[Assessment of cytotoxicity and biosafety of polyphenolic extracts from olive pits]. / Evaluación de la citotoxicidad y bioseguridad de un extracto de polifenoles de huesos de aceitunas.

UNLABELLED: The olive tree is a source of bioactive compounds, both its fruit and its by-products. Some of its compounds have shown health benefits, being objective of this work the evaluation of biosafety in-vitro and in vivo of extracts of olive stones rich in polyphenols. MATERIAL AND METHODS: He has been evaluated for cytotoxicity by addition of lyophilized extract dissolved in PBS(0-400 mg/l) to a culture of the cell line THP1-XBlue- CD14 and evaluation of cell viability by the reaction of reduction of resazurin by living cells. Biosecurity has been evaluated in zebrafish, incubating eggs fertilized in 0 to 100 mg/l extract for 24 to 72 hours and measuring parameters: a) lethal (dead embryos, coagulated eggs), b) sublethal (spontaneous movements, pigmentation, edemas) and c) teratogenic (malformations, retardation development). RESULTS: Cytotoxicity (toxic effect when less than 75% viability) extract bones of olive in the cell line THP1- XBlue-CD14, is in concentrations higher than 50 mg/l, calculating a LD50 (dose lethality 50) more than 800 mg/l. The biosafety of zebrafish embryos exposed to concentrations of extract from 0-100 mg/l showed total viability at 24, 48 and 72 hours post fertilization (hpf), not observed mortality or appreciated embryos with sublethal effects, teratogenic, or advancement or delay in hatching. It can be concluded that the bones of olive extract is highly biosecured until at least 100 mg/l concentrations.

El olivo constituye una fuente de compuestos bioactivos, tanto en su fruto, como en sus subproductos. Algunos de sus compuestos han mostrado beneficios para la salud, siendo objetivo de este trabajo la evaluación de la bioseguridad in vitro e in vivo de extractos de huesos de aceituna ricos en polifenoles. Material y métodos: Se ha evaluado la citotoxicidad mediante adición de extracto de hueso de olivas disuelto en PBS(0-400 mg/l) a un cultivo de la línea celular THP1- XBlue-CD14 y evaluación de la viabilidad celular mediante la reacción de reducción de la resazurina por las células vivas. La bioseguridad se ha evaluado en pez cebra, incubando huevos fecundados en extracto de 0 a 100 mg/l durante 24 a 72 horas y midiendo los parámetros: a) letales (embriones muertos, huevos coagulados), b) subletales (movimientos espontáneos, pigmentación, edemas) y c) teratogénicos (malformaciones, retraso desarrollo). Resultados: La citotoxicidad (efecto tóxico cuando viabilidad inferior al 75%) del extracto de huesos de oliva en la línea celular THP1-XBlue-CD14, está en concentraciones superiores a 50 mg/l de extracto (viabilidad 77,5%), calculando una LD50 (dosis de letalidad 50%) superior a 800 mg/l. La bioseguridad in vivo con los embriones de pez cebra expuestos a concentraciones de extracto de 0- 100 mg/l mostró total viabilidad a 24, 48 y 72 horas post fecundación (hpf), no observándose mortalidad ni se apreciaron embriones con efectos subletales, teratógenos, ni adelanto o retraso en la eclosión. Se puede concluir que el extracto de huesos de olivas es altamente bioseguro hasta al menos concentraciones de 100 mg/l.

Evaluación de la citotoxicidad y bioseguridad de un extracto de polifenoles de huesos de aceitunas / Assessment of cytotoxicity and biosafety of polyphenolic extracts from olive pits

El olivo constituye una fuente de compuestos bioactivos, tanto en su fruto, como en sus subproductos. Algunos de sus compuestos han mostrado beneficios para la salud, siendo objetivo de este trabajo la evaluación de la bioseguridad in vitro e in vivo de extractos de huesos de aceituna ricos en polifenoles. Material y métodos: Se ha evaluado la citotoxicidad mediante adición de extracto de hueso de olivas disuelto en PBS (0-400 mg/l) a un cultivo de la línea celular THP1- XBlue-CD14 y evaluación de la viabilidad celular mediante la reacción de reducción de la resazurina por las células vivas. La bioseguridad se ha evaluado en pez cebra, incubando huevos fecundados en extracto de 0 a 100 mg/l durante 24 a 72 horas y midiendo los parámetros: a) letales (embriones muertos, huevos coagulados), b) subletales (movimientos espontáneos, pigmentación, edemas) y c) teratogénicos (malformaciones, retraso desarrollo). Resultados: La citotoxicidad (efecto tóxico cuando viabilidad inferior al 75%) del extracto de huesos de oliva en la línea celular THP1-XBlue-CD14, está en concentraciones superiores a 50 mg/l de extracto (viabilidad 77,5%), calculando una LD50 (dosis de letalidad 50%) superior a 800 mg/l. La bioseguridad in vivo con los embriones de pez cebra expuestos a concentraciones de extracto de 0- 100 mg/l mostró total viabilidad a 24, 48 y 72 horas post fecundación (hpf), no observándose mortalidad ni se apreciaron embriones con efectos subletales, teratógenos, ni adelanto o retraso en la eclosión. Se puede concluir que el extracto de huesos de olivas es altamente bioseguro hasta al menos concentraciones de 100 mg/l (AU)

The olive tree is a source of bioactive compounds, both its fruit and its by-products. Some of its compounds have shown health benefits, being objective of this work the evaluation of biosafety in-vitro and in vivo of extracts of olive stones rich in polyphenols. Material and methods: He has been evaluated for cytotoxicity by addition of lyophilized extract dissolved in PBS (0-400 mg/l) to a culture of the cell line THP1-XBlueCD14 and evaluation of cell viability by the reaction of reduction of resazurin by living cells. Biosecurity has been evaluated in zebrafish, incubating eggs fertilized in 0 to 100 mg/l extract for 24 to 72 hours and measuring parameters: a) lethal (dead embryos, coagulated eggs), b) sublethal (spontaneous movements, pigmentation, edemas) and c) teratogenic (malformations, retardation development). Results: Cytotoxicity (toxic effect when less than 75% viability) extract bones of olive in the cell line THP1- XBlue-CD14, is in concentrations higher than 50 mg/l, calculating a LD50 (dose lethality 50) more than 800 mg/l. The biosafety of zebrafish embryos exposed to concentrations of extract from 0-100 mg/l showed total viability at 24, 48 and 72 hours post fertilization (hpf), not observed mortality or appreciated embryos with sublethal effects, teratogenic, or advancement or delay in hatching. It can be concluded that the bones of olive extract is highly biosecured until at least 100 mg/l concentrations (AU)

Olive oil mill wastewater toxicity in the marine environment: alterations of stress indices in tissues of mussel Mytilus galloprovincialis.

This study investigated the impact of olive mill wastewater (OMW) as a pollutant of the marine environment, via the detection of stress indice alterations in mussels Mytilus galloprovincialis. Due to the absence of data concerning the levels of OMW in the receiving waters, mortality test (96h) was first performed in order to estimate the range of OMW concentration where no mortality occurs. OMW concentrations ranging from 0.01 to 0.1% (v/v) showed no increased mortality and thus were used for the determination of pre-pathological alterations in tissues of mussels. In particular, mussels exposed to either 0.1 or 0.01% (v/v) OMW for 5 days showed significant alterations of stress indices in their tissues. Specifically, decreased neutral red retention (NRR) assay time values, inhibition of acetylcholinesterase (AChE) activity, as well as a significant increase of micronucleus (MN) frequency and DNA damage were detected in haemolymph/haemocytes and gills, compared with values measured in tissues of control mussels. The results of the present study showed that OMW disposal into the marine environment could induce pre-pathological alterations in marine organisms, before severe disturbances, such as disease, mortality, or population changes occur.

Enzyme and fungal treatments and a combination thereof reduce olive mill wastewater phytotoxicity on Zea mays L. seeds.

The phytotoxicity of olive-mill wastewater (OMW) has been suggested to be mainly due to its phenolic components. This study investigated the impact of three different low-cost dephenolization treatments on the wastewater phytotoxicity. To this aim, germinability of maize (Zea mays L.) seeds sown on a sandy-loamy soil which had been spread with different volumes (from 40 to 160m(3)ha(-1)) of either biologically-treated OMW or relative incubation control was determined. Biological treatments included either Panus tigrinus liquid cultures or incubation with commercial laccase (1UIml(-1)) or an innovative sequential combination of laccase and P. tigrinus cultures. All treatments markedly reduced phytotoxicity and promising results were obtained with commercial laccase. In fact, germinability and mean germination times in soil spread with laccase-treated OMW, did not significantly differ from those observed in soil irrigated with tap water (control) up to OMW volumes of 120m(3)ha(-1). Although the highest phenol reduction (ca. 81%) was obtained by the sequential use of laccase and P. tigrinus, the feasibility of the enzyme treatment is undoubtedly more convincing under the technological point of view.

In vitro antifungal and anti-elastase activity of some aliphatic aldehydes from Olea europaea L. fruit.

Olea europaea preparations are traditionally employed in a variety of troubles, including skin infections. Olive extracts and some of their pure compounds have shown antimicrobial activity in vitro. The present study deals with the antifungal activity of some aliphatic aldehydes from olive fruit [hexanal, nonanal, (E)-2-hexenal, (E)-2-heptenal, (E)-2-octenal, (E)-2-nonenal] against Tricophyton mentagrophytes (6 strains), Microsporum canis (1 strains) and Candida spp. (7 strains). The capability of these substances to inhibit elastase, a virulence factor essential for the dermatophytes colonization, and their cytotoxicity on cultures of reconstructed human epidermis, are also described. Aldehydes tested, inhibited the growth of T. mentagrophytes and M. canis in the range of concentration between <1.9 and 125 microg/ml; the unsaturated aldehydes showed the most broad spectrum of activity in that inhibited all strains tested. None of the aldehydes exhibited activity against Candida spp. strains. (E)-2-octenal and (E)-2-nonenal inhibited the elastase activity in a concentration-dependent manner; the anti-elastase activity suggests an additional target of the antimicrobial activity of these compounds. Aldehydes were devoid of cytotoxicity on cultures of human reconstructed epidermis. The antifungal activity of the aldehydes from olive fruit here reported, substantiates the use of olive and olive oil in skin diseases and suggests that these natural compounds could be useful agents in the topical treatment of fungal cutaneous infections.

Detrimental effects of olive mill wastewater on the composting process of agricultural wastes.

In order to study the suitability of composting olive mill wastewater (OMW-L) by repeated applications, OMW-L was added to one mixture of lawn trimmings and olive husks as bulking agents. The composting process of this mixture was compared with another pile having 35% of olive mill wastewater sludge (OMW-S) obtained from evaporation ponds and a third, as a control, without olive mill wastewater. The repeated applications of OMW-L resulted in a sharp decrease in respiration measurements after the first 20 days of composting and showed a re-increase after 40 days following the substituting of OMW-L by water. The OMW-L addition increased the rate of water-soluble phenols in the compost and caused the appearance of a phenol fraction of high molecular-mass (510 kDa) at the end of composting. OMW-L addition also caused a clear decrease in both thermophilic bacteria and thermophilic eumycete counts. A longer persistence of phytotoxicity was observed in comparison with the other piles. However, the OMW-S produced a compost with a high degree of maturity.

Contribution of hydrolytic enzymes produced by saprophytic fungi to the decrease in plant toxicity caused by water-soluble substances in olive mill dry residue.

We studied the influence of saprophytic fungi on the toxic effect that the water-soluble substances in dry residues from olive (ADOR) have on the growth of plants. All saprophytic fungi were able to decrease the phytotoxicity of ADOR, although the toxicity of this residue did not decrease in the same way. Penicillium chrysogenum was able to reduce the toxicity of ADOR when this residue was applied at the highest dose of 15%. Fusarium lateritum, F. graminearum and Mucor racemosus were able to reduce the toxicity of ADOR when this residue was applied at the intermediate doses. However, F. oxysporum decreased the phytotoxicity of ADOR only when the residue was applied at the lowest dose of 2.5%. All saprophytic fungi tested produce endoglucanase, endopolymetylgalacturonase and endoxiloglucanase when grown in the presence of ADOR. A close relationship was found between the decrease in the phytotoxicity of ADOR and the amount of hydrolytic enzymes produced by the saprophytic fungi. These results shows that hydrolytic enzymes can be important in the degradation of phytotoxic substances present in olive mill dry residue.