 0.0001). We then, investigated hepatocytes transcriptomic activity of genes related to lipid metabolism. Both DGAT2 and SREBP1c were significantly up-regulated upon palmitate incubation (Fig. 3 E and F). Such an up-regulation was blunted when hepatocytes were incubated with the enriched human serum fraction (-64%, p ≤ 0.001 and − 68%, p ≤ 0.0001 for DGAT2 and SREBP1c, respectively). Oxidative and inflammatory stress management barely account for TOTUM-448 benefit on human hepatocytes Along with lipotoxicity, the pro-inflammatory context drives altered hepatocyte function and subsequent MASLD onset. Using a DCFDA probe, we showed that palmitate promoted ROS production (Fig. 4 A). The presence of TOTUM-448 metabolites tends to limit this production(p > 0.05). Regarding the inflammatory status, while only trends appeared when expressions of the targeted genes were considered separately (suppl. figure S1), using a 2-way Anova approach to globalize the analysis, we observed a significant group effect (2-way ANOVA, p = 0.0008) and significant differences in post hoc comparisons of overall inflammatory gene expression between groups NHS and NHS + P (250µM), p < 0.05, EHS and EHS + P(250µM), p < 0.05, and NHS + P (250µM) and EHS + P (250µM), p < 0.01, suggesting that palmitate consistently induced inflammatory gene expression while the presence of the metabolites significantly limited this induction (Fig. 4 B). Still, the presence of metabolites did not achieve a return to baseline, as it remained significantly different from the control group (without palmitate). TOTUM-448 human metabolites prevent from lipotoxicity-induced ER stress in human hepatocytes Abnormal lipid accumulation in steatotic livers coincides with perturbed endoplasmic reticulum (ER) proteostasis in hepatocytes. Therefore, we checked for Unfolded Protein Response (UPR) markers in palmitate stressed hepatocytes. As expected, palmitate markedly and significantly promoted both CHOP and XBP-1 expression (Fig. 5 A and B). Such an induction was abolished when cells were treated with TOTUM-448 metabolites (-36%, p ≤ 0.01 and − 66%, p ≤ 0.0001 for CHOP and XBP1, respectively. However, neither the naïve (NHS) nor the enriched human serum (EHS) fraction had any impact on those markers in the absence of a lipotoxic context. To support these data, we analyzed ATF-6 activity as the result of the presence of ATF-6 protein in the nucleus. Accordingly, ATF-6 was increased upon palmitate stress. However, limitation by TOTUM-448 metabolites remained a trend (-29%, p = 0.2). Discussion In this manuscript, we first demonstrated that the ingestion of TOTUM-448 (1) led to bioavailable circulating metabolites and that these metabolites were able to drive the attenuation of palmitate-induced intracellular lipid storage including (2) TG (-27%, p ≤ 0.001) and (3) cholesterol (-52%, p = 0.0001), as well as the inhibition of (4) DGAT2 (-64%, p ≤ 0.001) and (5) SREBP1-c (-68%, p ≤ 0.0001) gene expression. TOTUM-448’s metabolites also inhibited (6) palmitate-induced inflammatory gene expression (p ≤ 0.01). A major effect was related to ER stress. While palmitate potently induced both (7) CHOP, (p ≤ 0.001) and (8) XBP1 mRNA expression (p ≤ 0.0001), (9) ATF-6 activity (p = 0.0053) and ultimately (10) Caspase-3 activity (p ≤ 0.0001), the presence of TOTUM-448’s metabolites normalized these markers (-36%, p ≤ 0.01; -66%, p ≤ 0.0001 and − 31%, p ≤ 0.01, for CHOP, XBP1 and Caspase-3, respectively; a trend was observed for ATF-6 (-29%, p = 0.2)). First, the nutritional dimension of the dose used may be questioned. Each volunteer was exposed once per clinical phase to 4284 mg of TOTUM-448 representing an approximative global amount of 372 mg of polyphenols (see suppl. Table S1 for chemical characterization of TOTUM-448). The recommended dose for polyphenol daily intakes is about 1g, thus the nutritional dimension of the dose is quite relevant. Besides, the rational of the dose also echoes with paralleled clinical trials we have launched. Indeed, the dose chosen is also the one tested for long term supplementation and investigation in humans. The clinical trial is ongoing: NCT06704321. Then, the bioavailability of the ingredient was investigated. In this study, we were able to detect 12 different circulating metabolites resulting from the ingestion of the blend including 2 oleuropein glucuronide isomers (olive leaf 30 ), 3 luteolin glucuronide isomers (metabolites that may derive from luteolin-7-O-glucoside, luteolin-7-O-glucuronide and luteolin presents in chrysanthellum, in both artichoke and olive leaves 31 ), 1 ferulic acid sulfate (metabolite that may derive from either chlorogenic acid present in artichoke leaf and, chrysanthellum or caffeoylquinic acid also present in artichoke leaf 32 , 33 ), 3 hydroxytyrosol sulfate isomers, 1 hydroxytyrosol glucuronide, 1 tyrosol glucuronide, 1 homovanilic acid sulfate (metabolites that may derive from both hydroxytyrosol and oleuropein present in olive leaf 30 ). These metabolomic analyses evidenced a clear and diverse bioavailability of the components of the blend and supported the rational for further investigation of the bioactivity of the serum. One may also question the ex vivo model. In our clinical ex vivo approach, volunteers should be seen as metabolites producers rather than patients expecting health benefits with measurable clinical scores. Thus, this protocol is designed to collect circulating metabolites in a standardized way and only male healthy volunteers with no treatment or pathology, aged from 18 to 35, BMI ranged from 20 to 28, with normal kidney and liver functions are recruited. The collected serum, either naïve (control baseline) or enriched (with circulating metabolites of interest) are further used/incubated on cell cultures according to the patent DIRV#18–0058 (written invention disclosure by the French National Institute for Agronomic, Food and Environment Research INRAE) to investigate a wide range of cell activities/markers that represent the final readout/score. In this study we targeted hepatocytes as a major protagonist in lipid metabolism and MASDL onset. Hepatocytes are responsible for glucose, xenobiotic metabolism and for lipids metabolism as well. In our hands, palmitate consistently promoted lipids accumulation in hepatocytes including triglycerides and cholesterol mimicking a relevant MASLD onset. The presence of metabolites resulting from TOTUM-448 ingestion potently prevented these MASLD hallmarks. From a mechanistic point of view, these results match with the inhibition of both, DGAT2, an enzyme involved in triglycerides synthesis 34 and SREBP1c, involved in the de novo lipogenesis in the liver 35 . Besides, these results are fully consistent with published observations regarding the effect of the different components of the blend when studied separately. In patients with diagnosed NAFLD, supplementation with artichoke leaf extracts (Cynara scolymus L.) (600 mg daily for 9 weeks) reduced liver size and decreased serum total cholesterol and triglyceride concentrations 36 . In a high-fat diet rat model, artichoke leaf extract supplementation limited hepatic steatosis 37 . A clinical study on a cohort of steatotic patients showed that supplementation with Chrysanthellum americanum extract decreased fibrosis and hepatic steatosis 38 . Decreased choline intake is significantly associated with increased liver fibrosis in post-menopausal women with NAFLD 39 . In MASLD, the lipotoxic context fuels inflammation and oxidative stress by stimulating ROS production, pro-inflammatory cytokine release and by promoting the recruitment of immune cells. In light of this, our cellular model fully addresses these criteria as palmitate increases DCF-DA staining and inflammatory gene expressions. Consistent with this, we observed an increase in global inflammatory gene expression in response to palmitate exposure. This detrimental effect was blunted in hepatocytes incubated with Totum-448-enriched serum. This observation must be tempered by the fact that the induction of the expression of CXCL1, IL-1β, IL-6, MCP-1 and TNFα did not reach significance when analyzed separately, suggesting a global effect on inflammatory status rather than a specific effect on a target gene. Consistently, in a recent randomized, controlled clinical study, Guo et al. demonstrated that NLRP3 inflammasome activation is significantly increased in patients with NAFLD, but that this activation can be strongly repressed by anthocyanins provided here by blueberry extract (Vaccinium myrtillus L.) 40 . Several polyphenols present in the enriched serum have already demonstrated antioxidant properties through scavenging of free radicals or inhibition of oxidative enzymes 41 42 including oleuropein in a similar HepG2 model 43 . Preclinically, olive leaf extracts have shown anti-inflammatory, antioxidant, and antifibrotic effects 44 . Finally, a 2021 randomized double-blind clinical study shows that supplementation with olive leaf extract (Olea europaea L.), characterized by its oleuropein and hydroxytyrosol content, shows a reduction in oxidized LDL 45 . Hepatocytes are also responsible for de novo lipids biogenesis along with lipid metabolism as well. Thus, the role of both, the smooth and the rough endoplasmic reticulum (ER), is pivotal. In light of this, dysregulation of lipid metabolism, increased inflammation or oxidative stress may either be a starting point or a consequence of the unbalance of the ER metabolism. This drives the establishment of a vicious cycle triggering the UPR (Unfold Protein Response) and leading to the activation of IRE1, PKR-like ER kinase (PERK) and ATF-6 to reset ER homeostasis 46 . Of interest, PERK activation results in an antioxidant response 47 , 48 while both PERK and ATF-6 participate to the recovery from liver’s condition as demonstrated by knockout studies 49 – 51 In this study, the influence of the TOTUM-448 metabolites on ER-stress management seems to be the cornerstone. Palmitate enhanced both XBP-1 52,53 and CHOP 54 expressions and significantly stimulates ATF-6 activity. XBP-1 and CHOP up-regulation result from the activation of IRE1 and PKR-like ER kinase (PERK), respectively. This up-regulation is fully consistent with literature data. Association between chronic ER and steatotic liver was previously established in both obese mouse models (genetic and high-fat diet models) and obese patients 55 . Palmitate also significantly enhanced caspase-3 activity. Such an enhancement was previously described in liver biopsies from patients suffering from NASH 56 . In rats fed a high fat diet, CHOP expression and active form of caspase-3 were found increased in liver 57 . The presence of the human metabolites from TOTUM-448 potently limited this palmitate-induced ER stress and normalized the studied markers (-36%, p ≤ 0.01; -66%, p ≤ 0.0001 and − 31%, p ≤ 0.01, for CHOP, XBP1 and Caspase-3, respectively; a trend was observed for ATF-6 (-29%, p = 0.2)) supporting the relevance of this nutritional strategy to manage MASLD. The protective effects of the serum enriched with TOTUM-448-derived metabolites against palmitate-induced endoplasmic reticulum (ER) stress may, at least in part, be attributed to the action of specific polyphenols known to be present in the plant extracts composing TOTUM-448. Several of these compounds have already been reported to mitigate ER stress. For example, hydroxytyrosol, a major compound of olive leaf extract, has been shown to protect HepG2 cells from tunicamycin-induced ER stress 58 . Similarly, luteolin, found in artichoke and Chrysanthellum extracts, was reported to prevent palmitate-induced ER stress, autophagy and apoptosis in AML12 hepatocytes 59 . In addition, chlorogenic acid, which is present in artichoke leaf extract, demonstrated protective effects against palmitate-induced ER stress in primary rat hepatocytes 60 . Inflammation, oxidative stress and ER stress being widely imbricated 61 , 62 we are facing a chicken-and-egg situation and, the question regarding the way the TOTUM-448’s metabolites support hepatoprotection remains. Do they improve hepatocyte behavior and lipid metabolism by preserving ER homeostasis or do they limit ER stress by improving lipid metabolism? Indeed, it has been reported that caloric restriction improves steatosis and alleviates ER stress 63 while normalization of ER stress markers was correlated with reduced hepatic lipid content 64 . ER is the major site of lipid synthesis in hepatocytes including triglyceride synthesis and storage. Both, the sterol regulatory element-binding proteins (SREBP1c for fatty acid synthesis and SREBP2 for sterol synthesis) and the diacylglycerol acyltransferase (DGAT) are ER-localized transcription factors and enzymes, respectively, involved in the regulation and the catalysis of de novo lipogenesis 46 . Accordingly, TOTUM-448’s metabolites were found to reduce SREBP1c and DGAT2 expression to blunt the vicious cycle of MASLD and promote hepatocyte homeostasis. Notably, the metabolites had no effect on their own but showed protective effects in the presence of palmitate, supporting the preventive role of this ingredient in maintaining hepatocyte function and homeostasis (Fig. 6 ). Methods Study Product VALBIOTIS has developed TOTUM-448 (priority French patent: FR1460064), a food ingredient composed of the combination of different plant aforementioned. Selection of the extracts was originally driven by published data regarding their possible support in preventing liver conditions. The formulation of the product meets the requirements of European regulations relating to food supplements (European directive n°2002/46/EC and French decree n°2006 − 352 of March 20, 2006 which transposes it). Ethics Preclinical Study The study was conducted in accordance with the Declaration of Helsinki of 1975 ( https://www.wma.net/what-we-do/medical-ethics/declaration-of-helsinki (accessed on November, 12th 2021)) revised in 2013. The human study was approved by the French Ethical Committee (NCT06047847/ N° ID RCB: 2023-A00579-36/ Comité de Protection des Personnes CPP Est III; approved 25th July 2023). The volunteers were informed of the objectives and the potential risks of the present study and provided their written informed consent before they participated in the study. Human Study Design and Pharmacokinetic of Absorption Ten healthy men (age: 24.5 years old, +/− 3.48; BMI: 23.35 kg/m2, +/−1.72; >60 kg) were enrolled for this study. Only male healthy volunteers with no treatment or pathology, aged from 18 to 35, BMI ranged from 20 to 28, with normal kidney and liver functions were recruited. They were checked for normal blood formulation, renal (urea and creatinine), and liver functions (aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma-glutamyltransferase (GGT) activities). Serum samples from all participants were collected in plain tubes. Unfortunately, female volunteers were not to be recruited to avoid hormonal variation and subsequent bias. The first phase of this clinical project was dedicated to determine TOTUM-448’s metabolites absorption profile. Prior to receiving the tested product, volunteers were fasted for 12 h and then, were given 4284 mg of TOTUM-448 as eight capsules. Nine milliliters of venous blood (median cubital vein) were collected before the ingestion of TOTUM-448 and then every 20 min for 240 min after the ingestion of the ingredient. Collected serum was aliquoted in sterile tubes and then stored until analyses at − 80°C. Location of storage: Clinical Investigation Center – Inserm 1405, University Hospital of Clermont-Ferrand. This dedicated research department is fully compliant with regulatory and ethical clinical obligations (certification according to the French standard NF S 96900). Circulating metabolites resulting from TOTUM-448 ingestion were quantified and characterized by ultra-high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS). Upon characterization of the absorption profile, volunteers underwent a second visit to the clinical center for the second phase of the project in order to collect serum containing TOTUM-448 metabolites at Tmax/Cmax. Context was similar to phase 1. Prior to receiving the tested product, volunteers were fasted for 12 h and then, were given 4284 mg of TOTUM-448 as eight capsules. Forty-eight milliliters of venous blood were collected before the product ingestion as a control baseline naïve serum. Then, at Cmax in the post-absorptive phase, forty-eight milliliters of blood were collected for circulating bioactive collection. Collection, aliquoting and storage conditions were similar to phase 1. Phenolic Compounds Extraction from Serum To extract phenolic compounds from serum, 0.9 mL of serum was combined with 2.7 mL of 100% methanol and mixed for one minute. The mixture was then centrifuged at 20,000× g for 15 minutes at room temperature. The supernatant was collected in separate tubes and evaporated to dryness using a SpeedVac Concentrator (Thermo Fisher Scientific, Illkirch, France). The dried material was reconstituted in 80 µL of a 50:50 methanol/water solution. After agitation for one minute, ultrasonication for one minute, and centrifugation at 20,000× g for 15 minutes at room temperature, the supernatant was stored at − 20°C until analysis by ultra-high performance liquid chromatography with tandem mass spectrometry detection. Ultra-High Performance Liquid Chromatography-Mass Spectrometry (UHPLC-MS/MS) Phenolic compounds were analyzed using a 1260 Infinity UHPLC system (Agilent Technologies) coupled to a 6430 triple quadrupole mass spectrometer (Agilent Technologies, Les Ulis, France). Four microliters of the sample were injected into a Zorbax SB-C18 column (2.1 × 100 mm, 1.8 µm) (Agilent Technologies, Les Ulis, France). The mobile phase consisted of two solvents: solvent A (water/formic acid 99.9:0.1, v/v) and solvent B (acetonitrile/formic acid 99.9:0.1, v/v), with a flow rate of 0.3 mL/min. The gradient for solvent A was as follows: 0 min 1% B, 2 min 5% B, 3 min 25% B, 6 min 25% B, 8 min 40% B, 11.5 min 95% B, 14 min 95% B, and 16 min 1% B. The MS/MS parameters were set to negative ion mode, with a capillary tension of 3000 V, a nebulizer pressure of 15 psi, a dry gas flow rate of 11 L/min, a dry temperature of 350°C, and acquisition in multiple reaction monitoring (MRM) mode. Data were processed using MassHunter software (Agilent Technologies). Human Hepatocyte Cultures The HepG2 human hepatocyte cell line was obtained from the European Collection of Authenticated Cell Cultures and purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France − 85011430). During the maintenance phase, HepG2 cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin/streptomycin (Life Technologies, Villebon-Sur-Yvette, France). All cell cultures were maintained at 37°C in an atmosphere of 5% CO2/95% air. To analyze the effects of TOTUM-448 metabolites, the cells were preincubated for 24 hours in DMEM with 10% human serum (either naïve or containing circulating metabolites) according to the Clinic’n’Cell methodology (DIRV INRAE 18–0058), followed by an additional 48-hour incubation in a palmitate-induced lipidic stress environment (palmitate 250 µM; Sigma, Saint-Quentin-Fallavier, France). Preparation of Palmitate Solution Palmitate (Sigma, Saint-Quentin-Fallavier, France) was first coupled to bovine serum albumin (BSA; Sigma, Saint-Quentin-Fallavier, France) and then fully dissolved in pure ethanol at 70°C to achieve a final concentration of 500 mmol/L. This stock solution was mixed with a prewarmed BSA solution (10% w/w, 37°C) to reach a final concentration of 5 mmol/L. The mixture was clarified by incubating at 55°C for 15 minutes twice. The final palmitate:BSA molar ratio was set at 3.2:1. The control vehicle was prepared under the same conditions. A 10% w/w BSA solution was added to an equivalent volume of ethanol to match the final palmitate solution. The final ethanol concentration was less than 0.05% by volume in all experiments. Cell Viability Cell viability was assessed using an XTT-based method (Cell Proliferation Kit II, Sigma-Aldrich, Saint-Quentin-Fallavier, France). Experimental procedures were followed according to the supplier’s recommendations. Optical density was measured at 450 nm, with measurements performed in hexaplicate for each sample condition of the ten volunteers. DCF-DA Staining HepG2 cells were seeded on a 96-well dark-wall clear-bottom plate at a density of 12,000 cells/cm². Twenty-four hours after H 2 O 2 stimulation, cells were washed and incubated with 5 µM of 2′,7′-dichlorofluorescin diacetate (DCF-DA) solution (ab113851, Abcam) for 45 minutes at 37°C in the dark, then rinsed with the dilution buffer according to the manufacturer’s protocol. Fluorescence was measured using a fluorescence plate reader (Berthold − Mitras) at Ex/Em = 485/535 nm in end-point mode. Red Oil Staining Oil Red O solution (0.5% in isopropanol) was obtained from Sigma (Saint-Quentin-Fallavier, France) and staining was performed according to the supplier’s recommendations. The working solution (0.2% in 60% isopropanol) was prepared by mixing Oil Red O solution with distilled water in a 3:2 ratio. Prior to staining, cells were washed twice with PBS and fixed with 4% paraformaldehyde for 30 minutes at room temperature. After discarding the PFA solution, cells were washed twice with water, incubated with 60% isopropanol for 5 minutes, and then stained with the working Oil Red O solution for 20 minutes. After five additional washes with water, cells were observed under a microscope. Triglycerides Levels Triglyceride content in HepG2 cells was determined using a triglyceride assay kit (Abcam, Paris, France—ab65336) according to the manufacturer’s protocol. Triglycerides were converted to free fatty acids and glycerol, and the glycerol was oxidized to generate a colorimetric product measured at 570 nm. Cholesterol Levels in HepG2 Cells Cholesterol levels were evaluated in cell lysates using a cholesterol quantification kit (Sigma, Saint-Quentin-Fallavier, France—MAK043) according to the manufacturer’s recommendations. Total cholesterol concentration was measured by a coupled enzyme reaction resulting in a fluorometric (λex = 535 nm/λem = 587 nm) product related to the cholesterol content. Real-Time RT-qPCR mRNA from HepG2 cells were isolated using TRIzol™ Reagent (Ambion – Life Technologies) according to the supplier’s recommendations. The expression levels of DGAT2 (Diacylglycerol O-acyltransferase 2), SREBP1-c (Sterol regulatory element-binding protein 1c), CXCL-1 (CXC motif chemokine ligand 1), IL-1β, IL-6, MCP-1, TNFα, CHOP (C/EBP homologous protein), and XBP-1 mRNA were measured by RT-qPCR (PowerUp SYBRgreen, Applied Biosystems). β-Actin was used as a housekeeping gene. Primers were designed as follows: IL6-F: 5’- TTC TGT GCC TGC AGC TTC − 3’; IL6-R: 5’- GCA GAT GAG TAC AAA AGT CCT GA -3’; MCP-1-F: 5’- GCC TCT GCA CTG AGA TCT TC -3’; MCP-1-R: 5’- AGC AGC CAC CTT CAT TCC − 3’; TNFa-F: 5’- TCA GCT TGA GGG TTT GCT AC -3’; TNFa-R: 5’- TGC ACT TTG GAG TGA TCG G -3’; IL1b-F: 5’- GAA CAA GTC ATC CTC ATT GCC − 3’; IL1b-R: 5’- CAG CCA ATC TTC ATT GCT CAA G -3’; CXCL1-F: 5’- TCT CTC TTT CCT CTT CTG TTC CTA − 3’; CXCL1-R: 5’- CAT CCC CCA TAG TTA AGA AAA TCA TC -3’; DGAT2-F: 5’- TCA GCA GGT TGT GTG TCT TC -3’; DGAT2-R: 5’- GGC TGG TGT TTG ACT GGA A -3’; SREBP1c-F: 5’- GGA TGG TGT TCA CTC GGT A -3’; SREBP1c-R: 5’- GGT GAT ATG TGT CTG CGT C -3’; XBP1-F: 5’- CGC TGT CTT AAC TCC TGG TTC − 3’; XBP1-R: 5’- CTG GAA CAG CAA GTG GTA GA -3’; CHOP-F: 5’- CAA TGA CTC AGC TGC CAT CT -3’; CHOP-R: 5’- AGC GAC AGA GCC AAA ATC AG -3’; ACTβ-F: 5’-ATT GGC AAT GAG CGG TTC-3’; ACTβ-R: 5’-GGA TGC CAC AGG ACT CCA-3’. Cell Lysis The lysis buffer was prepared by mixing 50 mmol/L Tris pH 7.8, 150 mmol/L NaCl, 0.5% sodium deoxycholate, and 1% NP40. Cell lysates were stored at − 80°C until analysis. Protein Quantification Protein content was measured using the BCA Protein Assay Kit (Sigma-Aldrich, Saint-Quentin-Fallavier, France). The BCA protein assay is based on a biuret reaction, where the reduction of Cu2 + to Cu + in the presence of proteins in an alkaline environment is proportional to the protein concentration. The chromogenic reagent bicinchoninic acid chelates the reduced copper, forming a purple complex that absorbs at 562 nm. Caspase-3 activity Caspase-3 activity in cells was evaluated using the Caspase-3 Assay Kit (Abcam, Paris, France—ab39401) according to the manufacturer’s protocol. To lyse the frozen monolayer of HepG2 cells grown on a twelve-well plate, 200 µL of the provided lysis buffer was used per well. Fifty microliters of freshly prepared lysate were then mixed with 55 µL of reconstituted reaction mix. Optical density at 405 nm was measured every two minutes for 90 minutes at 37°C. ATF6 activity Nuclear extracts were prepared from HepG2 cells using the Nuclear Extraction Kit (Abcam, Paris, France—ab113474), and protein content was determined using the BCA Protein Assay Kit (Sigma-Aldrich, Saint-Quentin-Fallavier, France). ATF6 protein expression was assessed using the ATF6 ELISA Kit (Signosis, TE-0041) according to the manufacturer’s instructions. Statistics Statistical analyses and figure generation were performed using Prism V.10.4.1 (GraphPad Software). The statistical plan included a Shapiro–Wilk normality test to determine if the data followed a Gaussian distribution. If the data were not normally distributed, a Kruskal–Wallis non-parametric test was used, followed by Dunn's test for post hoc comparisons. For normally distributed data with equal variance, one-way or two-way ANOVA with Tukey’s test for multiple comparisons was applied. For inflammatory gene expression, a two-way ANOVA targeting a main column effect was followed by Tukey’s post hoc test. If data were missing, making it impossible to run a repeated-measure two-way ANOVA, a mixed-effects analysis was used instead. In Fig. 1 , values are presented as means ± SEM, with statistical significance indicated as follows: * for p < 0.05; ** for p < 0.01; *** for p < 0.001; **** for p 0.05. For Figs. 2 to 5 , values are presented as box plots indicating median and interquartile range (lower and upper), with whiskers indicating minimum and maximum values. Statistical significance is indicated as follows: * for p < 0.05; ** for p < 0.01; **** for p 0.05. Declarations Funding This study was sponsored by VALBIOTIS SA. This work was supported by the Bordeaux Metabolome Facility and the MetaboHUB (ANR11-INBS-0010) project. This work was funded by the Pack Ambition Recherche 2021 MICROMETiv (Region Auvergne Rhône Alpes) and the common laboratory MEDIS-VALBIOTIS Labcom MIMETiv (ANR, ANR-22-LCV1-0003-01). Author contributions statement Conceptualization, F.W. and Y.W.; design, V.C., Y.F.O., P.S., M.C., V.S.; methodology, Y.W., L.B.-W., and F.W.; formal analysis, S.K., J.V., F.L-J. and F.W.; clinical investigation, A.B.M, V.R., N.M., G.P. and L.B.-W.; writing F.W., V.C. and Y.W.; data interpretation and reviewing B.P., V.C., F.W.,Y.W., S.B-D. and A.B; project administration V.S., M.C., S.P. and L.B.-W. All authors have read and agreed to the published version of the manuscript. All authors have read and agreed to the published version of the manuscript. Additional information The human ex vivo methodology used in this study has been registered as a written invention disclosure by the French National Institute for Agronomic, Food and Environment Research (INRAE) (DIRV#18-0058). Clinic’n’Cell® has been registered as a mark 22-26 , 28 , 29 , 65-67 . F.W. and L.W.-B. work for Clinic’n’Cell SAS (Faculty of Medicine and Pharmacy Clermont-Ferrand- France); V.C., A.B.M., Y.F.O., F.L-J., V.S., M.C., S.P., P.S. work for VALBIOTIS and provided the ingredient. Y.W. provides scientific consulting for Clinic’n’Cell SAS; S.K., J.V., B.P., V.R., N.M., G.P., S.B-D and A.B. have no conflict of interest to declare. Data availability statement The datasets generated and analysed during the current study are available from the corresponding author on reasonable request. References Younossi, Z. et al. Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention. Nature reviews. Gastroenterology & hepatology 15 , 11-20, doi:10.1038/nrgastro.2017.109 (2018). Rinella, M. E. et al. A multisociety Delphi consensus statement on new fatty liver disease nomenclature. Hepatology 78 , 1966-1986, doi:10.1097/HEP.0000000000000520 (2023). Chan, W. K. et al. 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09:56:57","extension":"xml","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":169698,"visible":true,"origin":"","legend":"","description":"","filename":"80d64e54cd0d4a0c92b33e33e75d69e61structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7460979/v1/32ff11aa73c975a57dc3192c.xml"},{"id":92159697,"identity":"b0ce2125-52f1-4073-b857-2eaec934d746","added_by":"auto","created_at":"2025-09-25 09:48:58","extension":"html","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":191964,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7460979/v1/189c7efc21c218fd2b82cf26.html"},{"id":92159680,"identity":"f2c08bcf-d95a-40f6-a004-40f86565514d","added_by":"auto","created_at":"2025-09-25 09:48:57","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":482541,"visible":true,"origin":"","legend":"\u003cp\u003eMetabolomic profiles in human serum following TOTUM-448 ingestion.\u003c/p\u003e\n\u003cp\u003eA: Circulating metabolites resulting from TOTUM-448 ingestion were determined using Ultra-High Performance Liquid Chromatography-Mass Spectrometry (UHPLC-MS/MS).Twelve detectable circulating human metabolites (2 oleuropein glucuronide isomers, 3 luteolin glucuronide isomers, 1 ferulic acid sulfate, 3 hydroxytyrosol sulfate isomers, 1 hydroxytyrosol glucuronide, 1 tyrosol glucuronide, and 1 homovanilic acid sulfate. B: Cumulative absorption for each metabolite family. AUC: Area under curve. Humans n=10 volunteers.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7460979/v1/05a929728e3f5db6ab8170b9.jpeg"},{"id":92160880,"identity":"cc636eec-aec9-4309-b02f-4b9408e5d703","added_by":"auto","created_at":"2025-09-25 10:04:57","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":315297,"visible":true,"origin":"","legend":"\u003cp\u003eCell culture experimental design and validation of the \u003cem\u003eex vivo\u003c/em\u003eprocedures (Human hepatocytes, HepG2).\u003c/p\u003e\n\u003cp\u003eA. Protocol design. B. Validation of the use of human serum for human hepatocyte cultures. Cell viability was determined using a XTT-based method. Fetal Bovine Serum (FBS) was used as the reference. C. Impact of palmitate on hepatocyte viability. D. Caspace-3 activity. NHS: Naïve human serum; EHS: Enriched human serum (serum collected at Cmax 100min and containing circulating bioactives resulting from TOTUM-448 ingestion); P(250µM): palmitate 250µM. Measurements were realized in hexaplicate (XTT) or triplicate (caspase-3 activity) for each volunteer (n=10 volunteers). Boxes indicate median and interquartile range (lower and upper), while whiskers indicate minimum and maximum *: p \u0026lt; 0.05; **: p \u0026lt; 0.01; ****: p \u0026lt; 0.0001; ns: p \u0026gt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7460979/v1/e42218918d0e269dd6d7f089.jpeg"},{"id":92160567,"identity":"7bc8810d-b5a6-44b6-b79f-3e0345ff0ee4","added_by":"auto","created_at":"2025-09-25 09:56:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":343114,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of TOTUM-448 metabolites on lipid storage in human hepatocytes\u003c/p\u003e\n\u003cp\u003eA. Red oil staining. B. Red oil staining quantification. C. Intracellular triglyceride content. D. Intracellular cholesterol content. E and F: mRNA relative expression of DGAT2 and SREBP1c (RTqPCR). NHS: Naïve human serum; EHS: Enriched human serum (containing circulating bioactives resulting from TOTUM-448 ingestion); P(250µM): palmitate 250µM. Measurements were realized in triplicate for each volunteer (n=10 volunteers). Boxes indicate median and interquartile range (lower and upper), while whiskers indicate minimum and maximum *: p \u0026lt; 0.05; **: p \u0026lt; 0.01; ***: p \u0026lt; 0.001; ****: p \u0026lt; 0.0001; ns: p \u0026gt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7460979/v1/84cf408178638e328ca3e9f3.png"},{"id":92159686,"identity":"656df416-bbb7-44ba-af5c-d2a2e7cafd3b","added_by":"auto","created_at":"2025-09-25 09:48:57","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":349062,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of TOTUM-448 human metabolites on redox and inflammatory status in human hepatocytes HepG2.\u003c/p\u003e\n\u003cp\u003eA: DCFDA staining (quantification expressed as the mean green pixel value); B: mRNA relative expression of CXCL1 IL-1β, IL-6, MCP-1 and TNFα. NHS: Naïve human serum; EHS: Enriched human serum (containing circulating bioactives resulting from TOTUM-448 ingestion); P(250µM): palmitate 250µM. Measurements were realized in triplicate for each volunteer (n=10 volunteers). Boxes indicate median and interquartile range (lower and upper), while whiskers indicate minimum and maximum *: p \u0026lt; 0.05; **: p \u0026lt; 0.01; ns: p \u0026gt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7460979/v1/117407c0227142157f6df1f7.jpeg"},{"id":92160569,"identity":"858b22f8-5e16-471a-93ec-286f579b7c0e","added_by":"auto","created_at":"2025-09-25 09:56:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":86386,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of TOTUM-448 human metabolites on ER stress level and UPR markers in a lipotoxic environment.\u003c/p\u003e\n\u003cp\u003eA and B. mRNA relative expression of CHOP and XBP-1, respectively. C. ATF-6 nuclear presence. NHS: Naïve human serum; EHS: Enriched human serum (containing circulating bioactives resulting from TOTUM-448 ingestion); P(250µM): palmitate 250µM. Measurements were realized in triplicate for each volunteer (n=10 volunteers). Boxes indicate median and interquartile range (lower and upper), while whiskers indicate minimum and maximum **: p \u0026lt; 0.01; ***: p \u0026lt; 0.001; ****: p \u0026lt; 0.0001; ns: p \u0026gt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7460979/v1/c5b368fa0e42e6216bb6eb9a.png"},{"id":92162020,"identity":"1d8cd2ba-e094-4969-8886-e25ccad198d4","added_by":"auto","created_at":"2025-09-25 10:12:57","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":390380,"visible":true,"origin":"","legend":"\u003cp\u003eBioavailable Human Metabolites from TOTUM-448 (Plant-Based, Polyphenol-Rich Ingredient) Maintain Liver Cell Functionality in a Lipotoxic Context that Drives MASLD Onset.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7460979/v1/57b23388b3f4cf8778678c21.jpeg"},{"id":98813969,"identity":"eb02375b-4adb-4f29-a480-23059cde81c3","added_by":"auto","created_at":"2025-12-22 16:08:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3081510,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7460979/v1/1e4ee938-b468-41a7-8b8f-c86c791462e1.pdf"}],"financialInterests":"Competing interest reported. The human ex vivo methodology used in this study has been registered as a written invention disclosure by the French National Institute for Agronomic, Food and Environment Research (INRAE) (DIRV#18-0058). \nF.W. and L.W.-B. work for Clinic’n’Cell SAS (Faculty of Medicine and Pharmacy Clermont-Ferrand- France); V.C., A.B.M., Y.F.O., F.L-J., V.S., M.C., S.P., P.S. work for VALBIOTIS and provided the ingredient. Y.W. provides scientific consulting for Clinic’n’Cell SAS; S.K., J.V., B.P., V.R., N.M., G.P., S.B-D and A.B. have no conflict of interest to declare.","formattedTitle":"Bioavailable Human Metabolites from TOTUM- 448 (Plant-Based, Polyphenol-Rich Ingredient) Maintain Liver Cell Functionality in a Lipotoxic Context that Drives MASLD Onset","fulltext":[{"header":"Introduction","content":"\u003cp\u003eChronic liver disease is a social and economic burden \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Among the most common liver disorders is metabolic dysfunction\u0026ndash;associated steatotic liver disease (MASLD), redefined in 2023 and previously described as non-alcoholic fatty liver disease (NAFLD)\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Its prevalence is estimated at about 30% of the adult population worldwide \u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e while it can reach up to 90% in obese individuals and 60% in people with diabetes \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Up to 20% of the normal-weight people are also concerned.\u003c/p\u003e\u003cp\u003eMASLD is characterized by the association of excessive fat accumulation in liver and at least one metabolic risk factor including overweight/obesity, type 2 diabetes, metabolic syndrome, hypertriglyceridemia andhypercholesterolemia. \u003csup\u003e2\u003c/sup\u003e. This steatosis results from the accumulation of lipids in liver parenchymal cells and triggers hepatic inflammation and fibrosis. Over time, it leads to cirrhosis and its associated complications \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Thus, MASLD is correlated with higher risks of cancer and cardiovascular disease mortality \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e and considered as the second most common reason for liver transplantation in the United States and Europe \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn light of this, strategies promoting hepatic lipid clearance are of particular interest. Different pharmacological approaches are acknowledged including statins, SGLT-2 inhibitors, GLP-1 agonists, pioglitazone, vitamin E and resmetirom \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Resmetirom, a liver-directed thyroid hormone receptor beta-selective agonist, has recently been approved by FDA for MASLD treatment \u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. However, these treatments designed to cure rather than to prevent evidence side effects (e.g., muscle related issues for statins; nausea and diarrhea for resmetirom) \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e and may be quite expensive for patients and healthcare systems \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. This highlights the need for alternative, safer, and preventive options. In this context, lifestyle interventions including dietary changes and exercise represent the initial step in the treatment as well as nutritional or nutraceutical strategies which have become a major field for therapeutic innovation \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAmong natural bioactives of interest, polyphenols are plant compounds known for their antioxidant and anti-inflammatory properties. Regarding their role in MASLD, the last five years have accounted for more than half of the total publications related to this theme, demonstrating the growing interest in nutritional approaches in the field. Yet, only about 7% of these investigations have been conducted at the clinical level. Certain plant extracts are already known for their hepato-protective properties. For example, artichoke leaf extract (Cynara scolymus) is widely described in the literature for its choleretic, hepatoprotective \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e and lipid metabolism regulation properties \u003csup\u003e\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Nevertheless, these strategies lack of relevant clinical data and optimization of the expected benefits, particularly through nutritional synergies, require further clinical validation.\u003c/p\u003e\u003cp\u003eTOTUM-448 is a patented polyphenol-rich blend of 5 different plant extracts (olive leaf (Olea europaea), bilberry (Vaccinium myrtillus), artichoke leaf (Cynara scolymus), chrysanthellum (Chrysanthellum indicum subsp. afroamericanum B.L. Turner), black pepper (Piper nigrum)) plus choline with potential synergistic properties on liver function. The aim of this work was to investigate whether and how this combination of different polyphenols can maintain, preserve or even improve hepatocyte metabolism in a lipotoxic context, supporting preventive strategies in the early stages of MASLD.\u003c/p\u003e\u003cp\u003eTo address this working hypothesis, we collected human serum containing circulating bioactive metabolites resulting from TOTUM-448 ingestion and according to the Clinic\u0026rsquo;n\u0026rsquo;Cell patented methodology \u003csup\u003e\u003cspan additionalcitationids=\"CR23 CR24 CR25 CR26 CR27 CR28\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, we evaluated their \u003cem\u003eex vivo\u003c/em\u003e influence on the behavior and function of human hepatocytes in a lipotoxic environment.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eKinetic of apparition of circulating metabolites resulting from TOTUM-448 ingestion in humans\u003c/h2\u003e\u003cp\u003eTOTUM-448 was ingested and digested by fasted healthy volunteers and the appearance of the circulating metabolites in the bloodstream was observed through a kinetic approach to determine the absorption profile. We found 12 detectable circulating human metabolites, including 2 oleuropein glucuronide isomers, 3 luteolin glucuronide isomers, 1 ferulic acid sulfate, 3 hydroxytyrosol sulfate isomers, 1 hydroxytyrosol glucuronide, 1 tyrosol glucuronide, and 1 homovanilic acid sulfate (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The Tmax ranged from 40 min to 160 min post-absorption depending on the targeted metabolite and the volunteer. In order to get a representative viewpoint of the diversity of the metabolites detected, we plotted, for each time point, the cumulative absorption for each metabolite family. The resulting curve rapidly reached a peak between 80 and 140 min (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Thus, to ensure the collection of serum fractions enriched with the highest diversity of metabolites, the 100 min time point (post-ingestion) was chosen for blood sampling during the second clinical phase.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eImpact on cell viability following incubation of HepG2 hepatocytes with either na\u0026iuml;ve or metabolites enriched human serum\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe final objective of this clinical \u003cem\u003eex vivo\u003c/em\u003e approach was to investigate whether and how these bioavailable human metabolites may preserve human hepatocyte function (in comparison with na\u0026iuml;ve serum) in a lipotoxic context mimicking MASLD. Therefore, blood was sampled before the ingestion of TOTUM-448 (na\u0026iuml;ve fraction) and at 100 min post ingestion (enriched fraction) for \u003cem\u003eex vivo\u003c/em\u003e investigations. Cell culture procedures were set as presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. Hepatocytes were pre-incubated in the presence of either na\u0026iuml;ve serum (NHS) or TOTUM-448 enriched serum (EHS), 24h prior to a lipotoxic stress induction by palmitate (250\u0026micro;M) that lasted for 48 additional hours (total serum incubation: 72 hours).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo ensure the biological soundness of this \u003cem\u003eex vivo\u003c/em\u003e approach, we first checked that human serum (either na\u0026iuml;ve or enriched) had no adverse impact on cell growth and viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). As expected, the absence of fetal calf serum stopped the cell proliferation, while its presence allowed normal cell behavior. The presence of human serum slightly limited cell growth but there was no significant difference between the na\u0026iuml;ve or the enriched fraction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The presence of palmitate had no impact on this parameter (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). However, when looking more precisely into the pro-apoptotic mechanisms, we found that palmitate markedly induced caspase-3 activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). This induction was significantly limited in the presence of the enriched serum fraction (-31%, p\u0026thinsp;\u0026le;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eTOTUM-448 human metabolites limit fat accumulation in human hepatocytes\u003c/h3\u003e\n\u003cp\u003eHepatocytes were stressed by the incubation with 250\u0026micro;M of palmitate to mimic a MASLD context. Stress was performed in the presence of either the na\u0026iuml;ve or the enriched serum fractions to test whether metabolites were able to contribute to maintain hepatocyte function in such a lipotoxic environment. According to red oil staining, the presence of palmitate dramatically promoted lipid storage in hepatocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B). Neither the na\u0026iuml;ve nor the enriched human serum fraction had any impact on such lipid accumulation alone. In contrast, the impact of palmitate on the staining was significantly reduced when the cells were incubated with TOTUM-448 metabolites. To get more in-depth, we characterized the type of lipids involved in such a lipid accumulation. We found that red oil staining observation remarkably parallels with triglycerides and cholesterol contents (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and D). Palmitate significantly stimulated both triglycerides and cholesterol accumulation in human hepatocytes, while the presence of TOTUM-448 metabolites significantly prevented it (triglycerides (TG) -27%%, p\u0026thinsp;\u0026le;\u0026thinsp;0.001; cholesterol \u0026minus;\u0026thinsp;52%, p\u0026thinsp;\u0026le;\u0026thinsp;0.0001). We then, investigated hepatocytes transcriptomic activity of genes related to lipid metabolism. Both DGAT2 and SREBP1c were significantly up-regulated upon palmitate incubation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and F). Such an up-regulation was blunted when hepatocytes were incubated with the enriched human serum fraction (-64%, p\u0026thinsp;\u0026le;\u0026thinsp;0.001 and \u0026minus;\u0026thinsp;68%, p\u0026thinsp;\u0026le;\u0026thinsp;0.0001 for DGAT2 and SREBP1c, respectively).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eOxidative and inflammatory stress management barely account for TOTUM-448 benefit on human hepatocytes\u003c/h3\u003e\n\u003cp\u003eAlong with lipotoxicity, the pro-inflammatory context drives altered hepatocyte function and subsequent MASLD onset. Using a DCFDA probe, we showed that palmitate promoted ROS production (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The presence of TOTUM-448 metabolites tends to limit this production(p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Regarding the inflammatory status, while only trends appeared when expressions of the targeted genes were considered separately (suppl. figure S1), using a 2-way Anova approach to globalize the analysis, we observed a significant group effect (2-way ANOVA, p\u0026thinsp;=\u0026thinsp;0.0008) and significant differences in \u003cem\u003epost hoc\u003c/em\u003e comparisons of overall inflammatory gene expression between groups NHS and NHS\u0026thinsp;+\u0026thinsp;P (250\u0026micro;M), p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, EHS and EHS\u0026thinsp;+\u0026thinsp;P(250\u0026micro;M), p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, and NHS\u0026thinsp;+\u0026thinsp;P (250\u0026micro;M) and EHS\u0026thinsp;+\u0026thinsp;P (250\u0026micro;M), p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, suggesting that palmitate consistently induced inflammatory gene expression while the presence of the metabolites significantly limited this induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Still, the presence of metabolites did not achieve a return to baseline, as it remained significantly different from the control group (without palmitate).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eTOTUM-448 human metabolites prevent from lipotoxicity-induced ER stress in human hepatocytes\u003c/h3\u003e\n\u003cp\u003eAbnormal lipid accumulation in steatotic livers coincides with perturbed endoplasmic reticulum (ER) proteostasis in hepatocytes. Therefore, we checked for Unfolded Protein Response (UPR) markers in palmitate stressed hepatocytes. As expected, palmitate markedly and significantly promoted both CHOP and XBP-1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and B). Such an induction was abolished when cells were treated with TOTUM-448 metabolites (-36%, p\u0026thinsp;\u0026le;\u0026thinsp;0.01 and \u0026minus;\u0026thinsp;66%, p\u0026thinsp;\u0026le;\u0026thinsp;0.0001 for CHOP and XBP1, respectively. However, neither the na\u0026iuml;ve (NHS) nor the enriched human serum (EHS) fraction had any impact on those markers in the absence of a lipotoxic context. To support these data, we analyzed ATF-6 activity as the result of the presence of ATF-6 protein in the nucleus. Accordingly, ATF-6 was increased upon palmitate stress. However, limitation by TOTUM-448 metabolites remained a trend (-29%, p\u0026thinsp;=\u0026thinsp;0.2).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this manuscript, we first demonstrated that the ingestion of TOTUM-448 (1) led to bioavailable circulating metabolites and that these metabolites were able to drive the attenuation of palmitate-induced intracellular lipid storage including (2) TG (-27%, p ≤ 0.001) and (3) cholesterol (-52%, p = 0.0001), as well as the inhibition of (4) DGAT2 (-64%, p ≤ 0.001) and (5) SREBP1-c (-68%, p ≤ 0.0001) gene expression. TOTUM-448’s metabolites also inhibited (6) palmitate-induced inflammatory gene expression (p ≤ 0.01). A major effect was related to ER stress. While palmitate potently induced both (7) CHOP, (p ≤ 0.001) and (8) XBP1 mRNA expression (p ≤ 0.0001), (9) ATF-6 activity (p = 0.0053) and ultimately (10) Caspase-3 activity (p ≤ 0.0001), the presence of TOTUM-448’s metabolites normalized these markers (-36%, p ≤ 0.01; -66%, p ≤ 0.0001 and − 31%, p ≤ 0.01, for CHOP, XBP1 and Caspase-3, respectively; a trend was observed for ATF-6 (-29%, p = 0.2)).\u003c/p\u003e\u003cp\u003eFirst, the nutritional dimension of the dose used may be questioned. Each volunteer was exposed once per clinical phase to 4284 mg of TOTUM-448 representing an approximative global amount of 372 mg of polyphenols (see suppl. Table S1 for chemical characterization of TOTUM-448). The recommended dose for polyphenol daily intakes is about 1g, thus the nutritional dimension of the dose is quite relevant. Besides, the rational of the dose also echoes with paralleled clinical trials we have launched. Indeed, the dose chosen is also the one tested for long term supplementation and investigation in humans. The clinical trial is ongoing: NCT06704321.\u003c/p\u003e\u003cp\u003eThen, the bioavailability of the ingredient was investigated. In this study, we were able to detect 12 different circulating metabolites resulting from the ingestion of the blend including 2 oleuropein glucuronide isomers (olive leaf \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e), 3 luteolin glucuronide isomers (metabolites that may derive from luteolin-7-O-glucoside, luteolin-7-O-glucuronide and luteolin presents in chrysanthellum, in both artichoke and olive leaves \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e), 1 ferulic acid sulfate (metabolite that may derive from either chlorogenic acid present in artichoke leaf and, chrysanthellum or caffeoylquinic acid also present in artichoke leaf \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e), 3 hydroxytyrosol sulfate isomers, 1 hydroxytyrosol glucuronide, 1 tyrosol glucuronide, 1 homovanilic acid sulfate (metabolites that may derive from both hydroxytyrosol and oleuropein present in olive leaf \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e). These metabolomic analyses evidenced a clear and diverse bioavailability of the components of the blend and supported the rational for further investigation of the bioactivity of the serum.\u003c/p\u003e\u003cp\u003eOne may also question the ex vivo model. In our clinical ex vivo approach, volunteers should be seen as metabolites producers rather than patients expecting health benefits with measurable clinical scores. Thus, this protocol is designed to collect circulating metabolites in a standardized way and only male healthy volunteers with no treatment or pathology, aged from 18 to 35, BMI ranged from 20 to 28, with normal kidney and liver functions are recruited. The collected serum, either naïve (control baseline) or enriched (with circulating metabolites of interest) are further used/incubated on cell cultures according to the patent DIRV#18–0058 (written invention disclosure by the French National Institute for Agronomic, Food and Environment Research INRAE) to investigate a wide range of cell activities/markers that represent the final readout/score. In this study we targeted hepatocytes as a major protagonist in lipid metabolism and MASDL onset.\u003c/p\u003e\u003cp\u003eHepatocytes are responsible for glucose, xenobiotic metabolism and for lipids metabolism as well. In our hands, palmitate consistently promoted lipids accumulation in hepatocytes including triglycerides and cholesterol mimicking a relevant MASLD onset. The presence of metabolites resulting from TOTUM-448 ingestion potently prevented these MASLD hallmarks. From a mechanistic point of view, these results match with the inhibition of both, DGAT2, an enzyme involved in triglycerides synthesis \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e and SREBP1c, involved in the \u003cem\u003ede novo\u003c/em\u003e lipogenesis in the liver \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Besides, these results are fully consistent with published observations regarding the effect of the different components of the blend when studied separately. In patients with diagnosed NAFLD, supplementation with artichoke leaf extracts (Cynara scolymus L.) (600 mg daily for 9 weeks) reduced liver size and decreased serum total cholesterol and triglyceride concentrations \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In a high-fat diet rat model, artichoke leaf extract supplementation limited hepatic steatosis \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. A clinical study on a cohort of steatotic patients showed that supplementation with Chrysanthellum americanum extract decreased fibrosis and hepatic steatosis \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Decreased choline intake is significantly associated with increased liver fibrosis in post-menopausal women with NAFLD \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn MASLD, the lipotoxic context fuels inflammation and oxidative stress by stimulating ROS production, pro-inflammatory cytokine release and by promoting the recruitment of immune cells. In light of this, our cellular model fully addresses these criteria as palmitate increases DCF-DA staining and inflammatory gene expressions. Consistent with this, we observed an increase in global inflammatory gene expression in response to palmitate exposure. This detrimental effect was blunted in hepatocytes incubated with Totum-448-enriched serum. This observation must be tempered by the fact that the induction of the expression of CXCL1, IL-1β, IL-6, MCP-1 and TNFα did not reach significance when analyzed separately, suggesting a global effect on inflammatory status rather than a specific effect on a target gene. Consistently, in a recent randomized, controlled clinical study, Guo et al. demonstrated that NLRP3 inflammasome activation is significantly increased in patients with NAFLD, but that this activation can be strongly repressed by anthocyanins provided here by blueberry extract (Vaccinium myrtillus L.) \u003csup\u003e40\u003c/sup\u003e. Several polyphenols present in the enriched serum have already demonstrated antioxidant properties through scavenging of free radicals or inhibition of oxidative enzymes \u003csup\u003e41 42\u003c/sup\u003e including oleuropein in a similar HepG2 model \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Preclinically, olive leaf extracts have shown anti-inflammatory, antioxidant, and antifibrotic effects \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Finally, a 2021 randomized double-blind clinical study shows that supplementation with olive leaf extract (Olea europaea L.), characterized by its oleuropein and hydroxytyrosol content, shows a reduction in oxidized LDL \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eHepatocytes are also responsible for \u003cem\u003ede novo\u003c/em\u003e lipids biogenesis along with lipid metabolism as well. Thus, the role of both, the smooth and the rough endoplasmic reticulum (ER), is pivotal. In light of this, dysregulation of lipid metabolism, increased inflammation or oxidative stress may either be a starting point or a consequence of the unbalance of the ER metabolism. This drives the establishment of a vicious cycle triggering the UPR (Unfold Protein Response) and leading to the activation of IRE1, PKR-like ER kinase (PERK) and ATF-6 to reset ER homeostasis \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Of interest, PERK activation results in an antioxidant response \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e while both PERK and ATF-6 participate to the recovery from liver’s condition as demonstrated by knockout studies \u003csup\u003e\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e–\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eIn this study, the influence of the TOTUM-448 metabolites on ER-stress management seems to be the cornerstone. Palmitate enhanced both XBP-1 \u003csup\u003e52,53\u003c/sup\u003e and CHOP \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e expressions and significantly stimulates ATF-6 activity. XBP-1 and CHOP up-regulation result from the activation of IRE1 and PKR-like ER kinase (PERK), respectively. This up-regulation is fully consistent with literature data. Association between chronic ER and steatotic liver was previously established in both obese mouse models (genetic and high-fat diet models) and obese patients \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Palmitate also significantly enhanced caspase-3 activity. Such an enhancement was previously described in liver biopsies from patients suffering from NASH \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. In rats fed a high fat diet, CHOP expression and active form of caspase-3 were found increased in liver \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. The presence of the human metabolites from TOTUM-448 potently limited this palmitate-induced ER stress and normalized the studied markers (-36%, p ≤ 0.01; -66%, p ≤ 0.0001 and − 31%, p ≤ 0.01, for CHOP, XBP1 and Caspase-3, respectively; a trend was observed for ATF-6 (-29%, p = 0.2)) supporting the relevance of this nutritional strategy to manage MASLD. The protective effects of the serum enriched with TOTUM-448-derived metabolites against palmitate-induced endoplasmic reticulum (ER) stress may, at least in part, be attributed to the action of specific polyphenols known to be present in the plant extracts composing TOTUM-448. Several of these compounds have already been reported to mitigate ER stress. For example, hydroxytyrosol, a major compound of olive leaf extract, has been shown to protect HepG2 cells from tunicamycin-induced ER stress \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Similarly, luteolin, found in artichoke and Chrysanthellum extracts, was reported to prevent palmitate-induced ER stress, autophagy and apoptosis in AML12 hepatocytes \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. In addition, chlorogenic acid, which is present in artichoke leaf extract, demonstrated protective effects against palmitate-induced ER stress in primary rat hepatocytes \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eInflammation, oxidative stress and ER stress being widely imbricated \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e we are facing a chicken-and-egg situation and, the question regarding the way the TOTUM-448’s metabolites support hepatoprotection remains. Do they improve hepatocyte behavior and lipid metabolism by preserving ER homeostasis or do they limit ER stress by improving lipid metabolism?\u003c/p\u003e\u003cp\u003eIndeed, it has been reported that caloric restriction improves steatosis and alleviates ER stress \u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e while normalization of ER stress markers was correlated with reduced hepatic lipid content \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. ER is the major site of lipid synthesis in hepatocytes including triglyceride synthesis and storage. Both, the sterol regulatory element-binding proteins (SREBP1c for fatty acid synthesis and SREBP2 for sterol synthesis) and the diacylglycerol acyltransferase (DGAT) are ER-localized transcription factors and enzymes, respectively, involved in the regulation and the catalysis of \u003cem\u003ede novo\u003c/em\u003e lipogenesis \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Accordingly, TOTUM-448’s metabolites were found to reduce SREBP1c and DGAT2 expression to blunt the vicious cycle of MASLD and promote hepatocyte homeostasis. Notably, the metabolites had no effect on their own but showed protective effects in the presence of palmitate, supporting the preventive role of this ingredient in maintaining hepatocyte function and homeostasis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003c/div\u003e\u003c/div\u003e\n\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Methods","content":"\u003ch2\u003eStudy Product\u003c/h2\u003e\u003cp\u003eVALBIOTIS has developed TOTUM-448 (priority French patent: FR1460064), a food ingredient composed of the combination of different plant aforementioned. Selection of the extracts was originally driven by published data regarding their possible support in preventing liver conditions. The formulation of the product meets the requirements of European regulations relating to food supplements (European directive n°2002/46/EC and French decree n°2006 − 352 of March 20, 2006 which transposes it).\u003c/p\u003e\u003ch3\u003eEthics Preclinical Study\u003c/h3\u003e\u003cp\u003eThe study was conducted in accordance with the Declaration of Helsinki of 1975 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.wma.net/what-we-do/medical-ethics/declaration-of-helsinki\u003c/span\u003e\u003cspan address=\"https://www.wma.net/what-we-do/medical-ethics/declaration-of-helsinki\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (accessed on November, 12th 2021)) revised in 2013. The human study was approved by the French Ethical Committee (NCT06047847/ N° ID RCB: 2023-A00579-36/ Comité de Protection des Personnes CPP Est III; approved 25th July 2023). The volunteers were informed of the objectives and the potential risks of the present study and provided their written informed consent before they participated in the study.\u003c/p\u003e\u003ch2\u003eHuman Study Design and Pharmacokinetic of Absorption\u003c/h2\u003e\u003cp\u003eTen healthy men (age: 24.5 years old, +/− 3.48; BMI: 23.35 kg/m2, +/−1.72; \u0026gt;60 kg) were enrolled for this study. Only male healthy volunteers with no treatment or pathology, aged from 18 to 35, BMI ranged from 20 to 28, with normal kidney and liver functions were recruited. They were checked for normal blood formulation, renal (urea and creatinine), and liver functions (aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma-glutamyltransferase (GGT) activities). Serum samples from all participants were collected in plain tubes. Unfortunately, female volunteers were not to be recruited to avoid hormonal variation and subsequent bias.\u003c/p\u003e\u003cp\u003eThe first phase of this clinical project was dedicated to determine TOTUM-448’s metabolites absorption profile. Prior to receiving the tested product, volunteers were fasted for 12 h and then, were given 4284 mg of TOTUM-448 as eight capsules. Nine milliliters of venous blood (median cubital vein) were collected before the ingestion of TOTUM-448 and then every 20 min for 240 min after the ingestion of the ingredient. Collected serum was aliquoted in sterile tubes and then stored until analyses at − 80°C. Location of storage: Clinical Investigation Center – Inserm 1405, University Hospital of Clermont-Ferrand. This dedicated research department is fully compliant with regulatory and ethical clinical obligations (certification according to the French standard NF S 96900).\u003c/p\u003e\u003cp\u003eCirculating metabolites resulting from TOTUM-448 ingestion were quantified and characterized by ultra-high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS). Upon characterization of the absorption profile, volunteers underwent a second visit to the clinical center for the second phase of the project in order to collect serum containing TOTUM-448 metabolites at Tmax/Cmax. Context was similar to phase 1. Prior to receiving the tested product, volunteers were fasted for 12 h and then, were given 4284 mg of TOTUM-448 as eight capsules. Forty-eight milliliters of venous blood were collected before the product ingestion as a control baseline naïve serum. Then, at Cmax in the post-absorptive phase, forty-eight milliliters of blood were collected for circulating bioactive collection. Collection, aliquoting and storage conditions were similar to phase 1.\u003c/p\u003e\u003ch2\u003ePhenolic Compounds Extraction from Serum\u003c/h2\u003e\u003cp\u003eTo extract phenolic compounds from serum, 0.9 mL of serum was combined with 2.7 mL of 100% methanol and mixed for one minute. The mixture was then centrifuged at 20,000× g for 15 minutes at room temperature. The supernatant was collected in separate tubes and evaporated to dryness using a SpeedVac Concentrator (Thermo Fisher Scientific, Illkirch, France). The dried material was reconstituted in 80 µL of a 50:50 methanol/water solution. After agitation for one minute, ultrasonication for one minute, and centrifugation at 20,000× g for 15 minutes at room temperature, the supernatant was stored at − 20°C until analysis by ultra-high performance liquid chromatography with tandem mass spectrometry detection.\u003c/p\u003e\u003ch2\u003eUltra-High Performance Liquid Chromatography-Mass Spectrometry (UHPLC-MS/MS)\u003c/h2\u003e\u003cp\u003ePhenolic compounds were analyzed using a 1260 Infinity UHPLC system (Agilent Technologies) coupled to a 6430 triple quadrupole mass spectrometer (Agilent Technologies, Les Ulis, France). Four microliters of the sample were injected into a Zorbax SB-C18 column (2.1 × 100 mm, 1.8 µm) (Agilent Technologies, Les Ulis, France). The mobile phase consisted of two solvents: solvent A (water/formic acid 99.9:0.1, v/v) and solvent B (acetonitrile/formic acid 99.9:0.1, v/v), with a flow rate of 0.3 mL/min. The gradient for solvent A was as follows: 0 min 1% B, 2 min 5% B, 3 min 25% B, 6 min 25% B, 8 min 40% B, 11.5 min 95% B, 14 min 95% B, and 16 min 1% B. The MS/MS parameters were set to negative ion mode, with a capillary tension of 3000 V, a nebulizer pressure of 15 psi, a dry gas flow rate of 11 L/min, a dry temperature of 350°C, and acquisition in multiple reaction monitoring (MRM) mode. Data were processed using MassHunter software (Agilent Technologies).\u003c/p\u003e\u003ch2\u003eHuman Hepatocyte Cultures\u003c/h2\u003e\u003cp\u003eThe HepG2 human hepatocyte cell line was obtained from the European Collection of Authenticated Cell Cultures and purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France − 85011430). During the maintenance phase, HepG2 cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin/streptomycin (Life Technologies, Villebon-Sur-Yvette, France). All cell cultures were maintained at 37°C in an atmosphere of 5% CO2/95% air. To analyze the effects of TOTUM-448 metabolites, the cells were preincubated for 24 hours in DMEM with 10% human serum (either naïve or containing circulating metabolites) according to the Clinic’n’Cell methodology (DIRV INRAE 18–0058), followed by an additional 48-hour incubation in a palmitate-induced lipidic stress environment (palmitate 250 µM; Sigma, Saint-Quentin-Fallavier, France).\u003c/p\u003e\u003ch2\u003ePreparation of Palmitate Solution\u003c/h2\u003e\u003cp\u003ePalmitate (Sigma, Saint-Quentin-Fallavier, France) was first coupled to bovine serum albumin (BSA; Sigma, Saint-Quentin-Fallavier, France) and then fully dissolved in pure ethanol at 70°C to achieve a final concentration of 500 mmol/L. This stock solution was mixed with a prewarmed BSA solution (10% w/w, 37°C) to reach a final concentration of 5 mmol/L. The mixture was clarified by incubating at 55°C for 15 minutes twice. The final palmitate:BSA molar ratio was set at 3.2:1. The control vehicle was prepared under the same conditions. A 10% w/w BSA solution was added to an equivalent volume of ethanol to match the final palmitate solution. The final ethanol concentration was less than 0.05% by volume in all experiments.\u003c/p\u003e\u003ch2\u003eCell Viability\u003c/h2\u003e\u003cp\u003eCell viability was assessed using an XTT-based method (Cell Proliferation Kit II, Sigma-Aldrich, Saint-Quentin-Fallavier, France). Experimental procedures were followed according to the supplier’s recommendations. Optical density was measured at 450 nm, with measurements performed in hexaplicate for each sample condition of the ten volunteers.\u003c/p\u003e\u003ch2\u003eDCF-DA Staining\u003c/h2\u003e\u003cp\u003eHepG2 cells were seeded on a 96-well dark-wall clear-bottom plate at a density of 12,000 cells/cm². Twenty-four hours after H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e stimulation, cells were washed and incubated with 5 µM of 2′,7′-dichlorofluorescin diacetate (DCF-DA) solution (ab113851, Abcam) for 45 minutes at 37°C in the dark, then rinsed with the dilution buffer according to the manufacturer’s protocol. Fluorescence was measured using a fluorescence plate reader (Berthold − Mitras) at Ex/Em = 485/535 nm in end-point mode.\u003c/p\u003e\u003ch2\u003eRed Oil Staining\u003c/h2\u003e\u003cp\u003e Oil Red O solution (0.5% in isopropanol) was obtained from Sigma (Saint-Quentin-Fallavier, France) and staining was performed according to the supplier’s recommendations. The working solution (0.2% in 60% isopropanol) was prepared by mixing Oil Red O solution with distilled water in a 3:2 ratio. Prior to staining, cells were washed twice with PBS and fixed with 4% paraformaldehyde for 30 minutes at room temperature. After discarding the PFA solution, cells were washed twice with water, incubated with 60% isopropanol for 5 minutes, and then stained with the working Oil Red O solution for 20 minutes. After five additional washes with water, cells were observed under a microscope.\u003c/p\u003e\u003ch2\u003eTriglycerides Levels\u003c/h2\u003e\u003cp\u003eTriglyceride content in HepG2 cells was determined using a triglyceride assay kit (Abcam, Paris, France—ab65336) according to the manufacturer’s protocol. Triglycerides were converted to free fatty acids and glycerol, and the glycerol was oxidized to generate a colorimetric product measured at 570 nm.\u003c/p\u003e\u003ch2\u003eCholesterol Levels in HepG2 Cells\u003c/h2\u003e\u003cp\u003eCholesterol levels were evaluated in cell lysates using a cholesterol quantification kit (Sigma, Saint-Quentin-Fallavier, France—MAK043) according to the manufacturer’s recommendations. Total cholesterol concentration was measured by a coupled enzyme reaction resulting in a fluorometric (λex = 535 nm/λem = 587 nm) product related to the cholesterol content.\u003c/p\u003e\u003ch2\u003eReal-Time RT-qPCR\u003c/h2\u003e\u003cp\u003emRNA from HepG2 cells were isolated using TRIzol™ Reagent (Ambion – Life Technologies) according to the supplier’s recommendations. The expression levels of DGAT2 (Diacylglycerol O-acyltransferase 2), SREBP1-c (Sterol regulatory element-binding protein 1c), CXCL-1 (CXC motif chemokine ligand 1), IL-1β, IL-6, MCP-1, TNFα, CHOP (C/EBP homologous protein), and XBP-1 mRNA were measured by RT-qPCR (PowerUp SYBRgreen, Applied Biosystems). β-Actin was used as a housekeeping gene. Primers were designed as follows: IL6-F: 5’- TTC TGT GCC TGC AGC TTC − 3’; IL6-R: 5’- GCA GAT GAG TAC AAA AGT CCT GA -3’; MCP-1-F: 5’- GCC TCT GCA CTG AGA TCT TC -3’; MCP-1-R: 5’- AGC AGC CAC CTT CAT TCC − 3’; TNFa-F: 5’- TCA GCT TGA GGG TTT GCT AC -3’; TNFa-R: 5’- TGC ACT TTG GAG TGA TCG G -3’; IL1b-F: 5’- GAA CAA GTC ATC CTC ATT GCC − 3’; IL1b-R: 5’- CAG CCA ATC TTC ATT GCT CAA G -3’; CXCL1-F: 5’- TCT CTC TTT CCT CTT CTG TTC CTA − 3’; CXCL1-R: 5’- CAT CCC CCA TAG TTA AGA AAA TCA TC -3’; DGAT2-F: 5’- TCA GCA GGT TGT GTG TCT TC -3’; DGAT2-R: 5’- GGC TGG TGT TTG ACT GGA A -3’; SREBP1c-F: 5’- GGA TGG TGT TCA CTC GGT A -3’; SREBP1c-R: 5’- GGT GAT ATG TGT CTG CGT C -3’; XBP1-F: 5’- CGC TGT CTT AAC TCC TGG TTC − 3’; XBP1-R: 5’- CTG GAA CAG CAA GTG GTA GA -3’; CHOP-F: 5’- CAA TGA CTC AGC TGC CAT CT -3’; CHOP-R: 5’- AGC GAC AGA GCC AAA ATC AG -3’; ACTβ-F: 5’-ATT GGC AAT GAG CGG TTC-3’; ACTβ-R: 5’-GGA TGC CAC AGG ACT CCA-3’.\u003c/p\u003e\u003ch2\u003eCell Lysis\u003c/h2\u003e\u003cp\u003eThe lysis buffer was prepared by mixing 50 mmol/L Tris pH 7.8, 150 mmol/L NaCl, 0.5% sodium deoxycholate, and 1% NP40. Cell lysates were stored at − 80°C until analysis.\u003c/p\u003e\u003ch2\u003eProtein Quantification\u003c/h2\u003e\u003cp\u003eProtein content was measured using the BCA Protein Assay Kit (Sigma-Aldrich, Saint-Quentin-Fallavier, France). The BCA protein assay is based on a biuret reaction, where the reduction of Cu2 + to Cu + in the presence of proteins in an alkaline environment is proportional to the protein concentration. The chromogenic reagent bicinchoninic acid chelates the reduced copper, forming a purple complex that absorbs at 562 nm.\u003c/p\u003e\u003ch2\u003eCaspase-3 activity\u003c/h2\u003e\u003cp\u003eCaspase-3 activity in cells was evaluated using the Caspase-3 Assay Kit (Abcam, Paris, France—ab39401) according to the manufacturer’s protocol. To lyse the frozen monolayer of HepG2 cells grown on a twelve-well plate, 200 µL of the provided lysis buffer was used per well. Fifty microliters of freshly prepared lysate were then mixed with 55 µL of reconstituted reaction mix. Optical density at 405 nm was measured every two minutes for 90 minutes at 37°C.\u003c/p\u003e\u003ch2\u003eATF6 activity\u003c/h2\u003e\u003cp\u003eNuclear extracts were prepared from HepG2 cells using the Nuclear Extraction Kit (Abcam, Paris, France—ab113474), and protein content was determined using the BCA Protein Assay Kit (Sigma-Aldrich, Saint-Quentin-Fallavier, France). ATF6 protein expression was assessed using the ATF6 ELISA Kit (Signosis, TE-0041) according to the manufacturer’s instructions.\u003c/p\u003e\u003ch2\u003eStatistics\u003c/h2\u003e\u003cp\u003eStatistical analyses and figure generation were performed using Prism V.10.4.1 (GraphPad Software). The statistical plan included a Shapiro–Wilk normality test to determine if the data followed a Gaussian distribution. If the data were not normally distributed, a Kruskal–Wallis non-parametric test was used, followed by Dunn's test for post hoc comparisons. For normally distributed data with equal variance, one-way or two-way ANOVA with Tukey’s test for multiple comparisons was applied. For inflammatory gene expression, a two-way ANOVA targeting a main column effect was followed by Tukey’s post hoc test. If data were missing, making it impossible to run a repeated-measure two-way ANOVA, a mixed-effects analysis was used instead. In Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, values are presented as means ± SEM, with statistical significance indicated as follows: * for p \u0026lt; 0.05; ** for p \u0026lt; 0.01; *** for p \u0026lt; 0.001; **** for p \u0026lt; 0.0001; ns for p \u0026gt; 0.05. For Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e to \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, values are presented as box plots indicating median and interquartile range (lower and upper), with whiskers indicating minimum and maximum values. Statistical significance is indicated as follows: * for p \u0026lt; 0.05; ** for p \u0026lt; 0.01; **** for p \u0026lt; 0.0001; ns for p \u0026gt; 0.05.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis study was sponsored by VALBIOTIS SA. \u003c/p\u003e\n\u003cp\u003eThis work was supported by the Bordeaux Metabolome Facility and the MetaboHUB (ANR11-INBS-0010) project.\u003c/p\u003e\n\u003cp\u003eThis work was funded by the Pack Ambition Recherche 2021 MICROMETiv (Region Auvergne Rhône Alpes) and the common laboratory MEDIS-VALBIOTIS Labcom MIMETiv (ANR, ANR-22-LCV1-0003-01).\u003c/p\u003e\n\u003cp\u003eAuthor contributions statement\u003c/p\u003e\n\u003cp\u003eConceptualization, F.W. and Y.W.; design, V.C., Y.F.O., P.S., M.C., V.S.; methodology, Y.W., L.B.-W., and F.W.; formal analysis, S.K., J.V., F.L-J. and F.W.; clinical investigation, A.B.M, V.R., N.M., G.P. and L.B.-W.; writing F.W., V.C. and Y.W.; data interpretation and reviewing B.P., V.C., F.W.,Y.W., S.B-D. and A.B; project administration V.S., M.C., S.P. and L.B.-W. All authors have read and agreed to the published version of the manuscript. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003eAdditional information\u003c/p\u003e\n\u003cp\u003eThe human \u003cem\u003eex vivo \u003c/em\u003emethodology used in this study has been registered as a written invention disclosure by the French National Institute for Agronomic, Food and Environment Research (INRAE) (DIRV#18-0058). Clinic’n’Cell® has been registered as a mark \u003csup\u003e22-26\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e28\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e29\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e65-67\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eF.W. and L.W.-B. work for Clinic’n’Cell SAS (Faculty of Medicine and Pharmacy Clermont-Ferrand- France); V.C., A.B.M., Y.F.O., F.L-J., V.S., M.C., S.P., P.S. work for VALBIOTIS and provided the ingredient. Y.W. provides scientific consulting for Clinic’n’Cell SAS; S.K., J.V., B.P., V.R., N.M., G.P., S.B-D and A.B. have no conflict of interest to declare.\u003c/p\u003e\n\u003cp\u003eData availability statement \u003c/p\u003e\n\u003cp\u003eThe datasets generated and analysed during the current study are available from the corresponding author on reasonable request. \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYounossi, Z.\u003cem\u003e et al.\u003c/em\u003e Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention. \u003cem\u003eNature reviews. Gastroenterology \u0026amp; hepatology\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 11-20, doi:10.1038/nrgastro.2017.109 (2018).\u003c/li\u003e\n\u003cli\u003eRinella, M. 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J.\u003cem\u003e et al.\u003c/em\u003e Non-targeted and targeted analysis of collagen hydrolysates during the course of digestion and absorption. \u003cem\u003eAnalytical and bioanalytical chemistry\u003c/em\u003e \u003cstrong\u003e412\u003c/strong\u003e, 973-982, doi:10.1007/s00216-019-02323-x (2020).\u003c/li\u003e\n\u003cli\u003eWauquier, F.\u003cem\u003e et al.\u003c/em\u003e Human Enriched Serum Following Hydrolysed Collagen Absorption Modulates Bone Cell Activity: from Bedside to Bench and Vice Versa. \u003cem\u003eNutrients\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, doi:10.3390/nu11061249 (2019).\u003c/li\u003e\n\u003cli\u003eWauquier, F.\u003cem\u003e et al.\u003c/em\u003e TOTUM-854 Human Circulating Bioactives Preserve Endothelial Cell Function. \u003cem\u003eNutrients\u003c/em\u003e\u003cstrong\u003e17\u003c/strong\u003e, doi:10.3390/nu17081331 (2025).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"clinical trial, ex vivo, hypertension, plant extract, oxidative stress, lipotoxic stress, human metabolites, human endothelial cells","lastPublishedDoi":"10.21203/rs.3.rs-7460979/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7460979/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLipotoxic 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 \u003cem\u003eex vivo\u003c/em\u003e 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 (250\u0026micro;M). Hepatocyte responses were monitored to determine the effects of TOTUM-448\u0026rsquo;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\u0026rsquo;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\u0026rsquo;s metabolites normalized these ER stress markers.\u003c/p\u003e","manuscriptTitle":"Bioavailable Human Metabolites from TOTUM- 448 (Plant-Based, Polyphenol-Rich Ingredient) Maintain Liver Cell Functionality in a Lipotoxic Context that Drives MASLD Onset","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-25 09:48:52","doi":"10.21203/rs.3.rs-7460979/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-07T08:52:39+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-06T14:29:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-30T10:44:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-27T18:19:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-22T10:12:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"328199426592169665616963775460867204689","date":"2025-09-21T18:18:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"123281717111967520310633625595765766563","date":"2025-09-19T10:41:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"17317467609849830533160311612622545200","date":"2025-09-18T12:41:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"95603899993480304727540215560097643586","date":"2025-09-17T07:07:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"314285774673509779878237599171777901366","date":"2025-09-16T18:18:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-16T18:08:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-16T17:59:05+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-16T06:14:32+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-11T16:37:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-09-11T16:33:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"53d29967-63e0-4460-87dd-9be06a83abb4","owner":[],"postedDate":"September 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":55109780,"name":"Biological sciences/Biochemistry"},{"id":55109781,"name":"Biological sciences/Cell biology"}],"tags":[],"updatedAt":"2025-12-22T16:01:47+00:00","versionOfRecord":{"articleIdentity":"rs-7460979","link":"https://doi.org/10.1038/s41598-025-32556-z","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-12-16 15:57:45","publishedOnDateReadable":"December 16th, 2025"},"versionCreatedAt":"2025-09-25 09:48:52","video":"","vorDoi":"10.1038/s41598-025-32556-z","vorDoiUrl":"https://doi.org/10.1038/s41598-025-32556-z","workflowStages":[]},"version":"v1","identity":"rs-7460979","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7460979","identity":"rs-7460979","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}