Complementarity of Raman and Infrared spectroscopy for rapid characterization of fucoidan extracts.

BACKGROUND: Fucoidans are sulfated polysaccharides from the cell-wall of brown algae. They have a wide range of applications in medicine, including regenerative medicine, ophthalmology, cancer, and autoimmune disease. Biological activity of fucoidans directly depends on their structure, which remains poorly understood. This is primarily because the polymeric nature of these molecules limits the use of nuclear magnetic resonance and mass spectrometry, classical tools of structural biology for their structural characterization. Raman and Infrared spectroscopies are non-invasive and non-destructive techniques that can be used to probe the structural organization of biological specimens. In this study, we investigate the potential of Raman and Infrared spectroscopy for structural analysis of several fucoidan extracts. RESULTS: Our results show that Infrared and Raman provide different but complimentary information about the structure of crude extracts of fucoidans, revealing the presence of minor impurities from co-extractants. We also found that at high extraction temperatures acidic conditions limit formation of melanoidins, while also yielding relatively high sulfate ester fucoidan. However, at high temperatures, water extraction may potentially result in formation of advanced glycation end products. Their presence could be problematic for fucoidan extracts intended for medicinal use, as advanced glycation end products have been linked to endocrine interruption mechanisms in vivo by crosslinking to and permanently altering extracellular matrix proteins. CONCLUSION: Raman and Infrared can be used as complementary tools for rapid screening of crude fucoidan extracts, which can be a valuable tool for assessing impurities that remain after extraction.

Evaluation of the Effects of Fucoidans from Fucus Species and Laminaria hyperborea against Oxidative Stress and Iron-Dependent Cell Death.

Fucoidans are algal polysaccharides that exhibit protective properties against oxidative stress. The aim of this study was to investigate different fucoidans from brown seaweeds for their ability to protect against iron-dependent oxidative stress (ferroptosis), a main hallmark of retinal and brain diseases, including hemorrhage. We investigated five new high-molecular weight fucoidan extracts from Fucus vesiculosus, F. serratus, and F. distichus subsp. evanescens, a previously published Laminaria hyperborean extract, and commercially available extracts from F. vesiculosus and Undaria pinnatifida. We induced oxidative stress by glutathione depletion (erastin) and H2O2 in four retinal and neuronal cell lines as well as primary cortical neurons. Only extracts from F. serratus, F. distichus subsp. evanescens, and Laminaria hyperborea were partially protective against erastin-induced cell death in ARPE-19 and OMM-1 cells, while none of the extracts showed beneficial effects in neuronal cells. Protective fucoidans also attenuated the decrease in protein levels of the antioxidant enzyme GPX4, a key regulator of ferroptosis. This comprehensive analysis demonstrates that the antioxidant abilities of fucoidans may be cell type-specific, besides depending on the algal species and extraction method. Future studies are needed to further characterize the health-benefiting effects of fucoidans and to determine the exact mechanism underlying their antioxidative abilities.

Influence of Fucoidan Extracts from Different Fucus Species on Adult Stem Cells and Molecular Mediators in In Vitro Models for Bone Formation and Vascularization.

Fucoidans, sulfated polysaccharides extracted from brown algae, are marine products with the potential to modulate bone formation and vascularization processes. The bioactivity and safety of fucoidans are highly associated with their chemical structure, which may vary with algae species and extraction method. Thus, in depth evaluation of fucoidan extracts in terms of endotoxin content, cytotoxicity, and their detailed molecular biological impact on the individual cell types in bone is needed. In this study, we characterized fucoidan extracts from three different Fucus species including Fucus vesiculosus (Fv), Fucus serratus (Fs), and Fucus distichus subsp. evanescens (Fe) for their chemical features, endotoxin content, cytotoxicity, and bioactive effects on human outgrowth endothelial cells (OEC) and human mesenchymal stem cells (MSC) as in vitro models for bone function and vascularization. Extracts contained mainly high molecular weight (HMW) fucoidans and were free of endotoxins that may cause inflammation or influence vascularization. OEC tolerated fucoidan concentrations up to 200 µg/mL, and no indication of cytotoxicity was observed. The inflammatory response, however, investigated by real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) and endothelial barrier assessed by impedance measurement differed for the individual extracts. MSC in comparison with endothelial cells were more sensitive to fucoidans and showed partly reduced metabolic activity and proliferation at higher doses of fucoidans. Further results for MSC indicated impaired osteogenic functions in alkaline phosphatase and calcification assays. All tested extracts consistently lowered important molecular mediators involved in angiogenesis, such a VEGF (vascular endothelial growth factor), ANG-1 (angiopoietin 1), and ANG-2 (angiopoietin 2), as indicated by RT-PCR and ELISA. This was associated with antiangiogenic effects at the functional level using selected extracts in co-culture models to mimic bone vascularization processes during bone regeneration or osteosarcoma.

The Permeation of Acamprosate Is Predominantly Caused by Paracellular Diffusion across Caco-2 Cell Monolayers: A Paracellular Modeling Approach.

In drug development, estimating fraction absorbed (Fa) in man for permeability-limited compounds is important but challenging. To model Fa of such compounds from apparent permeabilities (Papp) across filter-grown Caco-2 cell monolayers, it is central to elucidate the intestinal permeation mechanism(s) of the compound. The present study aims to refine a computational permeability model to investigate the relative contribution of paracellular and transcellular routes to the Papp across Caco-2 monolayers of the permeability-limited compound acamprosate having a bioavailability of ∼11%. The Papp values of acamprosate and of several paracellular marker molecules were measured. These Papp values were used to refine system-specific parameters of the Caco-2 monolayers, that is, paracellular pore radius, pore capacity, and potential drop. The refined parameters were subsequently used as an input in modeling the permeability (Pmodeled) of the tested compounds using mathematical models collected from two published permeability models. The experimental data show that acamprosate Papp across Caco-2 monolayers is low and similar in both transport directions. The obtained acamprosate Papp, 1.56 ± 0.28 × 10-7 cm·s-1, is similar to the Papp of molecular markers for paracellular permeability, namely, mannitol (2.72 ± 0.24 × 10-7 cm·s-1), lucifer yellow (1.80 ± 0.35 × 10-7 cm·s-1), and fluorescein (2.10 ± 0.28 × 10-7 cm·s-1), and lower than that of atenolol (7.32 ± 0.60 × 10-7 cm·s-1; mean ± SEM, n = 3-6), while the end-point amount of acamprosate internalized by the cell monolayer, Qmonolayer, was lower than that of mannitol. Acamprosate did not influence the barrier function of the monolayers since it altered neither the Papp of the three paracellular markers nor the transepithelial electrical resistance (TEER) of the cell monolayer. The Pmodeled for all the paracellular markers and acamprosate was dominated by the Ppara component and matched the experimentally obtained Papp. Furthermore, acamprosate did not inhibit the uptake of probe substrates for solute carriers PEPT1, TAUT, PAT1, EAAT1, B0,+AT/rBAT, OATP2B1, and ASBT expressed in Caco-2 cells. Thus, the Pmodeled estimated well Ppara, and the paracellular route appears to be the predominant mechanism for acamprosate Papp across Caco-2 monolayers, while the alternative transcellular routes, mediated by passive diffusion or carriers, are suggested to only play insignificant roles.

Combined bioavailable isoflavones and probiotics improve bone status and estrogen metabolism in postmenopausal osteopenic women: a randomized controlled trial.

Background: Female age-related estrogen deficiency increases the risk of osteoporosis, which can be effectively treated with the use of hormone replacement therapy. However, hormone replacement therapy is demonstrated to increase cancer risk. Bioavailable isoflavones with selective estrogen receptor affinity show potential to prevent and treat osteoporosis while minimizing or eliminating carcinogenic side effects.Objective: In this study, we sought to determine the beneficial effects of a bioavailable isoflavone and probiotic treatment against postmenopausal osteopenia.Design: We used a novel red clover extract (RCE) rich in isoflavone aglycones and probiotics to concomitantly promote uptake and a favorable intestinal bacterial profile to enhance isoflavone bioavailability. This was a 12-mo, double-blind, parallel design, placebo-controlled, randomized controlled trial of 78 postmenopausal osteopenic women supplemented with calcium (1200 mg/d), magnesium (550 mg/d), and calcitriol (25 µg/d) given either RCE (60 mg isoflavone aglycones/d and probiotics) or a masked placebo [control (CON)].Results: RCE significantly attenuated bone mineral density (BMD) loss at the L2-L4 lumbar spine vertebra (P < 0.05), femoral neck (P < 0.01), and trochanter (P < 0.01) compared with CON (-0.99% and -2.2%; -1.04% and -3.05%; and -0.67% and -2.79, respectively). Plasma concentrations of collagen type 1 cross-linked C-telopeptide was significantly decreased in the RCE group (P < 0.05) compared with CON (-9.40% and -6.76%, respectively). RCE significantly elevated the plasma isoflavone concentration (P < 0.05), the urinary 2-hydroxyestrone (2-OH) to 16α-hydroxyestrone (16α-OH) ratio (P < 0.05), and equol-producer status (P < 0.05) compared with CON. RCE had no significant effect on other bone turnover biomarkers. Self-reported diet and physical activity were consistent and differences were nonsignificant between groups throughout the study. RCE was well tolerated with no adverse events.Conclusions: Twice daily RCE intake over 1 y potently attenuated BMD loss caused by estrogen deficiency, improved bone turnover, promoted a favorable estrogen metabolite profile (2-OH:16α-OH), and stimulated equol production in postmenopausal women with osteopenia. RCE intake combined with supplementation (calcium, magnesium, and calcitriol) was more effective than supplementation alone. This trial was registered at clinicaltrials.gov as NCT02174666.

Dietary polyacetylenes, falcarinol and falcarindiol, isolated from carrots prevents the formation of neoplastic lesions in the colon of azoxymethane-induced rats.

Falcarinol (FaOH) and falcarindiol (FaDOH) are found in many food plants of the Apiaceae family. Carrots are a major dietary source of these polyacetylenes. Feeding azoxymethane (AOM)-induced rats with carrots and purified FaOH have previously been shown to inhibit neoplastic transformations in the colon. FaOH and FaDOH have also shown to have a synergistic effect in vitro, resulting in a significant increased cytotoxic activity. Based on these findings the antineoplastic effect of FaOH and FaDOH (purity > 99%) was investigated in the AOM-induced rat model. Twenty rats received rat diet containing 7 µg FaOH per g feed and 7 µg FaDOH per g feed and 20 rats were controls receiving only rat diet. Then carcinogenesis was induced in all 40 rats with the carcinogen AOM. All animals received the designated diet for 2 weeks before AOM induction and continued on the designated diet throughout the experiment. Rats were euthanized 18 weeks after the first AOM injection and macroscopic polyp/cancers were measured, harvested and stained for histology. The difference in sizes of aberrant crypt foci (ACF) were analysed in a Wilcoxon rank sum test, in which the median number of small ACF was 218 in controls and 145 in polyacetylene treated rats (P < 0.001). Fifteen control rats and 8 treated rats had macroscopic tumors (P = 0.027). The number of tumors larger than 3 mm were 6 and 1 in control and treated rats, respectively (P = 0.032). In conclusion dietary supplements with FaOH and FaDOH reduced the number of neoplastic lesions as well as the growth rate of the polyps suggesting a preventive effect of FaOH and FaDOH on the development of colorectal cancer.

A safflower oil based high-fat/high-sucrose diet modulates the gut microbiota and liver phospholipid profiles associated with early glucose intolerance in the absence of tissue inflammation.

SCOPE: Omega-6 (n-6) PUFA-rich diets are generally considered obesogenic in rodents. Here, we examined how long-term intake of a high-fat/high-sucrose (HF/HS) diet based on safflower oil affected metabolism, inflammation, and gut microbiota composition. METHODS AND RESULTS: We fed male C57BL/6J mice a HF/HS diet based on safflower oil-rich in n-6 PUFAs-or a low-fat/low-sucrose diet for 40 wk. Compared to the low-fat/low-sucrose diet, intake of the safflower-based HF/HS diet only led to moderate weight gain, while glucose intolerance developed at week 5 prior to signs of inflammation, but concurrent with increased levels of linoleic acid and arachidonic acid in hepatic phospholipids. Intake of the HF/HS diet resulted in early changes in the gut microbiota, including an increased abundance of Blautia, while late changes coincided with altered inflammatory profiles and increased fasting plasma insulin. Analysis of immune cells in visceral fat and liver revealed no differences between diets before week 40, where the number of immune cells decreased in the liver of HF/HS-fed mice. CONCLUSION: We suggest that a diet-dependent increase in the n-6 to omega-3 (n-3) PUFA ratio in hepatic phospholipids together with gut microbiota changes contributed to early development of glucose intolerance without signs of inflammation.

Guaianolides and a seco-Eudesmane from the Resinous Exudates of Cushion Bush (Leucophyta brownii) and Evaluation of Their Cytostatic and Anti-inflammatory Activity.

A detailed phytochemical investigation of a dichloromethane extract of the resinous exudates of the cushion bush plant (Leucophyta brownii) resulted in the isolation of the new 8,12-guaianolides leucophytalins A (5) and B (6), the new 1,10-seco-eudesmane leucophytalin C (10), six rare 8,12-guaianolides (1-4, 7, and 8), and the xanthanolide tomentosin (9). The structures of all isolated compounds were elucidated on the basis of spectroscopic and spectrometric analyses. The structures of compounds isolated in crystalline form, including leucophytalins A and C, were further confirmed by X-ray crystallography. The crude extract exhibited moderate cytostatic activity against a breast cancer (MCF-7) and human colon cancer (HT-29) cell line with IC50 values of 9.3 and 18 µg/mL, respectively, and anti-inflammatory activity against the macrophage-like cell line RAW 264.7 with IC50 values of 3.9 and 6.1 µg/mL for thromboxane B2 and prostaglandin E2 production, respectively. The isolated compounds were evaluated for their cytostatic activity against MCF-7 and HT-29 cells (1, 3-10) and their anti-inflammatory activity against RAW 264.7 cells (1-10). All isolated compounds are most likely derived from (+)-germacrene A, and a biosynthetic pathway is proposed for these sesquiterpenoids.

Spectral effects of supplementary lighting on the secondary metabolites in roses, chrysanthemums, and campanulas.

To investigate the effect of the light spectrum on photosynthesis, growth, and secondary metabolites Rosa hybrida 'Scarlet', Chrysanthemum morifolium 'Coral Charm', and Campanula portenschlagiana 'BluOne' were grown at 24/18°C day/night temperature under purpose-built LED arrays yielding approximately 200 µmol m(-2)s(-1) at plant height for 16 h per day. The four light treatments were (1) 40% Blue/60% Red, (2) 20% Blue/80% Red, (3) 100% Red, and (4) 100% White (Control). The plant height was smallest in 40% Blue/60% Red in roses and chrysanthemums, while the biomass was smallest in the white control in roses and in 100% Red in chrysanthemums. The total biomass was unaffected by the spectrum in campanulas, while the leaf area was smallest in the 40% Blue/60% Red treatment. In 100% Red curled leaves and other morphological abnormalities were observed. Increasing the blue to red ratio increased the stomatal conductance though net photosynthesis was unaffected, indicating excess stomatal conductance in some treatments. With higher blue light ratio all phenolic acids and flavonoids increased. In view of the roles of these secondary metabolites as antioxidants, anti-pathogens, and light protectants, we hypothesize that blue light may predispose plants to better cope with stress.

Systemic allergic dermatitis caused by Apiaceae root vegetables.

BACKGROUND: Immediate hypersensitivity reactions to root vegetables of the Umbelliferae plant family (Apiaceae) is well known. Delayed-type hypersensitivity is rarely reported. OBJECTIVE: To report the first case of systemic contact dermatitis caused by root vegetables and some chemical implications. MATERIALS AND METHODS: Prick and patch testing were performed with fresh vegetables and selected allergens, and this was followed by high-performance liquid chromatography-mass spectrometry (MS)/MS analysis of the falcarinol syringe. RESULTS: The patient was contact-sensitive to celeriac, parsnip, and carrot, but tested negative to falcarinol. Subsequent analysis showed that the syringe contained falcarinol. CONCLUSION: The non-occupational sensitization resulting from both direct and systemic contact with Apiaceae root vegetables was apparently not caused by falcarinol.

Chitosan oligosaccharides promote the content of polyphenols in Greek oregano (Origanum vulgare ssp. hirtum).

Greek oregano is commonly used as a spice and in traditional medicine in Eurasia. The plant is rich in secondary metabolites, such as volatile organic compounds (VOC) and polyphenols. Chitosan oligosaccharides (COS) are used as a plant elicitor. The objectives of this study were to determine the effects of COS on the growth and content of secondary metabolites in Greek oregano. Four COS treatments (50, 200, 500, and 1000 ppm) were used in a field experiment. The 200 and 500 ppm COS treatments promoted plant height growth, whereas 50 and 200 ppm COS upregulated the content of polyphenols significantly (38 and 29%, respectively). The COS treatments induced H(2)O(2) generation in Greek oregano leaves; thus, the effect of H(2)O(2) treatment was studied to investigate the possible role of H(2)O(2) in growth and polyphenol production. A low concentration of H(2)O(2) also promoted plant height growth, but only tendencies to higher polyphenol content were seen.

Estrogenic activity of bovine milk high or low in equol using immature mouse uterotrophic responses and an estrogen receptor transactivation assay.

BACKGROUND: Cow's milk contain phytoestrogens especially equol depending on the composition of the feed ration. However, it is unknown whether milk differing in equol exhibits different estrogenicity in model systems and thereby potentially in humans as milk consumers. METHODS: The estrogenicity of high and low equol milk (HEM and LEM, respectively) and purified equol was investigated in immature female mice including mRNA expression of six estrogen-sensitive genes in uterine tissue. Extracts of HEM and LEM were also tested for estrogenicity in vitro in an estrogen receptor (ER) reporter gene assay with MVLN cells. RESULTS: The total content of phytoestrogens was approximately 10 times higher in HEM compared with LEM, but levels of endogenous milk estrone and 17beta-estradiol were similar in the two milk types (503-566 and 60-64.6pg/ml, respectively). There was no difference in uterine weight between mice receiving LEM and HEM, and no difference from controls. Equol (50 times the concentration in HEM) was not uterotrophic. The ERbeta mRNA expression was down-regulated in the uteri of HEM mice compared with LEM and controls, but there was no difference between milk types for any of the other genes. Extracts of HEM showed a higher estrogenicity than extracts of LEM in MVLN cells, and there was a dose-dependent increase in estrogenicity by equol. CONCLUSION: The higher in vitro estrogenicity of HEM was not reflected as a higher uterine weight in vivo although the down-regulation of ERbeta in uterine tissue of HEM mice could suggest some estrogenic activity of HEM at the gene expression level.

NMR-based metabonomic studies reveal changes in the biochemical profile of plasma and urine from pigs fed high-fibre rye bread.

This study presents an NMR-based metabonomic approach to elucidate the overall endogenous biochemical effects of a wholegrain diet. Two diets with similar levels of dietary fibre and macronutrients, but with contrasting levels of wholegrain ingredients, were prepared from wholegrain rye (wholegrain diet (WGD)) and non-wholegrain wheat (non-wholegrain diet (NWD)) and fed to four pigs in a crossover design. Plasma samples were collected after 7 d on each diet, and 1H NMR spectra were acquired on these. Partial least squares regression discriminant analysis (PLS-DA) on spectra obtained for plasma samples revealed that the spectral region at 3.25 parts per million dominates the differentiation between the two diets, as the WGD is associated with higher spectral intensity in this region. Spiking experiments and LC-MS analyses of the plasma verified that this spectral difference could be ascribed to a significantly higher content of betaine in WGD plasma samples compared with NWD samples. In an identical study with the same diets, urine samples were collected, and 1H NMR spectra were acquired on these. PLS-DA on spectra obtained for urine samples revealed changes in the intensities of spectral regions, which could be ascribed to differences in the content of betaine and creatine/creatinine between the two diets, and LC-MS analyses verified a significantly lower content of creatinine in WGD urine samples compared with NWD urine samples. In conclusion, using an explorative approach, the present studies disclosed biochemical effects of a wholegrain diet on plasma betaine content and excretion of betaine and creatinine.