Patricia Morales

Tocopherol composition and antioxidant activity of Spanish wild vegetables

Traditional use of noncultivated vegetables has decreased with the development of agriculture and global supply chains. However, some species are still consumed as part of our traditional Mediterranean diet. Plants are among the most important sources of natural antioxidants for retarding lipid oxidative rancidity in foods or for pharmaceutical applications against chronic diseases related to free radicals production. The present study reports tocopherols composition and antioxidant activity of eight wild greens traditionally used in Spain. According to the edible part consumed, two groups were differentiated. Leafy vegetables whose young stems with leaves are consumed (Apium nodiflorum (L.) Lag., Foeniculum vulgare Mill., Montia fontana L. and Silene vulgaris (Moench) Garcke), and wild asparagus whose young shoots with leaf buds scarcely developed are eaten (Asparagus acutifolius L., Bryonia dioica Jacq., Humulus lupulus L. and Tamus communis L.). Among the leafy vegetables, Silene vulgaris and Apium nodiflorum presented the highest antioxidant capacity and antioxidants contents. Among the wild asparagus, the highest antioxidant capacity was obtained in Humulus lupulus.

A competitive enzyme-linked immunosorbent assay for detection of bovine milk in ovine and caprine milk and cheese using a monoclonal antibody against bovine beta-casein.

A competitive ELISA (enzyme-linked immunosorbent assay) was performed to detect and quantify bovine milk in ovine and caprine milk and cheese using a monoclonal antibody (AH4 MAb) against bovine beta-casein. Ovine or caprine milk and cheese containing bovine milk were added simultaneously with the AH4 MAb to the wells of a microtiter plate that had been previously sensitized with commercial bovine beta-casein. The bovine caseins in milk or cheese samples compete with the bovine beta-casein bound to the plate for the AH4 MAb binding sites. Further immunorecognition of AH4 MAb bound to the bovine beta-casein immobilized onto the plate was attained with rabbit anti-mouse immunoglobulin conjugated to peroxidase. Subsequent enzymic conversion of the substrate showed clear differences in absorbance values during assay of mixtures of ovine and caprine milk and cheese containing various amounts of bovine milk. The competitive ELISA developed in this work allows the quantitative detection of bovine milk in ovine and caprine milk and cheese samples in the range of 0.5 to 25% of substitution.

Fatty acids profiles of some Spanish wild vegetables

Polyunsaturated fatty acids play an important role in human nutrition, being associated with several health benefits. The analyzed vegetables, in spite of its low fat content, lower than 2%, present a high proportion of polyunsaturated fatty acids of n-3, n-6 and n-9 series, such as α-linolenic, linoleic and oleic acids, respectively. Wild edible plants contain in general a good balance of n-6 and n-3 fatty acids. The present study tries to contribute to the preservation and valorization of traditional food resources, studying the fatty acids profile of 20 wild vegetables by gas-liquid chromatography with flame ionization detection. Results show that species in which leaves are predominant in their edible parts have in general the highest polyunsaturated fatty acid/saturated fatty acid ratios: Rumex pulcher (5.44), Cichorium intybus (5.14) and Papaver rhoeas (5.00). Due to the low n-6/n-3 ratios of the majority of the samples, they can be considered interesting sources of n-3 fatty acids, especially those with higher total fat amount, such as Bryonia dioica, Chondrilla juncea or Montia fontana, with the highest contents of α-linolenic acid (67.78, 56.27 and 47.65%, respectively). The wild asparaguses of Asparagus acutifolius and Tamus communis stand out for their linoleic acid content (42.29 and 42.45%, respectively). All these features reinforce the interest of including wild plants in diet, as an alternative to the variety of vegetables normally used.

Myricetin, quercetin, (+)-catechin and (-)-epicatechin protect against N-nitrosamines-induced DNA damage in human hepatoma cells.

The aim of this study was to investigate the protective effect of myricetin, quercetin, (+)-catechin and (-)-epicatechin, against N-nitrosodibutylamine (NDBA) and N-nitrosopiperidine (NPIP)-induced DNA damage in human hepatoma cells (HepG2). DNA damage (strand breaks and oxidized purines/pyrimidines) was evaluated by the alkaline single-cell gel electrophoresis or Comet assay. (+)-Catechin at the lowest concentration (10 microM) showed the maximum reduction of DNA strand breaks (23%), the formation of endonuclease III (Endo III, 19-21%) and formamidopyrimidine-DNA glycosylase (Fpg, 28-40%) sensitive sites induced by NDBA or NPIP. (-)-Epicatechin also decreased DNA strand breaks (10 microM, 20%) and the oxidized pyrimidines/purines (33-39%) induced by NDBA or NPIP, respectively. DNA strand breaks induced by NDBA or NPIP were weakly reduced by myricetin at the lowest concentration (0.1 microM, 10-19%, respectively). Myricetin also reduced the oxidized purines (0.1 microM, 17%) and pyrimidines (0.1 microM, 15%) induced by NDBA, but not the oxidized pyrimidines induced by NPIP. Quercetin did not protect against NDBA-induced DNA damage, but it reduced the formation of Endo III and Fpg sensitive sites induced by NPIP (0.1 microM, 17-20%, respectively). In conclusion, our results indicate that (+)-catechin and (-)-epicatechin at the concentrations tested protect human derived cells against oxidative DNA damage effects of NDBA and NPIP. However, myricetin at the concentrations tested only protects human cells against oxidative DNA damage induced by NDBA and quercetin against oxidative DNA damage induced by NPIP.

Monoclonal antibody detection of porcine meat

A stable hybridoma cell line (DD9) has been produced secreting a monoclonal antibody specific for porcine muscle proteins. The DD9 monoclonal antibody (mAb) failed to show a significant cross-reactivity when tested against beef, horse, chicken, and soya proteins, as well as bovine caseins, gelatin, and bovine serum albumin. The DD9 mAb was further used in an indirect ELISA format for detection of defined amounts of porcine meat (1-100%) in beef and chicken meat mixtures immobilized on 96-well plates. Immunorecognition of monoclonal antibodies adsorbed to porcine meat was made with rabbit anti-mouse immunoglobulins conjugated to the enzyme horseradish peroxidase.

Wild Fragaria vesca L. fruits: a rich source of bioactive phytochemicals

Wild Fragaria vesca L. fruits were studied regarding nutritional and phytochemical compounds, and also antioxidant, antibacterial and biofilm formation inhibition activities. The fruits are good sources of carbohydrates (e.g., sucrose), soluble dietary fiber and polyunsaturated fatty acids, mainly linoleic and linolenic acids, as well as other components such as citric and succinic acids, and vitamins B9 and E (mainly γ-tocopherol). Significant amounts of soluble sugars, citric acid and some amounts of ascorbic acid, vitamins B9 and E (only α-tocopherol) were found also in the infusions. The hydromethanolic extracts revealed higher amounts of phenolic compounds, mainly ellagic acid derivatives and dihydroflavonol taxifolin-3-O-arabinofuranoside. Consistently, these extracts also showed higher antioxidant and antibacterial activities than the infusions, and were able to inhibit the formation of bacterial biofilms. Despite the lower content of bioactive compounds in the infusions compared to the fruits, both forms could be potentially applied in functional foods and/or nutraceuticals/pharmaceutical formulations.

Organosulfur compounds alone or in combination with vitamin C protect towards N-nitrosopiperidine- and N-nitrosodibutylamine-induced oxidative DNA damage in HepG2 cells.

The aim of this study was to investigate the protective effects of organosulfur compounds (OSCs) alone or in combination with vitamin C towards N-nitrosopiperidine (NPIP) and N-nitrosodibutylamine (NDBA)-induced oxidative DNA damage in the single cell gel electrophoresis (SCGE)/HepG2 assay. Diallyl sulfide (DAS) did not protect against NDBA-induced oxidized purines, but it reduced the oxidized purines induced by NPIP (1 microM, 29%). The formation of formamidopyridine-DNA glycosylase (Fpg) sensitive sites induced by NPIP or NDBA was prevented by dipropyl sulfide (DPS) at concentrations of 1-10 microM (55-24% and 66-15%, respectively). The maximum reduction of the formation of Fpg sensitive sites induced by NPIP was observed at the highest concentration of diallyl disulfide (DADS) (2.5 microM, 38%). However, the oxidative DNA damage induced by NDBA was strongly reduced by DADS at the lowest concentration tested (0.1 microM, 92%). The oxidative DNA damage induced by NPIP or NDBA was prevented by all the concentrations of dipropyl disulfide (DPDS) (0.1-2.5 microM, 59-80% and 51-64%, respectively). DADS and DPDS, in combination with vitamin C showed an overall protective effect towards the formation of Fpg sensitive sites induced by NPIP and NDBA. However, the contribution of OSCs to the protective effect found in combined experiments might not be relevant, because it could be caused by vitamin C alone. One feasible mechanism by which OSCs exert their protective effects towards N-nitrosamine-induced oxidative DNA damage could be by modulation of phase I and II enzyme activities. DADS and DPDS (0.1-2.5 microM) exerted greater inhibition on CYP2E1 and CYP2A6 activity than DAS and DPS (1-50 microM). However, DAS and DADS (1 microM) exerted greater inhibition on CYP1A1 activity than DPS and DPDS (1 microM). DAS/DPS (50 microM) and DADS (2.5 microM) exerted a moderate increase of UDP-glucuronyltransferase (UGT1A4) activity, whereas DPDS (2.5 microM) had the most pronounced effect.

Sandwich ELISA for detection of goats’ milk in ewes’ milk and cheese

A sandwich ELISA has been developed for the detection of defined amounts of goats’ milk (1–100%) in ewes’ milk and cheese. Polyclonal antibodies were raised in rabbits against goat caseins (GC). The resultant antibodies were affinity purified by immunoadsorption of the crude antiserum on to columns containing immobilized cow, ewe and goat caseins, followed by elution of the goats’ milk‐specific antibodies (anti‐GC) from the column containing the goat caseins. The anti‐GC antibodies bound to the wells of a microtiter plate were used to capture the goats’ caseins from milk or cheese mixtures. Further immunorecognition of the captured proteins was achieved with the same antibodies conjugated to biotin. ExtrAvidin‐peroxidase was used to detect biotinylated antibodies bound to their specific antigens. Subsequent enzymic conversion of the substrate gave clear absorbance differences when assaying mixtures of ewes’ milk and cheese containing variable amounts of goats’ milk.

Castanea sativa Mill. Flowers amongst the Most Powerful Antioxidant Matrices: A Phytochemical Approach in Decoctions and Infusions

Infusions and decoction of chestnut tree flowers have been used for different medical purposes, but their phytochemical profile and antioxidant activity are still mostly unknown. Herein, decoctions and infusions of flowers from the two most appreciated chestnut cultivars (longal and judia) in Trás-os-Montes, Portugal, were prepared and characterized with regard to their content in free sugars, organic acids, and phenolic compounds, such as flavonoids and hydrolyzable tannins, and their antioxidant activity. Overall, the decoction of the cultivar judia was the sample with both the highest quantity of flavonoids and antioxidant activity. The phenolic compound with the highest abundance in all samples was trigalloyl-HHDP-glucoside, followed by pentagalloyl glucoside. The sample with the highest quantity of total phenolic compounds was judia infusion, closely followed by longal decoction, which also gave the highest quantities of ellagitannins. Regarding sugars and organic acids, the profiles were more similar. These results corroborate ancestral claims of the health benefits of infusions and decoctions of chestnut flowers.

Mutagenicity and cytotoxicity of fruits and vegetables evaluated by the Ames test and 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay / Mutagenicidad y citotoxicidad de frutas y vegetales evaluadas por el test de Ames y el ensayo del bromuro de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolio (MTT)

The mutagenic and cytotoxic activity of the aqueous (H2O) and ethanolic (EtOH) extracts of fruits and vegetables were studied by the Ames test and the MTT assay. A significant mutagenic activity was found for three H2O extracts of broccoli (Brassica oleracea), carrot (Dacus carota) and licorice (Glycyrrhiza glabra) and for one EtOH extract of kiwi (Ananas sativus ) of the nine species analyzed. The frameshift tester strain TA98 was reverted by broccoli (2.4, 4.8 and 9.6 mg/mL; p < 0.01) and carrot (9.6 mg/mL; p < 0.001) aqueous extracts and by kiwi (9.6 mg/mL; p < 0.0001) EtOH extract, whereas strain TA100 was only sensitive to the mutagens of licorice H,O extract, within the nine fruits and vegetables tested. The mutagenic response of the extracts was not altered by the presence of S9 mix. Cytotoxicity was only found for three of the nine species tested. Percentage cytotoxic ac tivities at 4.8 mg/mL in pineapple (Actinidin diasinensis) and garlic (Allium sativum) H,O extracts were 77 and 91 %, respectively. Licorice EtOH extract was the only one that showed a cytotoxic activity at all of the concentrations used. The percentage of cytotoxic activity of licorice extract was 63% at 0.24 mg/mL and increased with increasing concentration of licorice up to 4.8 mg/mL. Thus, licorice (EtOH) extract was the most cytotoxic of the species tested.