Cell culture models for assessing the effects of bioactive compounds in common buckwheat (Fagopyrum esculentum): a systematic review.

Common buckwheat (CBW) is grown and consumed worldwide. In addition to its already established reputation as an excellent source of nutrients, CBW is gaining popularity as a possible component of functional foods. Whereas human studies remain the gold standard for evaluating the relationship between nutrition and health, the development of reliable in vitro or ex vivo models has made it possible to investigate the cellular and molecular mechanisms of CBW effects on human health. Herein is a systematic review of studies on the biological effect of CBW supplementation, as assessed on various types of cellular models. Although the studies reported here have been conducted in very different experimental conditions, the overall effects of CBW supplementation were found to involve a decrease in cytokine secretion and oxidation products, related mainly to CBW polyphenols and protein or peptide fractions. These chemical species also appeared to be involved in the modulation of cell signaling and hormone secretion. Although further studies are undoubtedly necessary, as is their extension to in vivo systems, these reports suggest that CBW-based foods could be relevant to maintaining and/or improving human health and the quality of life.

Docosahexaenoic Acid as Master Regulator of Cellular Antioxidant Defenses: A Systematic Review.

Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid that benefits the prevention of chronic diseases. Due to its high unsaturation, DHA is vulnerable to free radical oxidation, resulting in several unfavorable effects, including producing hazardous metabolites. However, in vitro and in vivo investigations suggest that the relationship between the chemical structure of DHA and its susceptibility to oxidation may not be as clear-cut as previously thought. Organisms have developed a balanced system of antioxidants to counteract the overproduction of oxidants, and the nuclear factor erythroid 2-related factor 2 (Nrf2) is the key transcription factor identified for transmitting the inducer signal to the antioxidant response element. Thus, DHA might preserve the cellular redox status promoting the transcriptional regulation of cellular antioxidants through Nrf2 activation. Here, we systematically summarize the research on the possible role of DHA in controlling cellular antioxidant enzymes. After the screening process, 43 records were selected and included in this review. Specifically, 29 studies related to the effects of DHA in cell cultures and 15 studies concerned the effects of consumption or treatment with DHA in animal. Despite DHA's promising and encouraging effects at modulating the cellular antioxidant response in vitro/in vivo, some differences observed among the reviewed studies may be accounted for by the different experimental conditions adopted, including the time of supplementation/treatment, DHA concentration, and cell culture/tissue model. Moreover, this review offers potential molecular explanations for how DHA controls cellular antioxidant defenses, including involvement of transcription factors and the redox signaling pathway.

Co-Administration of Propionate or Protocatechuic Acid Does Not Affect DHA-Specific Transcriptional Effects on Lipid Metabolism in Cultured Hepatic Cells.

Long-chain n-3 polyunsaturated fatty acids (n-3 LC-PUFAs) are collectively recognized triglyceride-lowering agents, and their preventive action is likely mediated by changes in gene expression. However, as most studies employ fish oil, which contains a mixture of n-3 LC-PUFAs, the docosahexaenoic acid (DHA)-specific transcriptional effects on lipid metabolism are still unclear. The aim of the present study was to further elucidate the DHA-induced transcriptional effects on lipid metabolism in the liver, and to investigate the effects of co-administration with other bioactive compounds having effects on lipid metabolism. To this purpose, HepG2 cells were treated for 6 or 24 h with DHA, the short-chain fatty acid propionate (PRO), and protocatechuic acid (PCA), the main human metabolite of cyanidin-glucosides. Following supplementation, we mapped the global transcriptional changes. PRO and PCA alone had a very slight effect on the transcriptome; on the contrary, supplementation of DHA highly repressed the steroid and fatty acid biosynthesis pathways, this transcriptional modulation being not affected by co-supplementation. Our results confirm that DHA effect on lipid metabolism are mediated at least in part by modulation of the expression of specific genes. PRO and PCA could contribute to counteracting dyslipidemia through other mechanisms.

Impact of processing on the nutritional and functional value of mandarin juice.

BACKGROUND: Although phenolic compounds have a role in the health benefits of fruit juice consumption, little is known about the effect of processing on their bioaccessibility. The release of phenolic compounds from the food matrix during digestion is an important pre-requisite for their effectiveness within the human body, and so it is fundamental to identify technological treatments able to preserve not only the concentration of phytochemicals, but also their bioaccessibility. In the present study, we investigated the impact of high-pressure homogenization (HPH), alone and in the presence of 100 g kg-1 trehalose or Lactobacillus salivarius, on the bioaccessibility of flavonoids in mandarin juice. In addition, digested mandarin juices were supplemented to liver cultured cells in basal and stressed conditions to evaluate their protective effect in a biological system. RESULTS: HPH reduced the concentration of total phenolics and main flavonoids but increased their bioaccessibility after in vitro digestion (P < 0.001). In the basal condition, supplementation with all digested juices significantly reduced intracellular reactive oxygen species (ROS) concentration (P < 0.001). Thiobarbituric acid reactive substances concentration in the medium was also reduced by supplementation with HPH-treated juices. Although pre-treatment with juices did not completely counteract the applied oxidative stress, it preserved cell viability, and cells pre-treated with juices submitted to HPH in the presence of probiotics showed the lowest ROS concentration. CONCLUSION: The present study represents an important step ahead in the evaluation of the impact of processing on the nutritional and functional value of food, which cannot simply be assessed based on chemical composition. © 2020 Society of Chemical Industry.

Olive oil by-product as functional ingredient in bakery products. Influence of processing and evaluation of biological effects.

Nowadays, the strong demand for adequate nutrition is accompanied by concern about environmental pollution and there is a considerable emphasis on the recovery and recycling of food by-products and wastes. In this study, we focused on the exploitation of olive pomace as functional ingredient in biscuits and bread. Standard and enriched bakery products were made using different flours and fermentation protocols. After characterization, they were in vitro digested and used for supplementation of intestinal cells (Caco-2), which underwent exogenous inflammation. The enrichment caused a significant increase in the phenolic content in all products, particularly in the sourdough fermented ones. Sourdough fermentation also increased tocol concentration. The increased concentration of bioactive molecules did not reflect the anti-inflammatory effect, which was modulated by the baking procedure. Conventionally fermented bread enriched with 4% pomace and sourdough fermented, not-enriched bread had the greatest anti-inflammatory effect, significantly reducing IL-8 secretion in Caco-2 cells. The cell metabolome was modified only after supplementation with sourdough fermented bread enriched with 4% pomace, probably due to the high concentration of tocopherol that acted synergistically with polyphenols. Our data highlight that changes in chemical composition cannot predict changes in functionality. It is conceivable that matrices (including enrichment) and processing differently modulated bioactive bioaccessibility, and consequently functionality.

A Dietary Intervention of Bioactive Enriched Foods Aimed at Adults at Risk of Metabolic Syndrome: Protocol and Results from PATHWAY-27 Pilot Study.

Around a quarter of the global adult population have metabolic syndrome (MetS) and therefore increased risk of cardiovascular mortality and diabetes. Docosahexaenoic acid, oat beta-glucan and grape anthocyanins have been shown to be effective in reducing MetS risk factors when administered as isolated compounds, but their effect when administered as bioactive-enriched foods has not been evaluated. OBJECTIVE: The overall aim of the PATHWAY-27 project was to evaluate the effectiveness of bioactive-enriched food consumption on improving risk factors of MetS. A pilot study was conducted to assess which of five bioactive combinations provided within three different food matrices (bakery, dairy or egg) were the most effective in adult volunteers. The trial also evaluated the feasibility of production, consumer acceptability and gastrointestinal tolerance of the bioactive-enriched food. METHOD: The study included three monocentric, parallel-arm, double-blind, randomised, dietary intervention trials without a placebo. Each recruiting centre tested the five bioactive combinations within a single food matrix. RESULTS: The study was completed by 167 participants (74 male, 93 female). The results indicated that specific bioactive/matrix combinations have effects on serum triglyceride or HDL-cholesterol level without adverse effects. CONCLUSION: The study evidenced that bioactive-enriched food offers a promising food-based strategy for MetS prevention, and highlighted the importance of conducting pilot studies.

Sourdough Fermentation Favorably Influences Selenium Biotransformation and the Biological Effects of Flatbread.

Although selenium is of great importance for the human body, in several world regions the intake of this essential trace element does not meet the dietary reference values. To achieve optimal intake, fortification of bread by using selenium-enriched flour has been put forward. Less is known on the potential effect of sourdough fermentation, which might be worth exploring as the biological effects of selenium strongly depend on its chemical form and sourdough fermentation is known to cause transformations of nutrients and phytochemicals, including the conversion of inorganic selenium into organic selenocompounds. Here we investigated the bio transformation of selenium by sourdough fermentation in a typical Italian flatbread (piadina) made with standard (control) or selenium-enriched flour. The different piadina were submitted to in vitro digestion, and the biological activity of the resulting hydrolysates was tested by means of cultured human liver cells exposed to an exogenous oxidative stress. The use of selenium-enriched flour and sourdough fermentation increased the total content of bioaccessible selenium in organic form, compared to conventional fermentation, and led to protective effects counteracting oxidative damage in cultured cells. The present study suggests that selenium-rich, sourdough-fermented bakery products show promise for improving human selenium nutrition whenever necessary.

Olive oil industry by-products. Effects of a polyphenol-rich extract on the metabolome and response to inflammation in cultured intestinal cell.

Over the past years, researchers and food manufacturers have become increasingly interested in olive polyphenols due to the recognition of their biological properties and probable role in the prevention of various diseases such as inflammatory bowel disease. Olive pomace, one of the main by-products of olive oil production, is a potential low-cost, phenol-rich ingredient for the formulation of functional food. In this study, the aqueous extract of olive pomace was characterized and used to supplement human intestinal cell in culture (Caco-2). The effect on the cell metabolome and the anti-inflammatory potential were then evaluated. Modification in the metabolome induced by supplementation clearly evidenced a metabolic shift toward a "glucose saving/accumulation" strategy that could have a role in maintaining anorexigenic hormone secretion and could explain the reported appetite-suppressing effect of the administration of polyphenol-rich food. In both basal and inflamed condition, supplementation significantly reduced the secretion of the main pro-inflammatory cytokine, IL-8. Thus, our data confirm the therapeutic potential of polyphenols, and specifically of olive pomace in intestinal bowel diseases. Although intervention studies are needed to confirm the clinical significance of our findings, the herein reported results pave the road for exploitation of olive pomace in the formulation of new, value-added foods. In addition, the application of a foodomics approach allowed observing a not hypothesized modulation of glucose metabolism.

Is cytotoxicity a determinant of the different in vitro and in vivo effects of bioactives?

BACKGROUND: Foodstuffs of both plant and animal origin contain a wide range of bioactive compounds. Although human intervention studies are mandatory to assess the health effects of bioactives, the in vitro approach is often used to select the most promising molecules to be studied in vivo. To avoid misleading results, concentration and chemical form, exposure time, and potential cytotoxicity of the tested bioactives should be carefully set prior to any other experiments. METHODS: In this study the possible cytotoxicity of different bioactives (docosahexaenoic acid, propionate, cyanidin-3-O-glucoside, protocatechuic acid), was investigated in HepG2 cells using different methods. Bioactives were supplemented to cells at different concentrations within the physiological range in human blood, alone or in combination, considering two different exposure times. RESULTS: Reported data clearly evidence that in vitro cytotoxicity is tightly related to the exposure time, and it varies among bioactives, which could exert a cytotoxic effect even at a concentration within the in vivo physiological blood concentration range. Furthermore, co-supplementation of different bioactives can increase the cytotoxic effect. CONCLUSIONS: Our results underline the importance of in vitro cytotoxicity screening that should be considered mandatory before performing studies aimed to evaluate the effect of bioactives on other cellular parameters. Although this study is far from the demonstration of a toxic effect of the tested bioactives when administered to humans, it represents a starting point for future research aimed at verifying the existence of a potential hazard due to the wide use of high doses of multiple bioactives.

Evidence of a DHA Signature in the Lipidome and Metabolome of Human Hepatocytes.

Cell supplementation with bioactive molecules often causes a perturbation in the whole intracellular environment. Omics techniques can be applied for the assessment of this perturbation. In this study, the overall effect of docosahexaenoic acid (DHA) supplementation on cultured human hepatocyte lipidome and metabolome has been investigated using nuclear magnetic resonance (NMR) in combination with traditional techniques. The effect of two additional bioactives sharing with DHA the lipid-lowering effect-propionic acid (PRO) and protocatechuic acid (PCA)-has also been evaluated in the context of possible synergism. NMR analysis of the cell lipid extracts showed that DHA supplementation, alone or in combination with PCA or PRO, strongly altered the cell lipid profile. The perfect discrimination between cells receiving DHA (alone or in combination) and the other cells reinforced the idea of a global rearrangement of the lipid environment induced by DHA. Notably, gas chromatography and fluorimetric analyses confirmed the strong discrimination obtained by NMR. The DHA signature was evidenced not only in the cell lipidome, but also in the metabolome. Results reported herein indicate that NMR, combined with other techniques, represents a fundamental approach to studying the effect of bioactive supplementation, particularly in the case of molecules with a broad spectrum of mechanisms of action.

PUFA and oxidative stress. Differential modulation of the cell response by DHA.

Although an increased dietary intake of long-chain n-3 PUFA is considered an effective preventive strategy, a theoretical concern related to the possible increase of lipid peroxidation induced by a PUFA-rich diet still remains a problem. In this study, the effects of different PUFA (linoleic, α-linolenic, arachidonic, eicosapentaenoic and docosahexaenoic acid) on cytotoxicity, lipid oxidation, and modulation of antioxidant defenses were evaluated in HepG2 cells submitted to an oxidative stress (H2O2). Results clearly evidenced that all supplemented PUFA, but DHA, enhanced cell susceptibility to H2O2. Overall, our results underline that PUFA cannot be considered as a single category but as individual compounds, and research on mechanisms of action and preventive effects should deal with the individual fatty acids, particularly in the case of DHA.

Sugar cane and sugar beet molasses, antioxidant-rich alternatives to refined sugar.

Molasses, the main byproduct of sugar production, is a well-known source of antioxidants. In this study sugar cane molasses (SCM) and sugar beet molasses (SBM) were investigated for their phenolic profile and in vitro antioxidant capacity and for their protective effect in human HepG2 cells submitted to oxidative stress. According to its higher phenolic concentration and antioxidant capacity in vitro, SCM exhibited an effective protection in cells, comparable to or even greater than that of α-tocopherol. Data herein reported emphasize the potential health effects of molasses and the possibility of using byproducts for their antioxidant activity. This is particularly important for consumers in developing countries, as it highlights the importance of consuming a low-price, yet very nutritious, commodity.

Pro- and anti-oxidant effects of polyunsaturated fatty acid supplementation in HepG2 cells.

PUFA are bioactive nutrients thought to be effective in the prevention of many chronic diseases. PUFA susceptibility to free radical oxidation represents the other side of the coin, and the role of PUFA as pro- or anti-oxidants is still an unanswered question. In this study we supplemented HepG2 cells with different PUFA, and observed different effects on cytotoxicity, oxidation and modulation of antioxidant defenses. These were not simply related to the length of carbon chain, or to the number and position of double bonds. ARA supply evidenced the induction of oxidative damage, while DHA supplemented cells appeared richer in antioxidant defenses. To our knowledge, our study is the first evidencing the different pro- or anti-oxidant effect of different fatty acids when supplemented to cells. Overall, this points out the importance of not generalizing dietary recommendations considering PUFA as one category, but to extend them to the individual fatty acids.

EPA or DHA supplementation increases triacylglycerol, but not phospholipid, levels in isolated rat cardiomyocytes.

It is well recognized that a high dietary intake of long-chain polyunsaturated fatty acids (LC-PUFA) has profound benefits on health and prevention of chronic diseases. In particular, in recent years there has been a dramatic surge of interest in the health effects of n-3 LC-PUFA derived from fish, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids. Notwithstanding, the metabolic fate and the effects of these fatty acids once inside the cell has seldom been comprehensively investigated. Using cultured neonatal rat cardiomyocytes as model system we have investigated for the first time, by means of high-resolution magic-angle spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy in combination with gas chromatography (GC), the modification occurring in the cell lipid environment after EPA and DHA supplementation. The most important difference between control and n-3 LC-PUFA-supplemented cardiomyocytes highlighted by HR-MAS NMR spectroscopy is the increase of signals from mobile lipids, identified as triacylglycerols (TAG). The observed increase of mobile TAG is a metabolic response to n-3 LC-PUFA supplementation, which leads to an increased lipid storage. The sequestration of mobile lipids in lipid bodies provides a deposit of stored energy that can be accessed in a regulated fashion according to metabolic need. Interestingly, while n-3 LC-PUFA supplementation to neonatal rat cardiomyocytes causes a huge variation in the cell lipid environment, it does not induce detectable modifications in water-soluble metabolites, suggesting negligible interference with normal metabolic processes.

n-3 and n-6 Polyunsaturated fatty acids suppress sterol regulatory element binding protein activity and increase flow of non-esterified cholesterol in HepG2 cells.

The plasma lipid-lowering effect of PUFA, one of their main beneficial effects, is considered to be related to the regulation of lipid biosynthesis through transcription factors including sterol regulatory element binding proteins (SREBP). In the present study, we compared the effect of different PUFA on SREBP activity in HepG2 cells, using a sterol regulatory element-luciferase reporter construct as a probe. Supplementation with different fatty acids reduced SREBP activity in the order 20 : 5n-3 = 18 : 2n-6 = 20 : 4n-6 " 18 : 3n-3 = 22 : 6n-3 = 22 : 5n-6 " 18 : 1n-9. The suppression of SREBP activity greatly depended on the degree of incorporation of the supplemented PUFA into cellular lipids, and correlated positively with the unsaturation index (r 0.831; P < 0.01) of total cell lipids. Supplemented PUFA were also metabolised to longer and more unsaturated species. These processing activities were higher for n-3 than n-6 PUFA (P < 0.01). We studied the effect of PUFA on the intracellular distribution of non-esterified cholesterol, using filipin staining and fluorescence microscopy with or without the cholesterol traffic blocker U18666A. The data show that the incorporation of PUFA increases non-esterified cholesterol flow from the plasma membrane to intracellular membranes. We conclude that suppression of SREBP activity by PUFA depends on the degree of incorporation into cellular lipids, and is associated with increased flow of non-esterified cholesterol between the plasma membrane and intracellular membranes.

n-3 PUFA as regulators of cardiac gene transcription: a new link between PPAR activation and fatty acid composition.

The fatty acids regulate gene expression directly binding to nuclear receptors or affecting the protein content of transcription factors. In this work, supplementing primary cultures of neonatal rat cardiomyocytes with 60 microM EPA or DHA, we demonstrated by an ELISA assay an increased PPAR beta/delta binding to DNA. n-3 PUFA supplementation deeply changed the acyl composition of both cytosolic and nuclear fractions. The high content of total fatty acids, particularly EPA and DHA, and its increase following supplementation suggested a selective accumulation of n-3 PUFAs in the nucleus, supporting the direct interaction of n-3 PUFA with PPAR. The activity of acyl-CoA thioesterase (ACOT), catalyzing the reaction leading to NEFA from acyl-CoA, increased in n-3 PUFA supplemented cells. The NEFA/acyl-CoA ratio is an important regulator of the fatty acid transport to the nucleus and consequent modulation of gene transcription, and although ACOT activity is not the only parameter of this ratio, it is important for the control of the NEFA pool composition. Our data further clarify what happens in cardiomyocytes following n-3 PUFA supplementation, linking the modification of acyl composition to ACOT activity and PPAR activation.

Green tea extract selectively activates peroxisome proliferator-activated receptor beta/delta in cultured cardiomyocytes.

Hypoxia/reoxygenation is one of the causes of the increased expression of inducible NO synthase in cardiomyocytes. In a recent study we demonstrated that a single, high dose of green tea extract (GT) supplemented to the medium of cultured cardiomyocytes just before hypoxia/reoxygenation is able to prevent the increased expression of inducible NO synthase, therefore reducing NO overproduction. In the present study we investigated the mechanism by which GT reduces NO production. Since a molecular mechanism for polyphenol activity has been postulated, and PPAR activation is related to the transcription of the inducible NO synthase gene, we evaluated the activation of PPAR by GT. A moderate GT concentration, supplemented to the cardiomyocyte medium since the initial seeding, selectively activated the PPAR-beta/delta isoform. Furthermore, we observed a reduction in NO production and an increase in total antioxidant activity, indicating that GT components may act on both reactive oxygen species, via an antioxidant mechanism, and NO overproduction. PPAR-beta/delta activation could represent the key event in the reduction of NO production by GT. Although PPAR activation by GT was lower than activation by fenofibrate, it is very interesting to note that it was selective for the beta/delta isoform, at least in neonatal cardiomyocytes.

Dietary Selenium for the counteraction of oxidative damage: fortified foods or supplements?

Since any significant modification in the Se status, leading to changes in the activity of the seleno-enzymes, may have important consequences on the susceptibility of tissues to oxidative stress, considerable efforts have been made upon increasing Se dietary intake. In this respect, an important debate is still open about the bioavailability and the effectiveness of Se, and more generally nutrients, in supplements compared with foods. Using male Wistar rats, we have compared the effectiveness of two different diets in which an adequate Se content (0.1 mg/kg) was achieved by adding the element as sodium selenite or as component of a lyophilized Se-enriched food, in the counteraction of an oxidative stress induced by intraperitoneal administration of adriamycin. Both Se-enriched diets were able to reduce the consequences of the oxidative stress in liver, mainly by increasing glutathione peroxidase activity. This increase was more evident in rats fed on the diet enriched with the lyophilized food, probably due to the different chemical forms of Se, or to other components of the food itself. Although further studies are needed, data herein presented may contribute to the characterization of the effectiveness of Se from different sources, foods or supplements, in the light of dietary advice to the population concerning improvement of Se intake.

N-3 PUFAs modulate global gene expression profile in cultured rat cardiomyocytes. Implications in cardiac hypertrophy and heart failure.

In cardiac cells the effects of n-3 PUFAs on the whole genome are still unknown despite their recognized cardioprotective effects and ability to modulate gene expression. We have evaluated the effects of n-3 PUFAs supplementation on the global gene expression profile in cultured neonatal rat cardiomyocytes, detecting many genes related to lipid transport and metabolism among the upregulated ones. Many of the downregulated genes appeared related to inflammation, cell growth, extracellular and cardiac matrix remodelling, calcium movements and ROS generation. Our data allow to speculate that the cardioprotective effect of n-3 PUFAs is related to a direct modulation of genes in cardiac cells.

Counteraction of adriamycin-induced oxidative damage in rat heart by selenium dietary supplementation.

Many reports indicate that dietary selenium, potentially increasing the activity of glutathione peroxidase, could offer protection against free-radical-induced damage. The effects of diets moderately enriched in selenium, as sodium selenite or as a lyophilized selenium-rich food, were studied in rats. Adriamycin, an anticancer drug causing a free-radical-mediated cardiotoxicity, was administered intraperitoneally to some rats. The onset of an oxidative damage was indicated by the increase in the plasma level of reactive oxygen metabolites coupled to a decrease in the total antioxidant activity but without modification of glutathione peroxidase activity, which were observed in all rats, independent of the dietary treatment. On the contrary, in the heart, selenium supplementation caused an increase in the total antioxidant activity, glutathione concentration, and glutathione peroxidase and catalase activities leading to a decreased generation of reactive oxygen metabolites. These results clearly indicate that a moderate Se dietary supplementation counteracts adriamycin-induced cardiotoxicity by preservation of endogenous antioxidants.