Resveratrol Metabolites Research Articles

Generation of Stilbene Glycoside with Promising Cell Rejuvenation Activity through Biotransformation by the Entomopathogenic Fungus Beauveria bassiana.

A stilbene glycoside (resvebassianol A) (1) with a unique sugar unit, 4-O-methyl-D-glucopyranose, was identified through biotransformation of resveratrol (RSV) by the entomopathogenic fungus Beauveria bassiana to obtain a superior RSV metabolite with enhanced safety. Its structure, including its absolute configurations, was determined using spectroscopic data, HRESIMS, and chemical reactions. Microarray analysis showed that the expression levels of filaggrin, HAS2-AS1, and CERS3 were higher, while those of IL23A, IL1A, and CXCL8 were lower in the resvebassianol A-treated group than in the RSV-treated group, as confirmed by qRT-PCR. Compound 1 exhibited the same regenerative and anti-inflammatory effects as RSV with no cytotoxicity in skin keratinocytes and TNF-α/IFN-γ-stimulated HIEC-6 cells, suggesting that compound 1 is a safe and stable methylglycosylated RSV. Our findings suggest that our biotransformation method can be an efficient biosynthetic platform for producing a broad range of natural glycosides with enhanced safety.

Intestinal pharmacokinetics of resveratrol and regulatory effects of resveratrol metabolites on gut barrier and gut microbiota

Resveratrol, a dietary polyphenol, has a variety of intestinal bioactivities. However, its material basis remains unknown. This study examined the intestinal pharmacokinetics of resveratrol using HPLC-MS/MS. After oral ingestion in mice, resveratrol and its sulfation metabolites were identified in copious amount in the entire intestinal tract and feces. The glucuronidation metabolites were found in major quantity only in the small intestine. The amount of resveratrol and its metabolites in the total intestine peaked at 4 h, with a concentration of 200 ± 74.8 μM, which corresponded to 14.0% of the administrated dose. During in vitro fermentation, resveratrol-3-O-sulfate, but not resveratrol, significantly promoted the growth of Lactobacillus reuteri (10-fold higher). During the incubation with Caco-2 cells, resveratrol-3-O-sulfate significantly up-regulated the mRNA expressions of tight junction and mucin-related proteins.In conclusion, the intestinal concentration of resveratrol could partially support its intestinal bioactivities, which may be mediated through the actions of its metabolites.

Measurement of Saccharin and trans-Resveratrol Metabolites in Urine as Adherence Markers for Small Quantity Lipid-Based Nutrient Supplement Consumption.

Saccharin and trans-resveratrol were incorporated into small quantity lipid-based nutritional supplements (SQ-LNS) to be evaluated as the markers of consumption for nutritional intervention studies. Forty-seven healthy women consumed a single supplement with either 8.6 mg of saccharin or 5 mg of trans-resveratrol, and urine was collected for 4 h. A rapid 11 min method employing multiple reaction monitoring and ultrahigh performance liquid chromatography coupled to a triple quadrupole mass spectrometer was developed to measure saccharin and resveratrol metabolites in urine simultaneously. The linear dynamic range of the method was from 3 to 1000 ng mL–1, with the correlation coefficient of 0.999 and limits of quantification from 15.28 to 53.03 ng mL–1. Sample preparation was simple dilution with an average recovery of 97.8%. Ion suppression was observed with urine concentrations >10%. Mean levels of saccharin and resveratrol-3-O-sulfate in urine were 5.481 ± 4.359 and 3.440 ± 4.160 nmol L–1, respectively. We developed and validated a method to measure saccharin and trans-resveratrol metabolites in urine to objectively corroborate the consumption of SQ-LNS for the first time in nutrition intervention studies.

Resveratrol administration prevents radiation-related changes in metabolic profiles of hearts 20 weeks after irradiation of mice with a single 2 Gy dose.

We aimed to evaluate whether resveratrol affects radiation-induced changes in metabolite profiles of the mouse heart. Hearts were irradiated in vivo with a single 2 Gy dose during the resveratrol administration and metabolite profiles of heart tissue were analyzed by the untargeted HR-MAS NMR approach twenty weeks after irradiation. The administration of resveratrol mitigated the radiation-induced decline in the content of choline-containing compounds and unsaturated lipids, which might reflect the stabilization of cell membrane structure against radiation-related damage. Results obtained with this mouse model suggest that the resveratrol supplementation may prevent metabolic changes related to radiation-induced damage in the heart.

Sera and lungs metabonomics reveals key metabolites of resveratrol protecting against PAH in rats

Pulmonary arterial hypertension (PAH) is a type of high morbidity and mortality disease. Currently, the intrinsic metabolic alteration and potential mechanism of PAH are still not fully uncovered. Previously, we have found that polyphenol resveratrol (Rev) reversed the remodeling of the pulmonary vasculature and decreased the number of mitochondria in pulmonary arterial smooth muscle cells (PASMCs) (Lei Yu et al. (2017)). However, potential effects of Rev on the changed metabolic molecules derived from lung tissue and serum have no fully elucidated. Thus, we conducted a systematic elaboration through the metabonomics method. Various of metabolites in different pathways including amino acid metabolism, tricarboxylic acid cycle (TCA), acetylcholine metabolism, fatty acid metabolism and biosynthesis in male Wistar rats’ sera and lung tissues were explored in three groups (normal group, PAH group, PAH and Rev treatment group). We found that leucine and isoleucine degradation, valine, leucine and isoleucine biosynthesis, tryptophan metabolism and aminoacyl-tRNA biosynthesis were involved in the development of PAH. Hydroxyphenyllactic, isopalmitic acid and cytosine might be significant key metabolites. Further work in this area may inform personalized treatment approaches in clinical practice of PAH through elucidating pathophysiology mechanisms of experimental verification.

Resveratrol Metabolites Are Able to Reduce Steatosis in Cultured Hepatocytes.

Steatosis is characterized primarily by excessive lipid accumulation in the form of triglycerides in the liver. Although resveratrol shows a low bioavailability, it has significant positive effects on steatosis. The aim of this study was to analyze whether some phase II and microbial resveratrol metabolites (trans-resveratrol-4′-O-glucuronide (R-4G); trans-resveratrol-3-O-glucuronide (R-3G); trans-resveratrol-3-O-sulfate (R-S) and dihydro-resveratrol (DH-R) were effective in reducing hepatocyte fat accumulation. An in vitro model mimicking the hepatocyte situation in fatty liver was developed by incubating mouse AML12 hepatocytes with palmitic acid (PA). For cell treatments, hepatocytes were incubated with 1, 10, or 25 µM resveratrol or its metabolites. Triglycerides and cell viability were assessed using commercial kits. Protein expression of enzymes and transporters involved in triglyceride metabolism were analyzed by western blot. We show for the first time that resveratrol and all the tested metabolites, at 1 µM, partially prevented lipid accumulation induced by the saturated fatty acid PA in AML12 hepatocytes. This effect was mainly due to the inhibition of de novo lipogenesis. This demonstrates that the low bioavailability of resveratrol is not as big a problem as it was thought to be, because resveratrol metabolites contribute to the delipidating effects of the parent compound.

Urinary Resveratrol Metabolites Output: Differential Associations with Cardiometabolic Markers and Liver Enzymes in House-Dwelling Subjects Featuring Metabolic Syndrome.

Metabolic syndrome (MetS) components are strongly associated with increased risk of non-alcoholic fatty liver disease (NAFLD) development. Several studies have supported that resveratrol is associated with anti-inflammatory and antioxidant effects on health status. The main objective of this study was to assess the putative associations between some urinary resveratrol phase II metabolites, cardiometabolic, and liver markers in individuals diagnosed with MetS. In this cross-sectional study, 266 participants from PREDIMED Plus study (PREvención con DIeta MEDiterránea) were divided into tertiles of total urinary resveratrol phase II metabolites (sum of five resveratrol conjugation metabolites). Urinary resveratrol metabolites were analyzed by ultra- performance liquid chromatography coupled to triple quadrupole mass spectrometry (UPLC-Q-q-Q MS), followed by micro-solid phase extraction (µ-SPE) method. Liver function markers were assessed using serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma-glutamyl transferase (GGT). Moreover, lipid profile was measured by triglycerides, very-low-density lipoprotein cholesterol (VLDL-c), and total cholesterol/high-density lipoprotein ratio (total cholesterol/HDL). Linear regression adjusted models showed that participants with higher total urine resveratrol concentrations exhibited improved lipid and liver markers compared to the lowest tertile. For lipid determinations: log triglycerides (βT3 = −0.15, 95% CI; −0.28, −0.02, p-trend = 0.030), VLDL-c, (βT3 = −4.21, 95% CI; −7.97, −0.46, p-trend = 0.039), total cholesterol/HDL ratio Moreover, (βT3 = −0.35, 95% CI; −0.66, −0.03, p-trend = 0.241). For liver enzymes: log AST (βT3 = −0.12, 95% CI; −0.22, −0.02, p-trend = 0.011, and log GGT (βT3 = −0.24, 95% CI; −0.42, −0.06, p-trend = 0.002). However, there is no difference found on glucose variables between groups. To investigate the risk of elevated serum liver markers, flexible regression models indicated that total urine resveratrol metabolites were associated with a lower risk of higher ALT (169.2 to 1314.3 nmol/g creatinine), AST (599.9 to 893.8 nmol/g creatinine), and GGT levels (169.2 to 893.8 nmol/g creatinine). These results suggested that higher urinary concentrations of some resveratrol metabolites might be associated with better lipid profile and hepatic serum enzymes. Moreover, urinary resveratrol excreted showed a reduced odds ratio for higher liver enzymes, which are linked to NAFLD.

Engineering the oleaginous yeast Yarrowia lipolytica for high-level resveratrol production

Resveratrol is a plant secondary metabolite with multiple health-beneficial properties. Microbial production of resveratrol in model microorganisms requires extensive engineering to reach commercially viable levels. Here, we explored the potential of the non-conventional yeast Yarrowia lipolytica to produce resveratrol and several other shikimate pathway-derived metabolites (p-coumaric acid, cis,cis-muconic acid, and salicylic acid). The Y. lipolytica strain expressing a heterologous pathway produced 52.1 ± 1.2 mg/L resveratrol in a small-scale cultivation. The titer increased to 409.0 ± 1.2 mg/L when the strain was further engineered with feedback-insensitive alleles of the key genes in the shikimate pathway and with five additional copies of the heterologous biosynthetic genes. In controlled fed-batch bioreactor, the strain produced 12.4 ± 0.3 g/L resveratrol, the highest reported titer to date for de novo resveratrol production, with a yield on glucose of 54.4 ± 1.6 mg/g and a productivity of 0.14 ± 0.01 g/L/h. The study showed that Y. lipolytica is an attractive host organism for the production of resveratrol and possibly other shikimate-pathway derived metabolites.

Comprehensive analysis of resveratrol metabolites in rats using ultra high performance liquid chromatography coupled with high resolution mass spectrometry

Resveratrol is an antitoxin secreted by plants such as Polygonum cuspidatum Sieb. et Zucc and Vitis vinifera L. when they are attacked by pathogens. In the present study, three methods were used to prepare biological samples, and then an efficient strategy based on ultra-high-performance liquid chromatography-linear ion trap-Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap MS) was developed to screen and identify resveratrol metabolites in rat urine, plasma and faeces. As a result, a total of 56 resveratrol metabolites were screened and characterized. Among them, 39 metabolites were found in rat urine, while 6 and 16 metabolites were characterized from rat plasma and faeces, respectively. In addition, 56, 12 and 15 metabolic products were screened by solid phase extraction (method I), methanol precipitation (method II) and acetonitrile precipitation (method III), respectively, indicating that method I could be adopted as the most acceptable method. The results also demonstrated that resveratrol mainly underwent glucuronidation, glucosylation, sulfation, hydroxylation, dehydrogenation, hydrogenation, methylation and their composite reactions. Moreover, these metabolic reactions occurred to form a possible metabolic network that is similar to a triangular pyramid model. In summary, this research provides an idea for the further study of drug metabolism.

Resveratrol Delivery from Implanted Cyclodextrin Polymers Provides Sustained Antioxidant Effect on Implanted Neural Probes

Intracortical microelectrodes are valuable tools used to study and treat neurological diseases. Due in large part to the oxidative stress and inflammatory response occurring after electrode implantation, the signal quality of these electrodes decreases over time. To alleviate this response, resveratrol, a natural antioxidant which elicits neuroprotective effects through reduction of oxidative stress, was utilized. This work compares traditional systemic delivery of resveratrol to the novel cyclodextrin polymer (pCD) local delivery approach presented herein, both in vitro and in vivo. The pCD displayed an extended resveratrol release for 100 days, as well as 60 days of free radical scavenging activity in vitro. In vivo results indicated that our pCD delivery system successfully delivered resveratrol to the brain with a sustained release for the entire short-duration study (up to 7 days). Interestingly, significantly greater concentrations of resveratrol metabolites were found at the intracortical probe implantation site compared to the systemic administration of resveratrol. Together, our pilot results provide support for the possibility of improving the delivery of resveratrol in an attempt to stabilize long-term neural interfacing applications.

Resveratrol reduces obesity in high-fat diet-fed mice via modulating the composition and metabolic function of the gut microbiota

Resveratrol (RSV) is a natural polyphenol with anti-obesity effects. However, the mechanisms of anti-obesity remain unclear due to its low bioavailability. Recent evidence demonstrates that gut microbiota plays a key role in obesity. This spurred us to investigate whether the anti-obesity effects of RSV are related to modulations in the gut microbiota and metabolic functions. Here, RSV significantly improved metabolic phenotype and intestinal oxidative stress in the high-fat diet (HFD)-fed mice. A multi-omics approach was used to systematically profile the microbial signatures at both the phylogenetic and functional levels using 16S rRNA gene sequencing and metagenome. At the phylogenetic level, RSV treatment significantly modulated the gut microbiota composition in HFD-fed mice, characterized with increased Blautia abundance and decreased Desulfovibrio and Lachnospiraceae_NK4A136_group abundance. At the functional level, RSV significantly decreased the enrichment of pathways linked to host metabolic disease and increased the enrichment of pathways involved in the generation of small metabolites. Besides, the fecal microbiota transplantation experiment showed anti-obesity and microbiota-modulating effects similar to those observed in the oral RSV-feeding experiment. Furthermore, metabolomic analysis and antibiotic treatment verified that 4-hydroxyphenylacetic acid (4-HPA) and 3-hydroxyphenylpropionic acid (3-HPP) were the two gut metabolites of RSV, which contribute to improving lipid metabolism in vitro. Moreover, the content of 4-HPA and 3-HPP exhibited strong correlation with the intestinal oxidative state. We concluded that the RSV-mediated alteration of gut microbiota, related gut metabolites and redox state of the intestinal environment contributed to prevention of metabolic syndrome in HFD-fed mice.

Resveratrol affects in vitro rumen fermentation, methane production and prokaryotic community composition in a time- and diet-specific manner.

SummaryThis study aimed to investigate the effect of resveratrol on methane production, rumen fermentation and microbial composition under high‐concentrate (HC) and high‐forage (HF) diets using the in vitro fermentation system. A total of 25 mg of resveratrol was supplemented into 300 mg of either HC or HF diet. Methane production, total volatile fatty acid (VFA) concentration, molar proportion of VFA, metabolites of resveratrol and prokaryotic community composition were measured after 12 and 24 h of in vitro fermentation. Resveratrol reduced methane production (ml per mg of dry matter degraded) by 41% and 60% under both HC and HF diets (P < 0.001), respectively, and this result could be associated with the lower abundance of Methanobrevibacter (P < 0.001) in response to resveratrol. The molar proportion of propionate was significantly higher in the resveratrol group only under the HC diet (P = 0.045). The relative abundance of 10 bacterial genera was affected by the three‐way interaction of treatment, diet and time (P < 0.05). Resveratrol was partly converted to dihydroresveratrol after 24 h of fermentation, and its degradation could be associated with microbes belonging to the order Coriobacteriales. Our results suggest that multiple factors (e.g. diet and time) should be considered in animal experiments to test the effect of polyphenol or other plant extracts on rumen fermentation, methane emission and microbial composition.

Piceatannol attenuates fat accumulation and oxidative stress in steatosis-induced HepG2 cells

Non-alcoholic fatty liver disease (NAFLD), which affects over 20% of the adult population, is the most common liver disease worldwide and can progress to inflammatory hepatitis, cirrhosis and liver cancer. The need to alleviate NAFLD is imperative, but there are limited pharmacological therapies available. Based on previous reports that piceatannol, a stilbenoid metabolite of resveratrol, exhibits anti-obesity, antioxidant and anti-inflammatory effects, the goal of this study was to determine the efficacy of piceatannol on prevention and/or treatment of NAFLD. The results showed that piceatannol significantly decreased fat accumulation and suppressed lipogenesis and fatty acids (FAs) uptake by decreasing sterol regulatory element-binding protein 1 (SREBP1) and cluster of differentiation 36 (CD36) in steatosis-induced HepG2 hepatocytes. Piceatannol treatment also promoted FAs β-oxidation by increasing farnesoid X receptor (FXR), peroxisome proliferator-activated receptor α (PPARα), and carnitine palmitoyltransferase 1α (CPT1α) under steatosis conditions. Moreover, piceatannol significantly suppressed FA-induced oxidative stress and inhibited phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular signal–regulated kinases 1/2 (ERK1/2). Overall, it is suggested that piceatannol reduced fat accumulation in steatosis-induced HepG2 cells by suppressing lipogenesis (SREBP1 and ACC) and FA uptake (CD36), and promoting FAs oxidation (FXR, PPARα and CPT1α).

Oxidative stress inhibition by resveratrol in alcohol-dependent mice.

Uncontrolled ingestion of alcohol has dramatic consequences on the entire organism that are also associated with the oxidation process induced by alcohol and elevate radical oxygen species. Resveratrol, a nonflavonoid phenol, shows well-documented antioxidant properties. We investigated the potential antioxidant ability of this natural compound in a mouse model of alcohol addiction. We administered (per os) for 60 d 10 mg · kg-1 · d-1 of resveratrol in alcoholic adult male mice. Oxidative stress was evaluated by measuring serum-free oxygen radicals defense and free oxygen radical levels. Resveratrol metabolites were measured in the serum of mice that were administered with resveratrol. Finally, the effect of resveratrol on the alcohol-induced alteration of brain-derived neurotrophic factors (BDNF) in the liver was investigated. Prolonged consumption of resveratrol strongly counteracts serum radical oxygen species formation caused by chronic alcohol intake without effects on natural, free oxygen radical defense. The presence of resveratrol metabolites in the serum only of animals supplemented with resveratrol potentiates the evidence that the antioxidant effect observed is due to the ingestion of the natural compound. Moreover, resveratrol supplementation can counteract alcohol-induced BDNF elevation in the liver, which is the main target of organ alcohol-induced damage. The consumption of resveratrol through metabolite formation may play a protective role by decreasing free radical formation and modulating the BDNF involved in hepatic disruption induced by chronic alcohol consumption. Further investigation into the mechanism underlying the protective effect could reinforce the potential use of resveratrol as a dietary supplement to prevent damage associated with chronic alcohol abuse.

Piceatannol Increases Antioxidant Defense and Reduces Cell Death in Human Periodontal Ligament Fibroblast under Oxidative Stress.

Piceatannol is a resveratrol metabolite that is considered a potent antioxidant and cytoprotector because of its high capacity to chelate/sequester reactive oxygen species. In pathogenesis of periodontal diseases, the imbalance of reactive oxygen species is closely related to the disorder in the cells and may cause changes in cellular metabolism and mitochondrial activity, which is implicated in oxidative stress status or even in cell death. In this way, this study aimed to evaluate piceatannol as cytoprotector in culture of human periodontal ligament fibroblasts through in vitro analyses of cell viability and oxidative stress parameters after oxidative stress induced as an injury simulator. Fibroblasts were seeded and divided into the following study groups: control, vehicle, control piceatannol, H2O2 exposure, and H2O2 exposure combined with the maintenance in piceatannol ranging from 0.1 to 20 μM. The parameters analyzed following exposure were cell viability by trypan blue exclusion test, general metabolism status by the 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) method, mitochondrial activity through the ATP production, total antioxidant capacity, and reduced gluthatione. Piceatannol was shown to be cytoprotective due the maintenance of cell viability between 1 and 10 μM even in the presence of H2O2. In a concentration of 0.1 μM piceatannol decreased significantly cell viability but increased cellular metabolism and antioxidant capacity of the fibroblasts. On the other hand, the fibroblasts treated with piceatannol at 1 μM presented low metabolism and antioxidant capacity. However, piceatannol did not protect cells from mitochondrial damage as measured by ATP production. In summary, piceatannol is a potent antioxidant in low concentrations with cytoprotective capacity, but it does not prevent all damage caused by hydrogen peroxide.

Conjugated Physiological Resveratrol Metabolites Induce Senescence in Breast Cancer Cells: Role of p53/p21 and p16/Rb Pathways, and ABC Transporters.

Recent evidence demonstrates that resveratrol (RSV) metabolites, but not free RSV, reach malignant tumors (MT) in breast cancer (BC) patients. Since these metabolites, as detected in MT, do not exert short-term antiproliferative or estrogenic/antiestrogenic activities, long-term tumor-senescent chemoprevention has been hypothesized. Consequently, here, for the first time, whether physiologically relevant RSV metabolites can induce senescence in BC cells is investigated. Human BC MCF-7 (wild-type p53) and MDA-MB-231 (mutant p53), and non-tumorigenic MCF-10A cells are treated with free RSV and physiological-derived metabolites (RSV 3-O-glucuronide, RSV 3-O-sulfate, RSV 4'-O-sulfate, dihydroresveratrol (DH-RSV), and DH-RSV 3-Oglucuronide). Cellular senescence is measured by SA-β-gal activity and senescence-associated markers (p53, p21Cip1/Waf1 , p16INK4a , and phosphorylation status of retinoblastoma (pRb/tRb)). Although no effect is observed in MDA-MB-231 and normal cells, RSV metabolites induce cellular senescence in MCF-7 cells by reducing their clonogenic capacity and arresting cell cycle at G2 M/S phase, but do not induce apoptosis. Senescence is induced through the p53/p21Cip1/Waf1 and p16INK4a /Rb pathways, depending on the RSV metabolite, and requires ABC transporters, but not estrogen receptors. These data suggest that RSV metabolites, as found in MT from BC patients, are not de-conjugated to release free RSV, but enter the cells and may exert long-term tumor-senescent chemoprevention.

&lt;p&gt;Metabolic profile and structure–activity relationship of resveratrol and its analogs in human bladder cancer cells&lt;/p&gt;

Purpose: Resveratrol (RV), a promising anti-cancer candidate, is limited in application for its poor bioavailability. However, the better bioavailability has been found in some RV derivatives. So in this paper, we explore the structure–activity relationship and the metabolic profiles of RV and its analogs (polydatin [PD], oxyresveratrol [ORV], acetylresveratrol [ARV]) in human bladder cancer T24 cells, and then evaluate their active forms and key chemical functional groups which may determine the fate of tumor cells.Methods: Drug sensitivity is evaluated by MTT assay, HE staining and flow cytometry analysis after T24 cells treated with RV, PD, ORV and ARV, respectively. Then the drug metabolites, in alive and dead T24 cells, also in T24 cell supernatant and lysates, are qualitatively and quantitatively analyzed by high-performance liquid chromatography, liquid chromatography coupled with tandem mass spectrum and high-resolution mass spectrometry technologies, respectively.Results: RV, ORV and ARV inhibit bladder cancer cells growth in a dose- and time-dependent manner, and exert the anti-tumor potency to T24 cells in order of ORV>ARV>RV>PD. Meanwhile, similar metabolic profiles of the above compounds are found not only in cell supernatant and lysate, but also in dead and alive T24 cells after drug treatment, and the main metabolites of RV, ORV and PD are their prototypes, but ARV is mainly metabolized to RV.Conclusion: The inhibitory potencies to T24 cells in the order of ORV>ARV>RV>PD are related to the structure and metabolism of RV and its analogs. Meanwhile, the number and position of free phenolic hydroxyl groups play a prominent role in antitumor activities. Therefore, protecting phenolic hydroxyl groups, and inhibiting drug metabolism to keep phenolic hydroxyl groups free would be the promising strategies to ensure the bioactivity of RV and its analogs, and thus to improve RV’s bioactivity and promote RV clinical translation.

Resveratrol, lunularin and dihydroresveratrol do not act as caloric restriction mimetics when administered intraperitoneally in mice

Resveratrol as well as caloric restriction were shown to extend lifespan in some model organisms and may possibly delay onset of ageing-related diseases in humans. Yet, resveratrol supplementation does not always extend lifespan of animal models or improve health status of humans. Because of interindividual differences in human microbiota, resveratrol metabolite production in the gut differs. While some individuals produce lunularin and dihydroresveratrol in their gut, others produce dihydroresveratrol only. Therefore, we addressed the question whether these metabolites differ in their biological impact on ageing and intraperitoneally injected 13-month-old C57BL/6JRj mice on an ad-libitum (AL) HFD with resveratrol, dihydroresveratrol or lunularin (24 mg/kg bodyweight; 3 times/week). Compared to mice injected with vehicle (AL-control), resveratrol and dihydroresveratrol did not change bodyweight and had no impact on insulin or glucose levels while lunularin slightly reduced feed intake and bodyweight gain. CR-mice showed lowered cholesterol, insulin and leptin levels, elevated adiponectin and phosphorylated AMPK levels in liver as well as increased transcription of Pck1 and Pgc1α when compared to the AL-control. In contrast, injections with the test substances did not change these parameters. We therefore conclude that in our model, resveratrol, lunularin and dihydroresveratrol did not act as CR mimetics.

Genomic sequencing, genome-scale metabolic network reconstruction, and in silico flux analysis of the grape endophytic fungus Alternaria sp. MG1

BackgroundAlternaria sp. MG1, an endophytic fungus isolated from grape, is a native producer of resveratrol, which has important application potential. However, the metabolic characteristics and physiological behavior of MG1 still remains mostly unraveled. In addition, the resveratrol production of the strain is low. Thus, the whole-genome sequencing is highly required for elucidating the resveratrol biosynthesis pathway. Furthermore, the metabolic network model of MG1 was constructed to provide a computational guided approach for improving the yield of resveratrol.ResultsFirstly, a draft genomic sequence of MG1 was generated with a size of 34.7 Mbp and a GC content of 50.96%. Genome annotation indicated that MG1 possessed complete biosynthesis pathways for stilbenoids, flavonoids, and lignins. Eight secondary metabolites involved in these pathways were detected by GC–MS analysis, confirming the metabolic diversity of MG1. Furthermore, the first genome-scale metabolic network of Alternaria sp. MG1 (named iYL1539) was reconstructed, accounting for 1539 genes, 2231 metabolites, and 2255 reactions. The model was validated qualitatively and quantitatively by comparing the in silico simulation with experimental data, and the results showed a high consistency. In iYL1539, 56 genes were identified as growth essential in rich medium. According to constraint-based analysis, the importance of cofactors for the resveratrol biosynthesis was successfully demonstrated. Ethanol addition was predicted in silico to be an effective method to improve resveratrol production by strengthening acetyl-CoA synthesis and pentose phosphate pathway, and was verified experimentally with a 26.31% increase of resveratrol. Finally, 6 genes were identified as potential targets for resveratrol over-production by the recently developed methodology. The target-genes were validated using salicylic acid as elicitor, leading to an increase of resveratrol yield by 33.32% and the expression of gene 4CL and CHS by 1.8- and 1.6-fold, respectively.ConclusionsThis study details the diverse capability and key genes of Alternaria sp. MG1 to produce multiple secondary metabolites. The first model of the species Alternaria was constructed, providing an overall understanding of the physiological behavior and metabolic characteristics of MG1. The model is a highly useful tool for enhancing productivity by rational design of the metabolic pathway for resveratrol and other secondary metabolites.

Endothelial Cells Deconjugate Resveratrol Metabolites to Free Resveratrol: A Possible Role in Tissue Factor Modulation.

The antithrombotic effects of resveratrol (RV) and its derivatives remain unknown. The objective is to evaluate the modulatory effects of RV, its glucoside form, piceid, and its biological metabolites (RV-3-O-β-d-glucuronide, RV-4'-O-d-glucuronide, and RV-3-O-sulfate) on tissue factor (TF). Moreover, the endothelial metabolism of RV is assessed. Human aortic endothelial cells (HAECs) are incubated with trans-piceid, trans-RV, or their biological metabolites and stimulated with tumor necrosis factor-α (TNF-α). TF activity, protein levels, and mRNA expression are determined in cell lysates. Moreover, RV conjugation (phase-II-metabolism) to its sulfated or glucuronidated metabolites and their deconjugation to their parent compound (free RV) are also assessed in cell lysates and culture media. RV decreased TF activity, protein levels, and mRNA expression, whereas piceid and RV metabolites (RVmet) had no effects. RV-3-O-sulfate was the main metabolite generated in the endothelium, while RVmet are deconjugated to free RV. Isomerization of trans-RV and its trans-metabolites to their cis-forms is observed. RV exerts antithrombotic effects by modulating TF. RVmet and piceid does not exert this effect. However, the capacity of endothelial cells to deconjugate RVmet to free RV indicates that RVmet function as an endothelial reservoir for RV regeneration, thus, contributing to the antithrombotic effects of RV.