Prooxidant effects of verbascoside, a bioactive compound from olive oil mill wastewater, on in vitro developmental potential of ovine prepubertal oocytes and bioenergetic/oxidative stress parameters of fresh and vitrified oocytes.

Verbascoside (VB) is a bioactive polyphenol from olive oil mill wastewater with known antioxidant activity. Oxidative stress is an emerging problem in assisted reproductive technology (ART). Juvenile ART is a promising topic because, in farm animals, it reduces the generation gap and, in human reproductive medicine, it helps to overcome premature ovarian failure. The aim of this study was to test the effects of VB on the developmental competence of ovine prepubertal oocytes and the bioenergetic/oxidative stress status of fresh and vitrified oocytes. In fresh oocytes, VB exerted prooxidant short-term effects, that is, catalase activity increase and uncoupled increases of mitochondria and reactive oxygen species (ROS) fluorescence signals, and long-term effects, that is, reduced blastocyst formation rate. In vitrified oocytes, VB increased ROS levels. Prooxidant VB effects in ovine prepubertal oocytes could be related to higher VB accumulation, which was found as almost one thousand times higher than that reported in other cell systems in previous studies. Also, long exposure times of oocytes to VB, throughout the duration of in vitro maturation culture, may have contributed to significant increase of oocyte oxidation. Further studies are needed to identify lower concentrations and/or shorter exposure times to figure out VB antioxidant effects in juvenile ARTs.

Synthesis and bioaccessibility of Fe-pheophytin derivatives from crude spinach extract.

Heme iron is recognized as a highly bioavailable source of iron suitable for treatment of iron deficiency anemia. However, the animal origin of purified heme limits its broad applicability due to religious, personal, and food safety issues. Development of chlorophyll-derived heme mimetics offers opportunities to expand current iron fortification strategies. The objective of this study was the synthesis of Fe-pheophytin (FePhe) derivatives from natural chlorophyll and subsequent evaluation of their digestive behavior and bioaccessibility in vitro. FePhe a and a' were synthesized from crude spinach extracts by treatment with 1.3 M iron(II)chloride and 0.25 M Na-acetate dissolved in glacial acetic acid at 80 degrees C for 30 min. FePhe-rich extracts (approximately 1 mM) were formulated into corn starch based test meals (7.5% lipid) and subjected to a 2-step in vitro digestion designed to simulate in vivo gastric and small intestinal conditions. Recovery of FePhe following digestion and transfer of FePhe and pheophytins (Phe) from test meal matrix to mixed micelles was assessed by RP C18-HPLC to determine the digestive stability and micellarization efficiency (bioaccessibility). FePhe a and a' derivatives were moderately stable to digestive conditions with recoveries of 52.3% and 58.7%, respectively. Residual Phe a was stable to digestion. Micellarization efficiency of FePhe a (4%) and a' (3.4%) was significantly (P < 0.05) lower than Phe a (25.8%) from test meals. While digestive stability and micellarization efficiency are limiting, the presence of lipophilic FePhe derivatives in mixed micelles suggests that these compounds would be available for subsequent absorption in the intestinal tract.

Interaction of environmental moisture with powdered green tea formulations: effect on catechin chemical stability.

Green tea and tea catechins must be stable in finished products to deliver health benefits; however, they may be adversely affected by tea processing/storage conditions and the presence of other components. The objective of this study was to determine the effects of storage relative humidity (RH) and addition of other ingredients on catechin stability in simulated dry beverage mixtures. Samples of green tea powder alone and mixed with sucrose, citric acid, and/or ascorbic acid were prepared and stored in desiccators at 22 degrees C and 0-85% RH for up to 3 months. Epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate were determined by high-performance liquid chromatography (HPLC). Formulation and the interaction of formulation and RH significantly promoted catechin degradation ( P < 0.0001). The chemical degradation of total and individual catechins in green tea powder formulations was significantly increased ( P < 0.0001) by exposure to increasing RH, and the degradation was exacerbated at > or = 58% RH by the presence of powdered citric acid and at > or = 75% RH by the presence of ascorbic acid. Catechins degraded the most in formulations containing both acids. Although catechin chemical stability was maintained at < or = 43% RH in all samples stored at 22 degrees C for 3 months, caking was observed in samples at these relative humidities. These results are the first to demonstrate that addition of other dry components to tea powders may affect catechin stability in finished dry blends and highlight the importance of considering the complex interplay between a multicomponent system and its environment for developing stable products.

Analysis of lycopene geometrical isomers in biological microsamples by liquid chromatography with coulometric array detection.

Methods of analysis for determining low quantities of lycopene cis-trans isomers in biological tissues are needed. Development of two liquid chromatography (LC) methods based on the polymeric C30 stationary phase equipped with coulometric electrochemical array detection (ED) is described. Separation of 13 lycopene isomers including prolycopene, (a novel tetra-cis-lycopene found in Tangerine tomatoes) was accomplished with both isocratic and gradient methods using different proportions of methanol, methyl tert.-butyl ether, water and 1 M ammonium acetate buffer. Carotenoids were detected at potential settings between 200 and 620 mV. Differences in generated current-voltage curves aided in tentative identification of trans carotenoid species and select cis isomers of lycopene. These methods were successfully applied in the analysis of small quantities of plasma, buccal mucosal cells, prostate and cervical tissues. Limits of detection for trans-lycopene by ED were found to be 50 fmol representing a 10- to 100-fold increase over conventional UV-Vis absorbance methods.

Assessment of degradation and intestinal cell uptake of carotenoids and chlorophyll derivatives from spinach puree using an in vitro digestion and Caco-2 human cell model.

Although numerous studies have demonstrated the health benefits of chlorophyll derivatives, information regarding the digestion, absorption, and metabolism of these phytochemicals is quite limited. To better understand the digestion of these pigments, green vegetables including fresh spinach puree (FSP), heat- and acid-treated spinach puree (HASP), and ZnCl(2)-treated spinach puree (ZnSP) were subjected to an in vitro digestion method which simulates both the gastric and small intestinal phases of the process. Native chlorophylls were converted to Mg-free pheophytin derivatives during digestion. Conversely, Zn-pheophytins were completely stable during the digestive process. Transfer of lipophilic chlorophyll derivatives, as well as the carotenoids lutein and beta-carotene, into the aqueous micellar fraction from the food matrix was quantified. Micellarization of total chlorophyll derivatives differed significantly (p < 0.05) for FSP (37.6%), HASP (17.2%), and ZnSP (8.7%). Micellarization of chlorophyll a derivatives was determined to be significantly more efficient than chlorophyll b derivatives in FSP and HASP (p < 0.01), but not in ZnSP (p > 0.05). Intestinal cell uptake of micellarized pigments was investigated using HTB-37 (parent) and clonal TC7 lines of human Caco-2 cells. Medium containing the pigment-enriched fraction generated during digestion was added to the apical surface of fully differentiated monolayers for 4 h. Pigments were then extracted from cells and analyzed by C18 HPLC with photodiode array detection. Both Caco-2 HTB-37 and TC7 clone cells accumulated 20-40% and 5-10% of micellarized carotenoid and chlorophyll derivatives, respectively. These results are the first to demonstrate uptake of chlorophyll derivatives by human intestinal cells and to support the potential importance of chlorophylls as health-promoting phytochemicals.

Cervical tissue and plasma concentrations of alpha-carotene and beta-carotene in women are correlated.

Results from epidemiologic studies suggest that a carotenoid-rich diet may reduce risk for cervical cancer, possibly by inhibiting the progression of cervical intraepithelial neoplasia, a preneoplastic lesion of the cervical tissue. Laboratory studies suggest that the mechanism may be linked to the metabolism of carotenoids to retinoic acid or retinoic acid-like compounds, which has been hypothesized to occur in the cervical tissue. The purpose of this study was to demonstrate the presence of provitamin A carotenoids in biopsied samples of this peripheral tissue in human subjects and to examine the relationship between baseline concentrations of these carotenoids in plasma and normal cervical tissue in subjects who were being evaluated for possible participation in a diet intervention trial. Subjects were 13 women aged 19-41 y. With the use of HPLC methodology, plasma concentrations of alpha-carotene, beta-carotene and beta-cryptoxanthin were determined with UV/visible light detection for plasma and electrochemical detection for cervical tissue. Relationships between plasma and cervical tissue were evaluated with Pearson correlation analysis. Adjusted for plasma cholesterol concentration, plasma alpha-carotene and beta-carotene were correlated with cervical tissue concentrations (r = 0.91, P < 0.001; r = 0.90, P < 0.001; respectively). Adjusted for plasma cholesterol concentration, plasma beta-cryptoxanthin tended to be correlated with cervical tissue concentrations (r = 0.62, P = 0.058). These findings suggest that plasma concentrations of alpha-carotene and beta-carotene are good predictors of cervical tissue concentrations of these compounds in human subjects and describe a first step toward demonstrating a biological link between provitamin A carotenoids and cervical cancer in vivo.

Carotenoid determination in biological microsamples using liquid chromatography with a coulometric electrochemical array detector.

Numerous epidemiological studies have linked carotenoids to cancer preventive processes, thereby increasing interest in levels of these micronutrients in human tissue and serum. Conventional analyses of these biological tissues employ liquid chromatography (LC) with ultraviolet and visible absorbance (UV-VIS) detection. However, this type of carotenoid analysis does not provide adequate sensitivity for very small sample sizes, such as microscale biopsies, when only small quantities of tissue are available. Electrochemical detection (ECD) is a useful alternative to conventional UV-VIS detection methods for LC analysis of carotenoids in cases where high sensitivity is necessary. Both hydrocarbon (beta-carotene and alpha-carotene) and oxygenated carotenoids (lutein, zeaxanthin, and beta-cryptoxanthin) were detected at electrical potential settings between 220 and 520 mV. The generated electrochemical array data (hydrodynamic voltammograms) can be used to identify carotenoids as well as to differentiate between trans and cis configurations. Detection limits for beta-carotene by ECD were measured at 10 fmol representing approximately a 100- to 1000-fold increase over conventional LC-UV-VIS techniques. The developed methodology was applied successfully to microscale analysis of biological tissues.