RESUMO
Tomato pomace (TP) is an underutilized source of bioactive compounds with potential application in the food sector. A factorial experiment was designed to compare three culinary techniques, Thermomix®, Roner®, and traditional pan-frying, for the preparation of tomato sauces, enriched or not with TP, applying two temperatures and two cooking times. A multivariate analysis was performed on all the results obtained for the metabolites. The addition of TP significantly increased the content of bioactive compounds, especially phenolic compounds. OPLS-DA models were generated using cooking technique, temperature, and time as discriminant factors. The cooking technique had a greater effect on the phenolic content than cooking temperature or time. Thermomix® released bioactive compounds from the tomato into the sauce to a similar extent as pan-frying. Roner® proved to be effective in preserving the volatile fraction of the sauce. The Thermomix® significantly increased the amount of bioactive compounds, while the Roner® increased the volatile compounds.
RESUMO
The enterolignans, enterolactone and enterodiol, the main metabolites produced from plant lignans by the gut microbiota, have enhanced bioavailability and activity compared to their precursors, with beneficial effects on metabolic and cardiovascular health. Although extensively studied, the biosynthesis, cardiometabolic effects, and other therapeutic implications of mammalian lignans are still incompletely understood. The aim of this review is to provide a comprehensive overview of these phytoestrogen metabolites based on up-to-date information reported in studies from a wide range of disciplines. Established and novel synthetic strategies are described, as are the various lignan precursors, their dietary sources, and a proposed metabolic pathway for their conversion to enterolignans. The methodologies used for enterolignan analysis and the available data on pharmacokinetics and bioavailability are summarized and their cardiometabolic bioactivity is explored in detail. The special focus given to research on the health benefits of microbial-derived lignan metabolites underscores the critical role of lignan-rich diets in promoting cardiovascular health.
RESUMO
Maternal breast milk plays a key role in providing newborns with passive immunity and stimulating the maturation of an infant's immune system, protecting them from many diseases. It is known that diet can influence the immune system of lactating mothers and the composition of their breast milk. The aim of this study was to establish if a supplementation during the gestation and lactation of Lewis rats with extra virgin olive oil (EVOO), due to the high proportion of antioxidant components in its composition, has an impact on the mother's immune system and on the breast milk's immune composition. For this, 10 mL/kg of either EVOO, refined oil (control oil) or water (REF group) were orally administered once a day to rats during gestation and lactation periods. Immunoglobulin (Ig) concentrations and gene expressions of immune molecules were quantified in several compartments of the mothers. The EVOO group showed higher IgA levels in both the breast milk and the mammary glands than the REF group. In addition, the gene expression of IgA in mammary glands was also boosted by EVOO consumption. Overall, EVOO supplementation during gestation and lactation is safe and does not negatively affect the mother's immune system while improving breast milk immune composition by increasing the presence of IgA, which could be critical for an offspring's immune health.
Assuntos
Lactação , Azeite de Oliva , Ratos Endogâmicos Lew , Animais , Feminino , Gravidez , Ratos , Fenômenos Fisiológicos da Nutrição Materna , Imunoglobulina A/metabolismo , Imunoglobulina A/análise , Sistema Imunitário/efeitos dos fármacos , Suplementos Nutricionais , Glândulas Mamárias Animais/imunologia , Glândulas Mamárias Animais/metabolismo , Leite/química , Leite/imunologia , Leite Humano/química , Leite Humano/imunologiaRESUMO
The human gut microbiota is a complex community of micro-organisms that play a crucial role in maintaining overall health. Recent research has shown that gut microbes also have a profound impact on brain function and cognition, leading to the concept of the gut-brain axis. One way in which the gut microbiota can influence the brain is through the bioconversion of polyphenols to other bioactive molecules. Phenolic compounds are a group of natural plant metabolites widely available in the human diet, which have anti-inflammatory and other positive effects on health. Recent studies have also suggested that some gut microbiota-derived phenolic metabolites may have neurocognitive effects, such as improving memory and cognitive function. The specific mechanisms involved are still being studied, but it is believed that phenolic metabolites may modulate neurotransmitter signaling, reduce inflammation, and enhance neural plasticity. Therefore, to exert a protective effect on neurocognition, dietary polyphenols or their metabolites must reach the brain, or act indirectly by producing an increase in bioactive molecules such as neurotransmitters. Once ingested, phenolic compounds are subjected to various processes (eg, metabolization by gut microbiota, absorption, distribution) before they cross the blood-brain barrier, perhaps the most challenging stage of their trajectory. Understanding the role of phenolic compounds in the gut-brain axis has important implications for the development of new therapeutic strategies for neurological and psychiatric disorders. By targeting the gut microbiota and its production of phenolic metabolites, it may be possible to improve brain function and prevent cognitive decline. In this article, the current state of knowledge on the endogenous generation of phenolic metabolites by the gut microbiota and how these compounds can reach the brain and exert neurocognitive effects was reviewed.
RESUMO
Olive mill waste (OMW) is a promising source of valuable compounds such as polyphenols, terpenes, sterols, and other bioactive compounds, which are of interest to the pharmaceuticals and cosmeceutical industries. This review examines the potential of OMW extracts for health and beauty applications based on evidence reports from human clinical trials. The results achieved to date indicate health-enhancing properties, but little is known about the underlying mechanisms of action, dose-response relationships, and long-term impacts. Therefore, while olive by-products, extracted using eco-friendly methods, present opportunities for the development of high-value health and cosmetic products, further studies are necessary to determine the full range of their effects and establish specific therapeutic strategies. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
Assuntos
Olea , Humanos , Olea/química , Azeite de Oliva/química , Polifenóis/análiseRESUMO
SCOPE: The rosehip (Rosa canina) is a perennial shrub with a reddish pseudofruit that has demonstrated antidiabetic, antiatherosclerotic, and antiobesogenic effects in rodent models but there is low information about the molecular mechanisms underlying these effects on the onset and progression of diet-induced obesity. METHODS AND RESULTS: Four-week-old C57BL/6J male mice are subjected to a high-fat diet (HFD)-supplemented or not with R. canina flesh for 18 weeks. The results indicated that the R. canina flesh exerts a preventive effect on HFD-induced obesity with a significant reduction in body-weight gain and an improvement of hyperglycemia and insulin resistance caused by a HFD. At the tissue level, subcutaneous white adipose tissue exhibits a higher number of smaller adipocytes, with decreased lipogenesis. On its side, the liver shows a significant decrease in lipid droplet content and in the expression of genes related to lipogenesis, fatty acid oxidation, and glucose metabolism. Finally, the data suggest that most of these effects agree with the presence of a putative Perosxisome proliferator-activated receptor gamma (PPARγ) antagonist in the R. canina flesh. CONCLUSIONS: R. canina flesh dietary supplementation slows down the steatotic effect of a HFD at least in part through the regulation of the transcriptional activity of PPARγ.
Assuntos
Fármacos Antiobesidade , Rosa , Animais , Camundongos , Dieta Hiperlipídica/efeitos adversos , PPAR gama/metabolismo , Rosa/metabolismo , Extratos Vegetais/farmacologia , Extratos Vegetais/metabolismo , Camundongos Endogâmicos C57BL , Obesidade/etiologia , Fármacos Antiobesidade/farmacologia , Fármacos Antiobesidade/metabolismo , Fígado/metabolismoRESUMO
The health benefits of chestnut (Castanea sativa) shells (CSs) have been ascribed to phytochemicals, mainly phenolic compounds. Nevertheless, an exhaustive assessment of their intestinal absorption is vital considering a possible nutraceutical application. This study evaluated the bioactivity of CSs extract prepared by Supercritical Fluid Extraction and untargeted metabolomic profile upon in-vitro intestinal permeation across a Caco-2/HT29-MTX co-culture model. The results demonstrated the neuroprotective, hypoglycemic, and hypolipidemic properties of CSs extract by inhibition of acetylcholinesterase, α-amylase, and lipase activities. The untargeted metabolic profiling by LC-ESI-LTQ-Orbitrap-MS unveiled almost 60 % of lipids and 30 % of phenolic compounds, with 29 metabolic pathways indicated by enrichment analysis. Among phenolics, mostly phenolic acids, flavonoids, and coumarins permeated the intestinal barrier with most metabolites arising from phase I reactions (reduction, hydrolysis, and hydrogenation) and a minor fraction from phase II reactions (methylation). The permeation rates enhanced in the following order: ellagic acid < o-coumaric acid < p-coumaric acid < ferulaldehyde ≤ hydroxyferulic acid ≤ dihydroferulic acid < ferulic acid < trans-caffeic acid < trans-cinnamic acid < dihydrocaffeic acid, with better outcomes for 1000 µg/mL of extract concentration and after 4 h of permeation. Taken together, these findings sustained a considerable in-vitro intestinal absorption of phenolic compounds from CSs extract, enabling them to reach target sites and exert their biological effects.
Assuntos
Acetilcolinesterase , Função da Barreira Intestinal , Humanos , Células CACO-2 , Absorção Intestinal , Intestinos/química , Fenóis/análiseRESUMO
The oxidative stability of extra virgin olive oil (EVOO) depends on its composition, primarily, phenolic compounds and tocopherols, which are strong antioxidants, but also carotenoids, squalene, and fatty acids contribute. The aim of this study was to evaluate the effect of malaxation conditions and olive storage on the composition of 'Corbella' EVOO produced in an industrial mill to determine which parameters and compounds could give more stable oils. Although a longer malaxation time at a higher temperature and olive storage had a negative effect on the content of α-tocopherol, squalene, flavonoids, lignans, phenolic acids, and phenolic alcohols, the antioxidant capacity and oxidative stability of the oil were improved because of an increase in the concentration of oleacein (56-71%) and oleocanthal (42-67%). Therefore, these two secoiridoids could be crucial for better stability and a longer shelf life of EVOOs, and their enhancement should be promoted. A synergistic effect between secoiridoids and carotenoids could also contribute to EVOO stability. Additionally, 'Corbella' cultivar seems to be a promising candidate for the production of EVOOs with a high oleic/linoleic ratio. These findings signify a notable advancement and hold substantial utility and significance in addressing and enhancing EVOO stability.
RESUMO
Chestnut shells (CSs) are an appealing source of bioactive molecules, and constitute a popular research topic. This study explores the effects of in vitro gastrointestinal digestion and intestinal permeability on the bioaccessibility and bioactivity of polyphenols from CS extract prepared by subcritical water extraction (SWE). The results unveiled higher phenolic concentrations retained after gastric and intestinal digestion. The bioaccessibility and antioxidant/antiradical properties were enhanced in the following order: oral < gastric ≤ intestinal digests, attaining 40% of the maximum bioaccessibility. Ellagic acid was the main polyphenol in the digested and undigested extract, while pyrogallol-protocatechuic acid derivative was only quantified in the digests. The CS extract revealed potential mild hypoglycemic (<25%) and neuroprotective (<75%) properties before and after in vitro digestion, along with upmodulating the antioxidant enzymes' activities and downregulating the lipid peroxidation. The intestinal permeation of ellagic acid achieved 22.89% after 240 min. This study highlighted the efficacy of the CS extract on the delivery of polyphenols, sustaining its promising use as nutraceutical ingredient.
RESUMO
Usually found bound to other complex molecules (e.g., lignin, hemicellulose), phenolic compounds (PC) are widely present in agro-industrial by-products, and their extraction is challenging. In recent times, research is starting to highlight the bioactive roles played by bound phenolics (BPC) in human health. This review aims at providing a critical update on recent advances in green techniques for the recovery of BPC, focusing on enzymatic-assisted (EAE) and fermentation-assisted extraction (FAE) as well as in the combination of technologies, showing variable yield and features. The present review also summarizes the most recent biological activities attributed to BPC extracts until now. The higher antioxidant activity of BPC-compared to FPC-coupled with their affordable by-product source make them medicinally potent and economically viable, promoting their integral upcycling and generating new revenue streams, business, and employment opportunities. In addition, EAE and FAE can have a biotransformative effect on the PC itself or its moiety, leading to improved extraction outcomes. Moreover, recent research on BPC extracts has reported promising anti-cancer and anti-diabetic activity. Yet further research is needed to elucidate their biological mechanisms and exploit the true potential of their applications in terms of new food products or ingredient development for human consumption.
RESUMO
The present study attempted for the first time to explore the effects of the daily oral intake of a phenolics-rich extract from chestnut shells (CS) on the metabolomic profiling of rat tissues by liquid chromatography coupled to Orbitrap-mass spectrometry (LC-ESI-LTQ-Orbitrap-MS) targeted to polyphenolics and their metabolites and screen potential oxidative stress biomarkers, validating its use as a promising nutraceutical ingredient with outstanding antioxidant properties for the prevention and co-therapy of lifestyle-related diseases triggered by oxidative stress. The results demonstrated new insights into the metabolomic fingerprinting of polyphenols from CS, confirming their absorption and biotransformation by phase I (hydrogenation) and II (glucuronidation, methylation, and sulfation) enzymes. Phenolic acids were the main polyphenolic class, followed by hydrolyzable tannins, flavanols, and lignans. In contrast to the liver, sulfated conjugates were the principal metabolites reaching the kidneys. The multivariate data analysis predicted an exceptional contribution of polyphenols and their microbial and phase II metabolites to the in-vivo antioxidant response of the CS extract in rats, recommending its use as an appealing source of anti-aging molecules for nutraceuticals. This is the first study that explored the relation between metabolomic profiling of rat tissues and in-vivo antioxidant effects after oral treatment with a phenolics-rich CS extract.
Assuntos
Antioxidantes , Fenóis , Animais , Ratos , Estresse Oxidativo , Suplementos Nutricionais , Polifenóis , BiomarcadoresRESUMO
Tomato pomace, a waste product consisting of peels, seeds, rich on fibrous matter, represents an underutilized source of bioactive compounds, such as polyphenols and carotenoids. Here we present a three-pronged review of the circular utilization of tomato waste in line with the United Nations Sustainable Development Goals. First, we explain why tomato waste is important, highlighting the processing techniques that generate it. The bioactive compounds in these by-products are then comprehensively reviewed, focusing especially on phenolic compounds and carotenoids and the methods used for their extraction. Finally, we examine the potential of these bioactive ingredients for application in food systems and pharmaceutical products.
RESUMO
BACKGROUND: Phenolic compounds have been associated with protective effects against type-2 diabetes (T2D). We used a metabolomics approach to determine urinary phenolic metabolites associated with T2D and fasting plasma glucose. METHODS: This case-control study within the PREDIMED trial included 200 participants at high cardiovascular risk, 102 of whom were diagnosed with T2D. A panel of urinary phenolic compounds were analysed using a novel method based on liquid chromatography coupled to mass spectrometry. Multivariate statistics and adjusted logistic regressions were applied to determine the most discriminant compounds and their association with T2D. The relationship between the discriminant phenolic compounds and plasma glucose was assessed using multivariable linear regressions. RESULTS: A total of 41 phenolic compounds were modeled in the orthogonal projection to latent structures discriminant analysis, and after applying adjusted logistic regressions two were selected as discriminant: dihydrocaffeic acid (OR = 0.22 (CI 95 %: 0.09; 0.52) per 1-SD, p-value = 0.021) and genistein diglucuronide (OR = 0.72 (CI 95%: 0.59; 0.88) per 1-SD, p-value = 0.021). Both metabolites were associated with a lower risk of suffering from T2D, but only dihydrocaffeic acid was inversely associated with plasma glucose (ß = -17.12 (95 % CI: -29.92; -4.32) mg/dL per 1-SD, p-value = 0.009). CONCLUSIONS: A novel method using a metabolomics approach was developed to analyse a panel of urinary phenolic compounds for potential associations with T2D, and two metabolites, dihydrocaffeic acid and genistein diglucuronide, were found to be associated with a lower risk of this condition.
Assuntos
Diabetes Mellitus Tipo 2 , Dieta Mediterrânea , Humanos , Biomarcadores , Glicemia , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/metabolismo , Genisteína , Metabolômica/métodos , Fenóis , Fatores de RiscoRESUMO
Phenolic compounds in peanuts may moderate inflammation and endothelial function. Thus, the aim of this study was to evaluate the association of urinary phenolic metabolites (UPMs) with vascular biomarkers after peanut product consumption. A three-arm parallel-group randomized controlled trial was conducted in 63 healthy young adults who consumed 25 g/day of skin roasted peanuts (SRP), 32 g/day of peanut butter (PB), or 32 g/day of a control butter for six months. UPMs were analyzed by liquid chromatography coupled to mass spectrometry. Additionally, urinary eicosanoids, prostacyclin I2 (PGI2), and thromboxane A2 (TXA2) were determined using two competitive enzyme-linked immunosorbent assay kits. Consumers of SRP and PB presented significantly higher excretion of UPMs (enterodiol glucuronide (p = 0.018 and p = 0.031), 3-hydroxybenzoic acid (p = 0.002 and p < 0.001), vanillic acid sulfate (p = 0.048 and p = 0.006), p-coumaric acid (p = 0.046 and p = 0.016), coumaric acid glucuronide I (p = 0.001 and p = 0.030) and II (p = 0.003 and p = 0.036), and isoferulic acid (p = 0.013 and p = 0.015) in comparison with the control group. An improvement in PGI2 (p = 0.037) levels and the TXA2:PGI2 ratio (p = 0.008) was also observed after the peanut interventions compared to the control. Interestingly, UPMs with significantly higher post-intervention levels were correlated with an improvement in vascular biomarkers, lower TXA2 (r from -0.25 to -0.48, p < 0.050) and TXA2:PGI2 ratio (r from -0.25 to -0.43, p < 0.050) and higher PGI2 (r from 0.24 to 0.36, p < 0.050). These findings suggest that the UPMs with higher excretion after peanut product consumption could have a positive impact on vascular health.
RESUMO
Chestnut (Castanea sativa) shells (CSs), an undervalued agro-industrial biowaste, have arisen as a source of bioactive compounds with promising health-promoting effects. This study attempted, for the first time, to develop a functional food, namely cookies, using a CS extract obtained by an eco-friendly technology (subcritical water extraction). The cookies were characterized regarding their nutritional composition, total phenolic and flavonoid contents (TPC and TFC, respectively), antioxidant/antiradical activities, phenolic profile, and sensory evaluation. The results demonstrated that the CS-extract-enriched cookies were mainly composed of carbohydrates (53.92% on dry weight (dw)), fat (32.62% dw), and fiber (5.15% dw). The phenolic profile outlined by HPLC-PDA revealed the presence of phenolic acids, flavonoids, and hydrolysable tannins, attesting to the high TPC and TFC. The in vitro antioxidant/antiradical effects proved the bioactivity of the functional cookies, while the sensory evaluation unveiled excellent scores on all attributes (≥6.25). The heatmap diagram corroborated strong correlations between the TPC and antioxidant/antiradical properties, predicting that the appreciated sensory attributes were closely correlated with high carbohydrates and phenolic compounds. This study encourages the sustainable recovery of antioxidants from CSs and their further employment as an active nutraceutical ingredient in functional cookies.
RESUMO
As they become more health conscious, consumers are paying increasing attention to food quality and safety. In coffee production, fraudulent strategies to reduce costs and maximize profits include mixing beans from two species of different economic value, the addition of other substances and/or foods, and mislabeling. Therefore, testing for coffee authenticity and detecting adulterants is required for value assessment and consumer protection. Here we provide an overview of the chromatography, spectroscopy, and single-nucleotide polymorphism-based methods used to distinguish between the major coffee species Arabica and Robusta. This review also describes the techniques applied to trace the geographical origin of coffee, based mainly on the chemical composition of the beans, an approach that can discriminate between coffee-growing regions on a continental or more local level. Finally, the analytical techniques used to detect coffee adulteration with other foods and/or coffee by-products are discussed, with a look at the practice of adding pharmacologically active compounds to coffee, and their harmful effects on health.
Assuntos
Coffea , Café , Café/química , Coffea/química , Sementes/química , Qualidade dos Alimentos , Manipulação de Alimentos/métodosRESUMO
Chestnut (Castanea sativa) shells (CSS) are a source of bioactive compounds with well demonstrated in-vitro antioxidant properties. Nevertheless, no in-vivo studies have already evaluated this effect. This study evaluated the effects of the oral daily administration of an eco-friendly CSS extract (50 and 100 mg/kg per body weight (b.w.)) to rats regarding in-vivo antioxidant activity, glucose and lipids levels, and metabolomic profiling of polyphenols by LC-ESI-LTQ-Orbitrap-MS. The results demonstrated the in-vivo antioxidant properties in the animals liver, kidney and blood serum, as well as protective effects against hemolysis and rising of blood glucose and lipids levels. New insights on metabolomic profiling of polyphenols proved their absorption and further biotransformation by phase I (hydrogenation and hydroxylation) and II reactions (glucuronidation, methylation and sulfation). This is the first study that attempted to validate a novel nutraceutical ingredient extracted from CSS byin-vivoassays, corroborating the outcomes screened by in-vitro assays.
Assuntos
Antioxidantes , Extratos Vegetais , Ratos , Animais , Antioxidantes/farmacologia , Extratos Vegetais/farmacologia , Fenóis/análise , Polifenóis/análise , Suplementos Nutricionais , LipídeosRESUMO
The challenge of sustainable agriculture is to increase yields and obtain higher quality products. Increased antioxidant compounds such as polyphenols in harvest products may be an added value for sustainable agriculture. The aim of the present study was to investigate whether three organic fertilization treatments with different levels of carbon and nitrogen, i.e., N-rich, N-rich+C, and N-poor+C, affected the phenolic content of different tomato varieties. The examined parameters were productivity, plant nutritional status, δ13C, and tomato phenolic content as an indication of the antioxidant capacity. The best production was obtained with 'Cornabel', a high-yielding Pebroter variety. The total phenolic content was highest in the traditional 'Cuban Pepper' variety regardless of treatment, while naringenin levels were high in all the Pebroter varieties. In N-poor+C fertilized plants, a lower N-NO3 content in leaves was correlated with higher levels of total polyphenols in the fruit. The high-water stress suffered by Montserrat varieties coincided with a low total phenolic content in the tomatoes. In conclusion, organic fertilization with reduced N did not influence the tomato yield but positively affected phenolic compound levels in varieties less sensitive to water stress.
RESUMO
(1) Background: To explore the association between microbial phenolic metabolites (MPM) and metabolic syndrome (MetS) and its clinical features in adolescents aged 12.02 ± 0.41 years. (2) Methods: a cross-sectional study was conducted in 560 participants at baseline in the SI! Program for Secondary Schools trial. The following MPM, coumaric acids (m-, o-, p-coumaric acids), dihydroxyphenylpropionic acid, dihydroresveratrol, enterolignans, gallic acid, hydroxybenzoic acids, hydroxyphenylacetic acid, hydroxytyrosol, protocatechuic acid, syringic acid, urolithins (A, B), and vanillic acid, were analyzed by HPLC-LTQ-Orbitrap-HRMS. MetS and its clinical features were defined in accordance with the International Diabetes Federation. (3) Results: Out of all MPM, urolithin A was inversely associated with the diastolic blood pressure z-score. Urolithin B was inversely associated with the MetS score and waist circumference z-score. Additionally, higher levels of gallic acid were associated with lower odds of presenting MetS (OR = 0.85, 95% CI: 0.77; 0.93) and abdominal obesity (OR = 0.93, 95% CI: 0.89; 0.98). Higher urolithin B levels were inversely associated with abdominal obesity (OR = 0.94, 95% CI: 0.89; 0.98) and high blood glucose (OR = 0.92, 95% CI:0.88; 0.96); (4) Conclusions: gallic acid, urolithin A and B were associated with lower odds of presenting MetS or some of its clinical features in adolescents. This is the first study that evaluates several MPM with MetS in adolescents, highlighting the importance of MPM on cardiometabolic health at early life stages.
RESUMO
The demand for high-quality extra virgin olive oil (EVOO) is growing due to its unique characteristics. The aroma and flavor of EVOO depend on its content of volatile organic compounds (VOCs), whose formation is affected by the olive variety and maturity index, and the oil production process. In this study, the sensory quality and VOC and fatty acid (FA) profiles were determined in Arbequina olive oils produced by applying different malaxation parameters (20, 25, and 30 °C, and 30 and 45 min). All the olive oils were classified as EVOO by a sensory panel, regardless of the production conditions. However, cold extraction at 20 °C resulted in more positive sensory attributes (complexity). The FA concentration increased significantly with the malaxation temperature, although the percentage profile remained unaltered. Finally, an OPLS-DA model was generated to identify the discriminating variables that separated the samples according to the malaxation temperature. In conclusion, the tested range of malaxation parameters appeared not to degrade the distinctive attributes/organoleptic profile of olive oil and could be applied to obtain an EVOO of high sensory quality, especially at 20 °C.