Pomegranate Extract Augments Energy Expenditure Counteracting the Metabolic Stress Associated with High-Fat-Diet-Induced Obesity.

Obesity is associated to a low grade of chronic inflammation leading to metabolic stress, insulin resistance, metabolic syndrome, dislipidemia, cardiovascular disease, and even cancer. A Mediterranean diet has been shown to reduce systemic inflammatory factors, insulin resistance, and metabolic syndrome. In this scenario, precision nutrition may provide complementary approaches to target the metabolic alterations associated to "unhealthy obesity". In a previous work, we described a pomegranate extract (PomE) rich in punicalagines to augment markers of browning and thermogenesis in human differentiated adipocytes and to augment the oxidative respiratory capacity in human differentiated myocytes. Herein, we have conducted a preclinical study of high-fat-diet (HFD)-induced obesity where PomE augments the systemic energy expenditure (EE) contributing to a reduction in the low grade of chronic inflammation and insulin resistance associated to obesity. At the molecular level, PomE promotes browning and thermogenesis in adipose tissue, reducing inflammatory markers and augmenting the reductive potential to control the oxidative stress associated to the HFD. PomE merits further investigation as a complementary approach to alleviate obesity, reducing the low grade of chronic inflammation and metabolic stress.

Acid hydrolysis of saponin-rich extracts of quinoa, lentil, fenugreek and soybean to yield sapogenin-rich extracts and other bioactive compounds.

BACKGROUND: Typical hydrolysis times of saponins generally do not take into consideration the effect of time on the degradation of the target compounds, namely sapogenins. When producing natural extracts, it should be borne in mind that conducting hydrolysis to yield a target compound might also affect the final composition of the extracts in terms of other bioactive compounds. In our study, saponin-rich extracts from fenugreek, quinoa, lentil, and soybean were produced and their acid hydrolysis to give sapogenin-rich extracts was conducted over different periods (0-6 h). The disappearance of saponins and appearance of sapogenins was analyzed using high-performance liquid chromatography-diode array detection-mass spectrometry (HPLC-DAD-MS) and gas chromatography-mass spectrometry (GC-MS), respectively. The impact of hydrolysis on the phytosterols and tocopherol in the extracts was also evaluated. RESULTS: Fenugreek showed the highest saponin content (169 g kg-1 ), followed by lentil (20 g kg-1 ), quinoa (15 g kg-1 ), and soybean (13 g kg-1 ). Hydrolysis for 1 h caused the complete disappearance of saponins and the greatest release of sapogenins. Hydrolyzed fenugreek and quinoa extracts contained the highest amounts of sapogenins and minor fractions of phytosterols and tocopherol. Hydrolyzed extracts of lentil and soybean contained a major fraction of phytosterols and a low fraction of sapogenins. In all cases, sapogenins decreased after 1 h of hydrolysis, phytosterols slightly decreased, and tocopherol was unaffected. Standards of diosgenin and oleanolic acid also showed this decreasing pattern under acid hydrolysis conditions. CONCLUSION: Hydrolysis times of 1 h for saponin-rich extracts from the assayed seeds guarantee the maximum transformation to sapogenin-rich extracts, along with phytosterols and tocopherol. Fenugreek and quinoa seeds are preferred for this. © 2018 Society of Chemical Industry.

Protein matrices ensure safe and functional delivery of rosmarinic acid from marjoram (Origanum majorana) extracts.

BACKGROUND: To understand the interactions between carriers and functional ingredients is crucial when designing delivery systems, to maximize bioefficacy and functionality. In this study, two different protein matrices were evaluated as means to protect the extract isolated from marjoram leaves (Origanum majorana), casein micelles from fresh skim milk and soy protein isolate (SPI). RESULTS: Marjoram extract was obtained from pressurization of ethanol and water solvent. Protein dispersions of casein and SPI (5 g L-1 each) with or without marjoram extract (0.1-3 mg mL-1 ) were prepared and homogenized. The physicochemical characterization of charge and entrapment efficiency were conducted. The results demonstrated that entrapment efficiency was highly dependent on the carrier itself where SPI formulations showed 20% higher affinity when compared to casein micelles. To investigate the physiological behaviour of the marjoram-protein dispersions, human macrophages were employed. A non-specific inflammatory response of macrophages stimulated with bacterial lipopolysaccharide was measured for TNF-α, IL-1ß and IL-6 cytokine secretion. CONCLUSION: Casein and SPI protein formulations warranted high bioefficacy of marjoram extract, showing their potential as safe carriers. © 2018 Society of Chemical Industry.

Targeting the lipid metabolic axis ACSL/SCD in colorectal cancer progression by therapeutic miRNAs: miR-19b-1 role.

An abnormal acyl-CoA synthetase/stearoyl-CoA desaturase (ACSL/SCD) lipid network fuels colon cancer progression, endowing cells with invasive and migratory properties. Therapies against this metabolic network may be useful to improve clinical outcomes. Because micro-RNAs (miRNAs/miRs) are important epigenetic regulators, we investigated novel miRNAs targeting this pro-tumorigenic axis; hence to be used as therapeutic or prognostic miRNAs. Thirty-one putative common miRNAs were predicted to simultaneously target the three enzymes comprising the ACSL/SCD network. Target validation by quantitative RT-PCR, Western blotting, and luciferase assays showed miR-544a, miR-142, and miR-19b-1 as major regulators of the metabolic axis, ACSL/SCD Importantly, lower miR-19b-1 expression was associated with a decreased survival rate in colorectal cancer (CRC) patients, accordingly with ACSL/SCD involvement in patient relapse. Finally, miR-19b-1 regulated the pro-tumorigenic axis, ACSL/SCD, being able to inhibit invasion in colon cancer cells. Because its expression correlated with an increased survival rate in CRC patients, we propose miR-19b-1 as a potential noninvasive biomarker of disease-free survival and a promising therapeutic miRNA in CRC.

Novel Polyphenols That Inhibit Colon Cancer Cell Growth Affecting Cancer Cell Metabolism.

New series of polyphenols with a hydrophilic galloyl-based head and a hydrophobic N-acyl tail, linked through a serinol moiety, have been synthesized and tested against colon cancer cell growth. Our structure activity relationship studies revealed that galloyl moieties are essential for growth inhibition. Moreover, the length of the N-acyl chain is crucial for the activity. Introduction of a (Z) double bond in the acyl chain increased the anticancer properties. Our findings demonstrate that 16, the most potent compound within this series, has inhibitory effects on colon cancer cell growth and metabolism (glycolysis and mitochondrial respiration) at the same time that it activates 5'AMP-activated kinase (AMPK) and induces apoptotic cell death. Based on these results, we propose that 16 might reprogram colon cancer cell metabolism through AMPK activation. This might lead to alterations on cancer cell bioenergy compromising cancer cell viability. Importantly, these antiproliferative and proapoptotic effects are selective for cancer cells. Accordingly, these results indicate that 16, with an unsaturated C18 chain, might be a useful prototype for the development of novel colon cancer cell growth inhibitors affecting cell metabolism.

Pressurized Liquid Extraction (PLE) as an Innovative Green Technology for the Effective Enrichment of Galician Algae Extracts with High Quality Fatty Acids and Antimicrobial and Antioxidant Properties.

Marine organisms are potentially prolific sources of high qualify fatty acids that represent useful leads in the development of new nutraceutical agents. In this work, we investigated the lipid composition of six algae species from the Northwest of Spain (Ulva intestinalis, Ulva lactuca, Fucus vesiculosus,Dictyota dichotoma, Cystoseira baccata and Himanthalia elongata) and compared the antioxidant and antibacterial activity of ethanolic extracts obtained by pressurized liquid extraction (PLE). Furthermore, Fucus vesiculosus (F. vesiculosus) PLE using five solvents of different polarities (hexane, ethyl acetate, acetone, ethanol and ethanol:water 50:50) at three temperatures (80 °C, 120 °C and 160 °C) was investigated. F. vesiculosus ethanolic PLE extract presents considerably higher capacity of inhibiting 50% of DPPH (1,1-diphenyl-2-picryl hydrazyl) (IC50 = 7.17 μg/mL) in comparison with the rest of macroalgae studied. Moreover, the potential antimicrobial activity tested on E. coli and S. aureus shows that F. vesiculosus extract produced the best inhibition (IC50 was 2.24 mg/mL (E. coli) and 1.27 mg/mL (S. aureus)). Furthermore, regarding the different solvents and temperatures used to investigate F. vesiculosus PLE, results showed that this technique using ethyl acetate is a selective method to enrich long chain fatty acids (oleic acid, arachidonic acid and eicosapentaenoic acid) with ω-6/ω-3 ratios close to 2.7.

Identification of antitumoral agents against human pancreatic cancer cells from Asteraceae and Lamiaceae plant extracts.

BACKGROUND: Pancreatic cancer is one of the most aggressive and mortal cancers. Although several drugs have been proposed for its treatment, it remains resistant and new alternatives are needed. In this context, plants and their derivatives constitute a relevant source of bioactive components which might efficiently inhibit tumor cell progression. METHODS: In this study, we have analyzed the potential anti-carcinogenic effect of different Asteraceae (Achillea millefolium and Calendula officinalis) and Lamiaceae (Melissa officinalis and Origanum majorana) plant extracts obtained by different green technologies (Supercritical CO2 Extraction -SFE- and Ultrasonic Assisted Extraction -UAE-) to identify efficient plant extracts against human pancreatic cancer cells that could constitute the basis of novel treatment approaches. RESULTS: Asteraceae extracts showed better results as antitumoral agents than Lamiaceae by inducing cytotoxicity and inhibiting cell transformation, and SFE extracts were most efficient than UAE extracts. In addition, SFE derived plant extracts from Achillea millefolium and Calendula officinalis displayed synergism with the chemotherapeutic 5-Fluororacil. CONCLUSION: These results show how Yarrow and Marigold SFE-derived extracts can inhibit pancreatic cancer cell growth, and could be proposed for a comprehensive study to determine the molecular mechanisms involved in their bioactivity with the final aim to propose them as potential adjuvants in pancreatic cancer therapy.

Biological Activities of Asteraceae (Achillea millefolium and Calendula officinalis) and Lamiaceae (Melissa officinalis and Origanum majorana) Plant Extracts.

Asteraceae (Achillea millefolium and Calendula officinalis) and Lamiaceae (Melissa officinalis and Origanum majorana) extracts were obtained by applying two sequential extraction processes: supercritical fluid extraction with carbon dioxide, followed by ultrasonic assisted extraction using green solvents (ethanol and ethanol:water 50:50). The extracts were analyzed in terms of the total content of phenolic compounds and the content of flavonoids; the volatile oil composition of supercritical extracts was analyzed by gas chromatography and the antioxidant capacity and cell toxicity was determined. Lamiaceae plant extracts presented higher content of phenolics (and flavonoids) than Asteraceae extracts. Regardless of the species studied, the supercritical extracts presented the lowest antioxidant activity and the ethanol:water extracts offered the largest, following the order Origanum majorana > Melissa officinalis ≈ Achillea millefolium > Calendula officinalis. However, concerning the effect on cell toxicity, Asteraceae (especially Achillea millefolium) supercritical extracts were significantly more efficient despite being the less active as an antioxidant agent. These results indicate that the effect on cell viability is not related to the antioxidant activity of the extracts.

Modulation of cholesterol-related gene expression by ergosterol and ergosterol-enriched extracts obtained from Agaricus bisporus.

PURPOSE: To investigate the effect of two extracts obtained from Agaricus bisporus on the mRNA expression of cholesterol-related genes. One of the extracts contained ergosterol and other fungal sterols (SFE) and the other contained ß-glucans and fungal sterols (EßG). METHODS: Firstly, the dietary mixed micelles (DMMs) generated after in vitro digestion of standards and SFE were applied to Caco2 cells. Then, the lower compartment after a Caco2-transport experiment was applied to HepG2 cells. The mRNA expression was assessed in both cell lines by low-density arrays (LDA). Mice received the extracts, ergosterol or control drugs after 4 weeks of a high-cholesterol diet. The lipid profile of plasma, liver and feces was determined. LDA assays were performed in liver and intestines. RESULTS: The DMM fraction of SFE up-regulated the LDLR mRNA expression in Caco2 cells. The lower compartment after Caco2-transport experiments up-regulated LDLR and modulated several other lipid-related genes in HepG2 cells. In mice, SFE decreased TC/HDL ratio and reduced hepatic triglycerides paralleled with down-regulation of Dgat1 expression, while EßG did it without transcriptional changes. Addition of SFE or ergosterol induced in jejunum a similar transcriptional response to simvastatin and ezetimibe; they all down-regulated Srebf2 and Nr1h4 (FXR) genes. CONCLUSION: Ergosterol-containing extracts from A. bisporus lowered hepatic triglyceride and modify the mRNA expression of cholesterol-related genes although the transcriptional regulation was unrelated to changes in plasma lipid profile. These extracts may be useful limiting hepatic steatosis and as bioactive ingredients to design novel functional foods preventing lifestyle-related diseases such as non-alcoholic fatty liver disease.

Dietary Strategies Implicated in the Prevention and Treatment of Metabolic Syndrome.

Metabolic syndrome (MetS) is established as the combination of central obesity and different metabolic disturbances, such as insulin resistance, hypertension and dyslipidemia. This cluster of factors affects approximately 10%-50% of adults worldwide and the prevalence has been increasing in epidemic proportions over the last years. Thus, dietary strategies to treat this heterogenic disease are under continuous study. In this sense, diets based on negative-energy-balance, the Mediterranean dietary pattern, n-3 fatty acids, total antioxidant capacity and meal frequency have been suggested as effective approaches to treat MetS. Furthermore, the type and percentage of carbohydrates, the glycemic index or glycemic load, and dietary fiber content are some of the most relevant aspects related to insulin resistance and impaired glucose tolerance, which are important co-morbidities of MetS. Finally, new studies focused on the molecular action of specific nutritional bioactive compounds with positive effects on the MetS are currently an objective of scientific research worldwide. The present review summarizes some of the most relevant dietary approaches and bioactive compounds employed in the treatment of the MetS to date.

The ellagic acid derivative 4,4'-di-O-methylellagic acid efficiently inhibits colon cancer cell growth through a mechanism involving WNT16.

Ellagic acid (EA) and some derivatives have been reported to inhibit cancer cell proliferation, induce cell cycle arrest, and modulate some important cellular processes related to cancer. This study aimed to identify possible structure-activity relationships of EA and some in vivo derivatives in their antiproliferative effect on both human colon cancer and normal cells, and to compare this activity with that of other polyphenols. Our results showed that 4,4'-di-O-methylellagic acid (4,4'-DiOMEA) was the most effective compound in the inhibition of colon cancer cell proliferation. 4,4'-DiOMEA was 13-fold more effective than other compounds of the same family. In addition, 4,4'-DiOMEA was very active against colon cancer cells resistant to the chemotherapeutic agent 5-fluoracil, whereas no effect was observed in nonmalignant colon cells. Moreover, no correlation between antiproliferative and antioxidant activities was found, further supporting that structure differences might result in dissimilar molecular targets involved in their differential effects. Finally, microarray analysis revealed that 4,4'-DiOMEA modulated Wnt signaling, which might be involved in the potential antitumor action of this compound. Our results suggest that structural-activity differences between EA and 4,4'-DiOMEA might constitute the basis for a new strategy in anticancer drug discovery based on these chemical modifications.

Rosemary (Rosmarinus officinalis L.) Extract as a Potential Complementary Agent in Anticancer Therapy.

Cancer remains an important cause of mortality nowadays and, therefore, new therapeutic approaches are still needed. Rosemary (Rosmarinus officinalis L.) has been reported to possess antitumor activities both in vitro and in animal studies. Some of these activities were attributed to its major components, such as carnosic acid, carnosol, ursolic acid, and rosmarinic acid. Initially, the antitumor effects of rosemary were attributed to its antioxidant activity. However, in recent years, a lack of correlation between antioxidant and antitumor effects exerted by rosemary was reported, and different molecular mechanisms were related to its tumor inhibitory properties. Moreover, supported by the U.S. Food and Drug Administration and the European Food and Safety Authority, specific compositions of rosemary extract were demonstrated to be safe for human health and used as antioxidant additive in foods, suggesting the potential easy application of this agent as a complementary approach in cancer therapy. In this review, we aim to summarize the reported anticancer effects of rosemary, the demonstrated molecular mechanisms related to these effects and the interactions between rosemary and currently used anticancer agents. The possibility of using rosemary extract as a complementary agent in cancer therapy in comparison with its isolated components is discussed.

Extraction of thymol from different varieties of thyme plants using green solvents.

BACKGROUND: Thymol (2-isopropyl-5-methylphenol) is the main monoterpene phenol found in thyme essential oil. This compound has revealed several biological properties, including antibacterial, anti-inflammatory and antioxidant activities. In this work, a comparison was made between the performance of different green solvents (ethanol, limonene and ethyl lactate), by pressurized liquid extraction (PLE) and supercritical fluid extraction (SFE) at different conditions, to extract thymol from three different varieties of thyme (Thymus vulgaris, Thymus zygis and Thymus citriodorus). Additionally, new solubility data of thymol in limonene and ethanol at ambient pressure and temperatures in the range 30-43 °C are reported. RESULTS: The highest thymol recoveries were attained with T. vulgaris (7-11 mg g(-1)). No thymol could be quantified in the PLE samples of T. citriodorus. The highest concentrations of thymol in the extracts were obtained with limonene. Thymol is very soluble in both solvents, particularly in ethanol (∼900 mg g(-1) at ∼40 °C), and is the main compound (in terms of peak area) present in the essential oil extracts obtained. CONCLUSION: The three solvents show good capacity to extract thymol from T. vulgaris and T. zygis by PLE. Although PLE proved to be a suitable technology to extract thymol from thyme plants, the highest concentrations of thymol were obtained by SFE with supercritical CO2 .

Extraction of functional ingredients from spinach (Spinacia oleracea L.) using liquid solvent and supercritical CO2 extraction.

BACKGROUND: In this work three different techniques were applied to extract dry leaves of spinach (Spinacia oleracea): solid-liquid extraction (SLE), pressurised liquid extraction (PLE) and supercritical fluid extraction (SFE) to investigate the influence of extraction solvent and technique on extracts composition and antioxidant activity. Moreover, the influence of carotenoids and phenolic compounds on the antioxidant and anti-inflammatory activities of spinach extracts was also studied. RESULTS: The higher concentrations of carotenoids and the lower content of phenolic compounds were observed in the supercritical CO2 extracts; whereas water and/or ethanol PLE extracts presented low amounts of carotenoids and the higher concentrations of phenolic compounds. PLE extract with the highest content of phenolic compounds showed the highest antioxidant activity, although SFE carotenoid rich extract also showed a high antioxidant activity. Moreover, both extracts presented an important anti-inflammatory activity. CONCLUSION: PLE seems to be a good technique for the extraction of antioxidant and anti-inflammatory compounds from spinach leaves. Moreover, spinach phenolic compounds and carotenoids present a high antioxidant activity, whereas spinach carotenoids seem to show a higher anti-inflammatory activity than phenolic compounds. It is worth noting that of our knowledge this is the first time the anti-inflammatory activity of lipophilic extracts from spinach leaves is reported.

Resveratrol metabolic fingerprinting after acute and chronic intakes of a functional beverage in humans.

The study of the bioavailability of active compounds in functional foods, such as polyphenol-rich beverages, is required before making nutritional claims. In this work, we aimed to study the urinary excretion of resveratrol (RV), taking into consideration its gut and microbial metabolites after consumption of a functional beverage (FB), applying a ultra performance liquid chromatography (UPLC)-MS/MS methodology. A randomized, crossover, placebo-controlled, double-blind intervention study was performed with 26 volunteers, who consumed 187 mL of a control placebo or a FB in an acute study, and twice a day during 15 days for a chronic consumption study. The whole profile of 21 RV metabolites increased after acute and chronic consumption of the FB with respect to the control-placebo beverage and to the baseline. Urinary excretion of RV and piceid phase II metabolites was similar after both consumption periods, but a later formation of microbial metabolites required urine sampling of up to 24 h after the consumption of the FB. In addition, the intervariability has been evaluated. This study allows the knowledge of the RV metabolites that reach target tissues where biological activity would be achieved in order to elucidate the beneficial effects of this grape extract FB.

Modulation of estrogen and epidermal growth factor receptors by rosemary extract in breast cancer cells.

Breast cancer is the leading cause of cancer-related mortality among females worldwide, and therefore the development of new therapeutic approaches is still needed. Rosemary (Rosmarinus officinalis L.) extract possesses antitumor properties against tumor cells from several organs, including breast. However, in order to apply it as a complementary therapeutic agent in breast cancer, more information is needed regarding the sensitivity of the different breast tumor subtypes and its effect in combination with the currently used chemotherapy. Here, we analyzed the antitumor activities of a supercritical fluid rosemary extract (SFRE) in different breast cancer cells, and used a genomic approach to explore its effect on the modulation of ER-α and HER2 signaling pathways, the most important mitogen pathways related to breast cancer progression. We found that SFRE exerts antitumor activity against breast cancer cells from different tumor subtypes and the downregulation of ER-α and HER2 receptors by SFRE might be involved in its antitumor effect against estrogen-dependent (ER+) and HER2 overexpressing (HER2+) breast cancer subtypes. Moreover, SFRE significantly enhanced the effect of breast cancer chemotherapy (tamoxifen, trastuzumab, and paclitaxel). Overall, our results support the potential utility of SFRE as a complementary approach in breast cancer therapy.

Precision nutrition impact on metabolic health and quality of life in aging population after a 3-month intervention: A randomized intervention.

OBJECTIVES: Innovative precision dietary procedures are required to promote healthy aging. This study aimed to evaluate the effects of a personalised strategy based on the inclusion of individualised foods and digital tools on overall health status and quality of life within a follow-up of 3 months in older adults with overweight or obesity. METHODS: 127 men and women aged between 50 and 80 years with overweight/obesity participated in the study-between January 2020 and September 2020 at the Center for Nutrition Research-University of Navarra and IMDEA-ALIMENTACIÓN-and were randomly assigned to a usual-care group (standard recommendations) or precision group (precision nutrition strategy based on the inclusion of individualised foods and a mobile application). Anthropometry, body fat percentage, biochemical parameters, diet, and quality of life (SF-36 Health Survey) were assessed at baseline and after 3 months. RESULTS: Both strategies were found to improve overall metabolic health; however, the precision approach demonstrated significantly better outcomes. The precision strategy reduced body weight at 3 months (-4.3 kg; p < 0.001) with significant improvements in body fat percentage, blood pressure and general metabolic health (glycated haemoglobin; alanine aminotransferase; aspartate aminotransferase; hepatic steatosis index) in comparison with the standard recommendations. The precision approach significantly enhanced the quality of life (SF-36) of individuals, with additional improvements in emotional well-being (p = 0.024) and vitality (p = 0.008). Adherence to the Mediterranean diet was significantly associated with a higher quality of life and vitality. CONCLUSION: These results support the benefit of precision nutrition approaches for promoting healthy aging and emotional well-being, enhancing the quality of life in aging populations, during the COVID-19 pandemic.

Antitumor effect of 5-fluorouracil is enhanced by rosemary extract in both drug sensitive and resistant colon cancer cells.

5-Fluorouracil (5-FU) is the most used chemotherapeutic agent in colorectal cancer. However, resistance to this drug is relatively frequent, and new strategies to overcome it are urgently needed. The aim of this work was to determine the antitumor properties of a supercritical fluid rosemary extract (SFRE), alone and in combination with 5-FU, as a potential adjuvant therapy useful for colon cancer patients. This extract has been recognized as a healthy component by the European Food Safety Authority (EFSA). The effects of SFRE both alone and in combination with 5-FU were evaluated in different human colon cancer cells in terms of cell viability, cytotoxicity, and cell transformation. Additionally, colon cancer cells resistant to 5-FU were used to assay the effects of SFRE on drug resistance. Finally, qRT-PCR was performed to ascertain the mechanism by which SFRE potentiates the effect of 5-FU. Our results show that SFRE displays dose-dependent antitumor activities and exerts a synergistic effect in combination with 5-FU on colon cancer cells. Furthermore, SFRE sensitizes 5-FU-resistant cells to the therapeutic activity of this drug, constituting a beneficial agent against both 5-FU sensitive and resistant tumor cells. Gene expression analysis indicates that the enhancement of the effect of 5-FU by SFRE might be explained by the downregulation of TYMS and TK1, enzymes related to 5-FU resistance.

Study on the 3-hydroxy-3-methyl-glutaryl CoA reductase inhibitory properties of Agaricus bisporus and extraction of bioactive fractions using pressurised solvent technologies.

BACKGROUND: Agaricus bisporus mushrooms were able to lower cholesterol levels in hypercholesterolaemic rats and it was suggested that dietary fibre might inhibit cholesterol absorption. However, A. bisporus extracts were also able to inhibit the 3-hydroxy-3-methyl-glutaryl CoA reductase (HMGCR, the key enzyme in the cholesterol biosynthetic pathway) and this might also contribute to the observed lowering of cholesterol levels in serum. RESULTS: The methanol-water extracts obtained from A. bisporus were able to inhibit up to 60% the HMGCR activity using an in vitro assay. The HMGCR inhibitory capacities depended on cultivation conditions, strains, etc. The potential inhibitors were not statins, they might be ß-glucans able to scavenge the substrate and impair the enzymatic reaction. They were present during all mushroom developmental stages and similarly distributed through all the tissues including the parts discarded as a by-product. Accelerated solvent extractions using 1:1 ethanol-water as pressurised solvent (10.7 MPa, 25°C, five cycles of 5 min) were more effective in the extraction of the HMGCiR inhibitor(s) than supercritical fluid extractions (9 MPa, 40°C) using CO2 with 10% ethanol. CONCLUSION: A mushroom cultivation and two extraction procedures were optimised to obtain fractions from A. bisporus with high HMGCR inhibitory activities to design novel ingredients for hypocholesterolaemic functional foodstuffs.

Why proanthocyanidins elute at increasing order of molecular masses when analysed by normal phase high performance liquid chromatography? Considerations of use.

Although it is widely known that proanthocyanidins elute at an increasing order of molecular masses when analysed by normal phase high performance liquid chromatography (NP-HPLC), there is no a consistent explanation of the mechanisms of their separation until now. Therefore, the aim of the present study was to give a reliable response to this question, using a complex procyanidin-rich grape seed extract. For this, an off-column static simulation of extract injection and a fragmented-column dynamic procyanidin location tests were studied to show their precipitation in an aprotic solvent, besides another off-column static simulation and multiple contact dynamic solubilisation tests to confirm procyanidin redissolution in an aprotic/protic solvent system. The results showed that separation of procyanidins in the aprotic/protic solvent system of Diol-NP-HPLC was governed by precipitation/redissolution mechanism, that could be extended to all known plant proanthocyanidin homopolymers, including hydrolysable tannins, if they are able to accomplish this condition. However, separation of monomer species, namely catechins and some hydroxybenzoic acids, was based on classic adsorption/partition mechanism. Other factors, such as analyte solubility, chromatographic conditions and sample preparation, that affect the viability of proanthocyanidin analysis by NP-HPLC were stand out and guidelines for its durable and reproducible use were defined.