The Perfect Cup? Coffee-Derived Polyphenols and Their Roles in Mitigating Factors Affecting Type 2 Diabetes Pathogenesis.

Type 2 diabetes (T2D) is a growing health concern with an estimated 462 million people having been diagnosed worldwide. T2D is characterized by chronically elevated blood glucose and insulin resistance, which culminate in a diminished function of the ß-cell mass in its later stages. This can be perpetuated by and result in inflammation, excess reactive oxygen species production, obesity, and the dysregulation of multiple cellular pathways. Many naturally occurring small molecules have been investigated in terms of their roles in modulating glucose homeostasis and ß-cell function. Many of these compounds can be found in commonly used sources of food and drink. Interestingly, a correlation has been observed between coffee consumption and T2D incidence. However, the specific compounds responsible for this correlation and their mechanisms are still somewhat undetermined. This paper reviews recent research findings on the effects of several polyphenols that are either found in coffee or are metabolites of compounds found in coffee (enterodiol, enterolactone, matairesinol, secoisolariciresinol, kaempferol, quercetin, and chlorogenic acid) on glucose homeostasis and health complications associated with glucose dysregulation, with a special emphasis on their potential anti-diabetic effects. The factors that affect polyphenol content in coffee are also addressed.

Impact of lignans in oilseed mix on gut microbiome composition and enterolignan production in younger healthy and premenopausal women: an in vitro pilot study.

BACKGROUND: Dietary lignans belong to the group of phytoestrogens together with coumestans, stilbenes and isoflavones, and themselves do not exhibit oestrogen-like properties. Nonetheless, the gut microbiota converts them into enterolignans, which show chemical similarity to the human oestrogen molecule. One of the richest dietary sources of lignans are oilseeds, including flaxseed. The aim of this pilot study was to determine the concentration of the main dietary lignans in an oilseed mix, and explore the gut microbiota-dependent production of enterolignans for oestrogen substitution in young and premenopausal women. The oilseed mix was fermented in a pH-controlled batch culture system inoculated with women's faecal samples. The lignan content and enterolignan production were measured by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), and the faecal-derived microbial communities were profiled by 16S rRNA gene-based next-generation sequencing. RESULTS: In vitro batch culture fermentation of faecal samples inoculated with oilseed mix for 24 h resulted in a substantial increase in enterolactone production in younger women and an increase in enterodiol in the premenopausal group. As for the gut microbiota, different baseline profiles were observed as well as different temporal dynamics, mainly related to Clostridiaceae, and Klebsiella and Collinsella spp. CONCLUSIONS: Despite the small sample size, our pilot study revealed that lignan-rich oilseeds could strongly influence the faecal microbiota of both younger and premenopausal females, leading to a different enterolignan profile being produced. Further studies in larger cohorts are needed to evaluate the long-term effects of lignan-rich diets on the gut microbiota and find out how enterolactone-producing bacterial species could be increased. Diets rich in lignans could potentially serve as a safe supplement of oestrogen analogues to meet the cellular needs of endogenous oestrogen and deliver numerous health benefits, provided that the premenopausal woman microbiota is capable of converting dietary precursors into enterolignans.

The flaxseed lignan secoisolariciresinol diglucoside decreases local inflammation, suppresses NFκB signaling, and inhibits mammary tumor growth.

PURPOSE: Exposure to the polyphenolic plant lignan secoisolariciresinol diglucoside (SDG) and its metabolite enterolactone (ENL) has been associated with reduced breast cancer progression, particularly for estrogen receptor alpha (ERα)-negative disease, and decreased preclinical mammary tumor growth. However, while preclinical studies have established that SDG and ENL affect measures of progression in models of triple-negative breast cancer (TNBC, a subset of ERα-negative disease), the molecular mechanisms underlying these effects remain unclear. METHODS: C57BL/6 mice were fed a control diet (control, 10% kcal from fat) or control diet + SDG (SDG, 100 mg/kg diet) for 8 weeks, then orthotopically injected with syngeneic E0771 mammary tumor cells (a model of TNBC); tumor growth was monitored for 3 weeks. The role of reduced NF-κB signaling in SDG's anti-tumor effects was explored in vitro via treatment with the bioactive SDG metabolite ENL. In addition to the murine E0771 cells, the in vitro studies utilized MDA-MB-231 and MCF-7 cells, two human cell lines which model the triple-negative and luminal A breast cancer subtypes, respectively. RESULTS: SDG supplementation in the mice significantly reduced tumor volume and expression of phospho-p65 and NF-κB target genes (P < 0.05). Markers of macrophage infiltration were decreased in the distal-to-tumor mammary fat pad of mice supplemented with SDG relative to control mice (P < 0.05). In vitro, ENL treatment inhibited viability, survival, and NF-κB activity and target gene expression in E0771, MDA-MB-231, and MCF-7 cells (P < 0.05). Overexpression of Rela attenuated ENL's inhibition of E0771 cell viability and survival. CONCLUSIONS: SDG reduces tumor growth in the E0771 model of TNBC, likely via a mechanism involving inhibition of NF-κB activity. SDG could serve as a practical and effective adjuvant treatment to reduce recurrence, but greater understanding of its effects is needed to inform the development of more targeted recommendations for its use.

Apoptotic effect of enterodiol, the final metabolite of edible lignans, in colorectal cancer cells.

BACKGROUND: Enterodiol (END) is transformed by human intestinal bacteria from lignans contained in various whole-grain cereals, nuts, legumes, flaxseed, and vegetables. It is known to have several physiological effects, but its effects on mitogen-activated protein kinase (MAPK) signaling and apoptosis in colorectal cancer (CRC) cells have not yet been elucidated. We therefore investigated the effects of END on apoptosis in CRC cells and whether these effects are mediated via MAPK signaling. RESULTS: Cell proliferation was decreased by END treatment in a time-dependent manner. In particular, END treatment resulted in an apoptosis rate of up to 40% in CT26 cells but showed no cytotoxicity toward RAW264.7 macrophages. Treatment with END also suppressed the migration of CRC cells in a concentration-dependent manner. The phosphorylation of extracellular signal-regulated kinase (ERK), jun N-terminal kinase (JNK), and p38 was down-regulated with END treatment. Furthermore, END decreased the expression levels of anti-apoptotic proteins in CRC cells. CONCLUSION: Enterodiol inhibited the growth of CRC cells by controlling the MAPK signaling pathway involved in proliferation and apoptosis. These results demonstrate that END has an apoptotic effect in CRC cells. © 2018 Society of Chemical Industry.

7-Hydroxymatairesinol improves body weight, fat and sugar metabolism in C57BJ/6 mice on a high-fat diet.

7-Hydroxymatairesinol (7-HMR) is a plant lignan abundant in various concentrations in plant foods. The objective of this study was to test HMRLignan™, a purified form of 7-HMR, and the corresponding Picea abies extract (total extract P. abies; TEP) as dietary supplements on a background of a high-fat diet (HFD)-induced metabolic syndrome in mice and in the 3T3-L1 adipogenesis model. Mice, 3 weeks old, were fed a HFD for 60 d. Subgroups were treated with 3 mg/kg body weight 7-HMR (HMRLignan™) or 10 mg/kg body weight TEP by oral administration. 7-HMR and TEP limited the increase in body weight (-11 and -13 %) and fat mass (-11 and -18 %) in the HFD-fed mice. Epididymal adipocytes were 19 and -12 % smaller and the liver was less steatotic (-62 and -65 %). Serum lipids decreased in TEP-treated mice (-11 % cholesterol, -23 % LDL and -15 % TAG) and sugar metabolism was ameliorated by both lignan preparations, as shown by a more than 70 % decrease in insulin secretion and insulin resistance. The expression of several metabolic genes was modulated by the HFD with an effect that was reversed by lignan. In 3T3-L1 cells, the 7-HMR metabolites enterolactone (ENL) and enterodiol (END) showed a 40 % inhibition of cell differentiation accompanied by the inhibited expression of the adipogenic genes PPARγ, C/EBPα and aP2. Furthermore, END and ENL caused a 10 % reduction in TAG uptake in HEPA 1-6 hepatoma cells. In conclusion, 7-HMR and TEP reduce metabolic imbalances typical of the metabolic syndrome and obesity in male mice, whereas their metabolites inhibit adipogenesis and lipid uptake in vitro.

Flaxseed-enriched diets change milk concentration of the antimicrobial danofloxacin in sheep.

BACKGROUND: Flaxseed is the most common and rich dietary source of lignans and is an acceptable supply of energy for livestock. Flaxseed lignans are precursors of enterolignans, mainly enterolactone and enterodiol, produced by the rumen and intestinal microbiota of mammals and have many important biological properties as phytoestrogens. Potential food-drug interactions involving flaxseed may be relevant for veterinary therapy, and for the quality and safety of milk and dairy products. Our aim was to investigate a potential food-drug interaction involving flaxseed, to explore whether the inclusion of flaxseed in sheep diet affects concentration of the antimicrobial danofloxacin in milk. RESULTS: Increased concentrations of enterodiol and enterolactone were observed in sheep plasma and milk after 2 weeks of flaxseed supplementation (P < 0.05). However, enterolactone and enterodiol conjugates were not detected in milk. Milk danofloxacin pharmacokinetics showed that area under the curve (AUC)0-24, maximum concentration (Cmax) and AUC0-24 milk-to-plasma ratios were reduced by 25-30% in sheep fed flaxseed-enriched diets (P < 0.05). Our results demonstrate, therefore, that flaxseed-enriched diets reduce the amount of danofloxacin in sheep milk and enrich the milk content of lignan-derivatives. CONCLUSION: These findings highlight an effect of flaxseed-enriched diets on the concentration of antimicrobials in ruminant's milk, revealing the potential of these modified diets for the control of residues of antimicrobial drugs in milk.

Bioactivation of Phytoestrogens: Intestinal Bacteria and Health.

Phytoestrogens are polyphenols similar to human estrogens found in plants or derived from plant precursors. Phytoestrogens are found in high concentration in soya, flaxseed and other seeds, fruits, vegetables, cereals, tea, chocolate, etc. They comprise several classes of chemical compounds (stilbenes, coumestans, isoflavones, ellagitannins, and lignans) which are structurally similar to endogenous estrogens but which can have both estrogenic and antiestrogenic effects. Although epidemiological and experimental evidence indicates that intake of phytoestrogens in foods may be protective against certain chronic diseases, discrepancies have been observed between in vivo and in vitro experiments. The microbial transformations have not been reported so far in stilbenes and coumestans. However, isoflavones, ellagitanins, and lignans are metabolized by intestinal bacteria to produce equol, urolithins, and enterolignans, respectively. Equol, urolithin, and enterolignans are more bioavailable, and have more estrogenic/antiestrogenic and antioxidant activity than their precursors. Moreover, equol, urolithins and enterolignans have anti-inflammatory effects and induce antiproliferative and apoptosis-inducing activities. The transformation of isoflavones, ellagitanins, and lignans by intestinal microbiota is essential to be protective against certain chronic diseases, as cancer, cardiovascular disease, osteoporosis, and menopausal symptoms. Bioavailability, bioactivity, and health effects of dietary phytoestrogens are strongly determined by the intestinal bacteria of each individual.

A Prospective Investigation of the Association Between Urinary Excretion of Dietary Lignan Metabolites and Weight Change in US Women.

Results from animal studies have consistently suggested that lignans play a role in the regulation of in body weight, but evidence from human studies has been limited. We examined the associations between urinary excretion of enterolactone and enterodiol, the major intestinal microbial metabolites of dietary lignans, and 10-year prospective weight change using data from 2 well-characterized cohort studies of US women: the Nurses' Health Study (2000-2010) and Nurses' Health Study II (1997-2007). Urinary excretion levels of enterolactone and enterodiol were measured at baseline. Associations with prospective weight change were analyzed using a multivariable-adjusted linear mixed-effects model. We observed that women in the highest quartile of urinary excretion of total lignans had significantly lower baseline body mass indices (weight in kilograms divided by height in meters squared) (mean, 24.6, 95% confidence interval (CI): 23.9, 25.2) than did those in the lowest quartile (mean, 27.7, 95% CI: 27.0, 28.4; P for trend < 0.01). Compared with women in the lowest quartile of enterodiol excretion, those in the highest quartile gained 0.27 kg/year less weight (95% CI: 0.12, 0.41; P for trend < 0.01) during the 10-year follow-up. The association was borderline significant for enterolactone (for the fourth vs. first quartile, least square mean of weight change rate = -0.14 kg/year, 95% CI: -0.29, 0.00). Our data suggest that higher urinary excretion of lignan metabolites, especially enterodiol, is associated with modestly slower weight gain.

Bioconversion of lignans in flaxseed cake by fermented tofu microbiota and isolation of Enterococcus faecium strain ZB26 responsible for converting secoisolariciresinol diglucoside to enterodiol.

Human intestinal microbiota plays a crucial role in converting secoisolariciresinol diglucoside, a lignan found in flaxseed, to enterodiol, which has a range of health benefits: antioxidative, antitumor, and estrogenic/anti-estrogenic effects. Given the high secoisolariciresinol diglucoside content in flaxseed cake, this study investigated the potential of co-fermenting flaxseed cake with fermented soybean product to isolate bacterial strains that effectively convert secoisolariciresinol diglucoside to enterodiol in a controlled environment (in vitro). The co-fermentation process with stinky tofu microbiota significantly altered the lignan, generating 12 intermediate lignan metabolites as identified by targeted metabolomics. One particular promising strain, ZB26, demonstrated an impressive ability to convert secoisolariciresinol diglucoside. It achieved a conversion rate of 87.42 ± 0.33%, with secoisolariciresinol and enterodiol generation rates of 94.22 ± 0.51% and 2.91 ± 0.03%, respectively. Further optimization revealed, under specific conditions (0.5 mM secoisolariciresinol diglucoside, pH 8, 30 °C for 3 days), ZB26 could convert an even higher percentage (97.75 ± 0.05%) of the secoisolariciresinol diglucoside to generate secoisolariciresinol (103.02 ± 0.16%) and enterodiol (3.18 ± 0.31%). These findings suggest that the identified strains ZB26 have promising potential for developing functional foods and ingredients enriched with lignans.

Diet Quality, Microbial Lignan Metabolites, and Cardiometabolic Health among US Adults.

The gut microbiome has been shown to play a role in the relationship between diet and cardiometabolic health. We sought to examine the degree to which key microbial lignan metabolites are involved in the relationship between diet quality and cardiometabolic health using a multidimensional framework. This analysis was undertaken using cross-sectional data from 4685 US adults (age 43.6 ± 16.5 years; 50.4% female) participating in the National Health and Nutrition Examination Survey for 1999-2010. Dietary data were collected from one to two separate 24-hour dietary recalls and diet quality was characterized using the 2015 Healthy Eating Index. Cardiometabolic health markers included blood lipid profile, glycemic control, adiposity, and blood pressure. Microbial lignan metabolites considered were urinary concentrations of enterolignans, including enterolactone and enterodiol, with higher levels indicating a healthier gut microbial environment. Models were visually examined using a multidimensional approach and statistically analyzed using three-dimensional generalized additive models. There was a significant interactive association between diet quality and microbial lignan metabolites for triglycerides, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, insulin, oral glucose tolerance, adiposity, systolic blood pressure, and diastolic blood pressure (all p < 0.05). Each of these cardiometabolic health markers displayed an association such that optimal cardiometabolic health was only observed in individuals with both high diet quality and elevated urinary enterolignans. When comparing effect sizes on the multidimensional response surfaces and model selection criteria, the strongest support for a potential moderating relationship of the gut microbiome was observed for fasting triglycerides and oral glucose tolerance. In this study, we revealed interactive associations of diet quality and microbial lignan metabolites with cardiometabolic health markers. These findings suggest that the overall association of diet quality on cardiometabolic health may be affected by the gut microbiome.

Urinary Phytoestrogen Metabolites Positively Correlate with Serum 25(OH)D Level Based on National Health and Nutrition Examination Survey 2009-2010.

Studies showed that vitamin D (25-hydroxyvitamin D) level in the human blood circulation could be affected by exogenous estrogen exposure. This study aims to explore the relationships between urinary phytoestrogens metabolites and serum total 25(OH)D in general population, urinary phytoestrogens metabolites (daidzein, enterodiol, enterolactone, equol, genistein and o-desmethylangolensin). Totally 2,609 adults ≥6 y old from the 2009-2010 National Health and Nutrition Examination Surveys (NHANES) were recruited into the cross-sectional analyses and information including demographic, socioeconomic, examinations and laboratory test were collected. All analyses were performed using Stata13.0, one-way analysis of variance and multivariable regression were utilised according to data characteristics, respectively. It showed that age, race, education level, body mass index (BMI), and sampling season had significant effects on serum 25(OH)D level (all p<0.001). In the whole population, urinary enterodiol and equol were significantly positively associated with serum total 25(OH)D level (ß=0.86, 95%CI=0.08-1.65, p<0.05; ß=1.68, 95%CI=0.91-2.45, p<0.001). Equol was also found significantly positively correlated with total 25(OH)D in both female and male separately (ß=1.69, 95%CI=0.51-2.87, p<0.05; ß=1.66, 95%CI=0.63-2.69, p<0.05). Phytoestrogen concentrations in the urinary and 25(OH)D levels in the serum had proved a positive correlation in our study, which provide theoretical basis and reference for the dietary nutrient intake in the population.

Influence of different lignan compounds on enterolignan production by Bifidobacterium and Lactobacillus strains.

Enterolignans, i.e. enterodiol and enterolactone, are polyphenols derived from the microbial metabolism of dietary lignans. They are considered phytoestrogens because of their estrogenic/antiestrogenic activity, which confers them benefits to human health when they reach sufficient levels in plasma. Hence, there is a great interest in studying the bacteria involved in enterolignan production. In the present study, three bifidobacterial strains (Bifidobacterium bifidum INIA P466, Bifidobacterium catenulatum INIA P732 and Bifidobacterium pseudolongum INIA P2) were found capable of producing low levels of enterodiol (2-11 µM) from lignan extracts; while another one (Bifidobacterium pseudocatenulatum INIA P946) was found to produce an important increment of the lignan secoisolariciresinol (SECO). Subsequently, the three enterodiol-producing bifidobacteria and another three Lactobacillus strains previously identified as enterolignans producers (Lactobacillus gasseri INIA P508, Lactobacillus salivarius INIA P448 and Lb. salivarius INIA P183), were tested on pure lignans yielding both enterodiol and enterolactone from secoisolariciresinol (SECO), while they did not metabolised the other lignan tested (i.e. matairesinol). B. catenulatum INIA P732 and Lb. gasseri INIA P508 were the strains that transformed the greatest percentage of SECO, yielding enterolactone concentrations above 2 mM. In addition, the formation of the intermediate compound dihydroxyenterodiol was observed as part of SECO transformation by all the strains. In this work, we have demonstrated for the first time how strains of Bifidobacterium and Lactobacillus are capable of carrying out the complete enterolignan metabolism, transforming a purified lignan (SECO) into enterodiol and enterolactone. The isolation and characterization of bacteria able to metabolize lignans and produce enterolignans, especially belonging to Bifidobacterium and Lactobacillus genera, is of biotechnological interest, because of their potential application in functional foods and as probiotics.

Polyphenol Microbial Metabolites Exhibit Gut and Blood⁻Brain Barrier Permeability and Protect Murine Microglia against LPS-Induced Inflammation.

Increasing evidence supports the beneficial effects of polyphenol-rich diets, including the traditional Mediterranean diet, for the management of cardiovascular disease, obesity and neurodegenerative diseases. However, a common concern when discussing the protective effects of polyphenol-rich diets against diseases is whether these compounds are present in systemic circulation in their intact/parent forms in order to exert their beneficial effects in vivo. Here, we explore two common classes of dietary polyphenols, namely isoflavones and lignans, and their gut microbial-derived metabolites for gut and blood-brain barrier predicted permeability, as well as protection against neuroinflammatory stimuli in murine BV-2 microglia. Polyphenol microbial metabolites (PMMs) generally showed greater permeability through artificial gut and blood-brain barriers compared to their parent compounds. The parent polyphenols and their corresponding PMMs were evaluated for protective effects against lipopolysaccharide-induced inflammation in BV-2 microglia. The lignan-derived PMMs, equol and enterolactone, exhibited protective effects against nitric oxide production, as well as against pro-inflammatory cytokines (IL-6 and TNF-α) in BV-2 microglia. Therefore, PMMs may contribute, in large part, to the beneficial effects attributed to polyphenol-rich diets, further supporting the important role of gut microbiota in human health and disease prevention.

Enterolactone has stronger effects than enterodiol on ovarian cancer.

BACKGROUND: Ovarian cancer is one of the three leading gynecological malignancies, characterized by insidious growth, highly frequent metastasis, and quick development of drug resistance. As a result, this disease has low 5-year survival rates. Estrogen receptor inhibitors were commonly used for the treatment, but only 7% to 18% of patients respond to anti-estrogen therapies. Therefore, more effective therapies to inhibit estrogen-related tumors are urgently needed. Recently, phytoestrogens, such as lignans with estrogen-like biological activities, have attracted attention for their potential effects in the prevention or treatment of estrogen-related diseases. Enterodiol (END) and enterolactone (ENL) are mammalian lignans, which can reduce the risk of various cancers. However, the effects of END and ENL on ovarian cancer are not adequately documented. METHODS: We used in vitro assays on the ES-2 cell line to evaluate the inhibiting effects of END and ENL on ovarian cancer cell proliferation, invasion and migration ability and in vivo xenograft experiments on nude mice to validate the anticancer effects of END and ENL. RESULTS: The in vitro assays demonstrated that high-dose END and ENL could obviously inhibit ovarian malignant properties, including cancerous proliferation, invasion, and metastasis. Compared to END, ENL behaved in a better time-dose dependent manner on the cancer cells. The in vivo experiments showed that END (1 mg/kg), ENL (1 mg/kg) and ENL (0.1 mg/kg) suppressed tumor markedly, and there were statistically significant differences between the experimental and control groups in tumor weight and volume. Compared to END, which have serious side effects to the animals at high concentration such as 1 mg/kg, ENL had higher anticancer activities and less side effects in the animals than END at the same concentrations, so it would be a better candidate for drug development. CONCLUSION: END and ENL both have potent inhibitory effects on ovarian cancer but ENL possesses a more effective anti-cancer capability and less side effects than END. Findings in this work provide novel insights into ovarian cancer therapeutics with phytoestrogens and encourage their clinical applications.

Enterolactone Reduces Telomerase Activity and The Level of Its Catalytic Subunit in Breast Cancer Cells.

OBJECTIVE: There is a positive correlation between higher serum phytoestrogen concentrations and lower risk of breast cancer. The activation of telomerase is crucial for the growth of cancer cells; therefore, the aim of this study was to examine the effects of enterolactone (ENL) and enterodiol (END) on this enzyme. MATERIALS AND METHODS: In this experimental study, we performed the viability assay to determine the effects of different concentrations of ENL and END on cell viability, and the effective concentrations of these two compounds on cell growth. We used western blot analysis to evaluate human telomerase reverse transcriptase catalytic subunit (hTERT) expression and polymerase chain reaction (PCR)-ELISA based on the telomeric repeat amplification protocol (TRAP) assay for telomerase activity. RESULTS: Both ENL and END, at 100 µM concentrations, significantly (P<0.05) reduced cell viability. However, only the 100 µM concentration of ENL significantly (P<0.05) decreased hTERT protein levels and telomerase activity. Lower concentrations of ENL did not have any significant effects on telomerase activity and hTERT protein levels. CONCLUSION: High concentration of ENL decreased the viability of MCF-7 breast cancer cells and inhibited the expression and activity of telomerase in these cells. Although END could reduce breast cancer cell viability, it did not have any effect on telomerase expression and activity.