Influence of olive polyphenols on glucose and cholesterol levels in medaka fish / Influencia de los polifenoles del olivo en los niveles de glucosa y colesterol en el pez medaka

BACKGROUND: Polyphenol-rich olive extracts are non-toxic and have anti-inflammatory, neuroprotective and antiadipogenic effects in cell and animal models. OBJECTIVE: To evaluate the potential influence of olive extracts on the mechanisms of digestion and absorption of polysaccharides and fats by quantifying amylase, glucose, phospholipase, and cholesterol in the medaka fish model. MATERIAL AND METHODS: For each assay, six adult fish were placed in a tank with an extract (0.01% concentration), performing three replicates per extract. A control group with standard feeding was used. The same procedure was followed to study glucose, adding a polysaccharide-rich diet and a corresponding overfed control. The fish were maintained under these conditions for five days. Five olive extracts were used without attempting to purify the polyphenols due to possible synergistic effects. Total concentrations were between 2-116mg/g (mainly oleuropein and hydroxytyrosol). On completion, amylase, phospholipase A2, glucose and cholesterol were quantified in each group. All assays were conducted in triplicate. Enzyme activities were also studied in juveniles. Non-parametric tests were used to determine possible differences, considering p < 0.05 to denote statistical significance. RESULTS: Polyphenol extracts were not toxic at a concentration of 0.1%, ten times higher than the concentration used. An overall decrease in glucose levels was observed in fish overfed with carbohydrates with the addition of the extracts, but without returning to the levels in the control group with standard feeding (between 15-40% decrease). There was no impact on amylase in adults or juveniles, an overall but not significant decrease in cholesterol, and an overall increase in phospholipase in the juveniles. CONCLUSION: Olive extracts rich in polyphenols lower glucose levels in overfed fish

ANTECEDENTES: Los extractos de aceitunas ricos en polifenoles no son tóxicos y tienen efectos antiinflamatorios, neuroprotectores y antiadipogénicos en modelos celulares y animales. OBJETIVO: Evaluar la influencia potencial de los extractos de aceituna en los mecanismos de digestión y absorción de polisacáridos y grasas mediante la cuantificación de amilasa, glucosa, fosfolipasa y colesterol en el modelo de pez medaka. MATERIAL Y MÉTODOS: Para cada ensayo, se colocaron seis peces adultos en un tanque con un extracto (al 0,01%), realizando tres repeticiones por extracto. Se usó un grupo control con alimentación estándar. Se siguió el mismo procedimiento para estudiar la glucosa, agregando una dieta rica en polisacáridos y un control de sobrealimentados. Los peces se mantuvieron en estas condiciones durante cinco días. Se usaron cinco extractos del olivo sin intentar purificar los polifenoles debido a posibles efectos sinérgicos. Las concentraciones totales fueron entre 2-116 mg/g (principalmente oleuropeína e hidroxitirosol). Al finalizar, se cuantificaron amilasa, fosfolipasa A2, glucosa y colesterol en cada grupo. Todos los ensayos se realizaron por triplicado. Las actividades enzimáticas también se estudiaron en alevines. Se utilizaron pruebas no paramétricas para determinar posibles diferencias, considerando p <0.05 para significación estadística. RESULTADOS: Los extractos de polifenoles no fueron tóxicos a una concentración de 0.1%, diez veces mayor que la concentración utilizada. Se observó una disminución general en los niveles de glucosa en peces sobrealimentados con carbohidratos con la adición de extractos, pero sin volver a los niveles del grupo control con alimentación estándar (disminución entre 15-40%). No hubo impacto sobre la amilasa en adultos o juveniles, se observó una disminución general pero no significativa del colesterol y un aumento general de la fosfolipasa en los juveniles. CONCLUSIÓN: Los extractos de aceitunas ricos en polifenoles reducen los niveles de glucosa en peces sobrealimentados

Influence of Olive Extracts on the Expression of Genes Involved in Lipid Metabolism in Medaka Fish.

Aims. To assess the possible effect of polyphenol-rich olive extracts on lipid metabolism in medaka fish by quantifying the expression of lipogenic and lipolytic genes. Materials and methods. Adult medaka fish were maintained in tanks for five days with five extracts at 0.01% in water, causing obesity through a diet rich in carbohydrates, with a control group maintained in water with a normal diet. The extracts contained polyphenols ranging between 7 and 116 mg/g (oleuropein, hydroxytyrosol) with an antioxidant power of 2-13 mmol of 2,4,6-tri(2-pyridyl)-1,3,5-triazine/100 g. After five days, the fish were sacrificed and the hepatic mRNA and its complementary DNA were extracted by reverse transcription. Complementary DNAs were quantified for three lipolytic and three lipogenic genes by real-time PCR. The relative gene expression was calculated from the amplification curves in reference to the control group. Results. The expression of genes involved in lipolysis, including peroxisome proliferator-activated receptor-±, acyl-CoA oxidase 1, and carnitine palmitoyltransferase 1, were clearly decreased in fish subjected to an obesogenic diet, and this situation could not be reversed in fish maintained with polyphenol-rich extracts. In contrast, lipogenic fatty acid synthase, acetyl-CoA carboxylase 1, and sterol regulatory element-binding protein 1 genes increased considerably with the obesogenic diet and reverted to the normal state with the olive extracts. The effect was not dependent on the total polyphenol content, the specific oleuropein or hydroxytyrosol concentration, or the antioxidant power, suggesting a synergistic effect. Conclusion. Olive polyphenols, acting as anti-lipogenic agents, have a positive effect on lipid metabolism, but their mechanism in each gene is different according to the extract, which supports synergistic mechanisms with the different proportions of polyphenols and accompanying phytochemicals in each extract.

Protection by polyphenol extract from olive stones against apoptosis produced by oxidative stress in human neuroblastoma cells

OBJECTIVE: We evaluated the protective activity of an extract from a by-product such as olive stones, through its ability to inhibit H202 induced apoptosis in the SH-SY5Y human neuroblastoma cell line. MATERIAL AND METHODS: To such end, 20,000 cells/well were cultivated and differentiation with retinoic acid was initiated. Once the cells were differentiated, apoptosis was induced with and without H2O2 extract. Finally, cDNA extraction was performed, and pro-apoptotic genes Bax and anti-apoptotic genes Bcl-2 were analyzed. Quantification of the gene expression was performed using the GAPDH gene marker. RESULTS: Cell viability with the extract is 97.6% (SD 5.7) with 10 mg/l and 62.8% (SD 1.2) to 50 mg/l, using 10 mg/l for the biomarker assay. The retinoic acid differentiated SH-S cell line (10 µM) shows a clear apoptosis when treated with H2O2 150 µM, with a Bax/Bcl-2 ratio of 3.75 (SD 0.80) in contrast to the differentiated control cells subjected to H2O2 and with extract, which have the same ratio of 1.02 (SD 0.01-0.03). CONCLUSION: The olive stone extract shows anti-apoptotic activity in the provoked cell death of SH-SY5Y human neuroblastoma cells in their normal state, defending them from oxidative stress which produces a significant increase in the apoptotic gene ratio in contrast to anti-apoptotic genes (Bax/Bcl-2).

Protection by polyphenol extract from olive stones against apoptosis produced by oxidative stress in human neuroblastoma cells / Protección de un extracto de polifenoles de huesos de aceitunas frente a la apoptosis producida por estrés oxidativo en células de neuroblastoma humano

Objective: We evaluated the protective activity of an extract from a by-product such as olive stones, through its ability to inhibit H2 02 induced apoptosis in the SH-SY5Y human neuroblastoma cell line. Material and methods: To such end, 20,000 cells/well were cultivated and differentiation with retinoic acid was initiated. Once the cells were differentiated, apoptosis was induced with and without H2 O2 extract. Finally, cDNA extraction was performed, and pro-apoptotic genes Bax and anti-apoptotic genes Bcl-2 were analyzed. Quantification of the gene expression was performed using the GAPDH gene marker. Results: Cell viability with the extract is 97.6% (SD 5.7) with 10 mg/l and 62.8% (SD 1.2) to 50 mg/l, using 10 mg/l for the biomarker assay. The retinoic acid differentiated SH-S cell line (10 µM) shows a clear apoptosis when treated with H2 O2 150 µM, with a Bax/Bcl-2 ratio of 3.75 (SD 0.80) in contrast to the differentiated control cells subjected to H2 O2 and with extract, which have the same ratio of 1.02 (SD 0.01-0.03). Conclusion: The olive stone extract shows anti-apoptotic activity in the provoked cell death of SH-SY5Y human neuroblastoma cells in their normal state, defending them from oxidative stress which produces a significant increase in the apoptotic gene ratio in contrast to anti-apoptotic genes (Bax/Bcl-2) (AU)

Objetivo: evaluar la actividad protectora del extracto de un subproducto como son los huesos de aceitunas, mediante su capacidad de inhibir la apoptosis en la línea celular humana de neuroblastoma SH-SY5Y inducida con H2 O2 . Material y métodos: se han cultivado 20.000 cel/pocillo, iniciando diferenciación con ácido retinoico y, una vez diferenciadas las células, se ha inducido la apoptosis con H2 O2 con extracto y sin presencia del mismo. Finalmente se efectúa la extracción de cDNA y el análisis de los genes proapoptótico Bax y antiapoptótico Bcl-2. La cuantificación de la expresión génica se realiza frente al gen marcador GAPDH. Resultados: la viabilidad celular con el extracto es del 97,6% (SD 5,7) con 10 mg/l y 62,8% (SD 1,2) a 50 mg/l, utilizando 10 mg/l para el ensayo de biomarcadores. Las células de la línea SH-S diferenciadas con ácido retinoico (10 µM), muestran una clara apoptosis al ser tratadas con H2 O2 150 µM, con una relación Bax/Bcl2 de 3,75 (SD 0,80) frente a las células diferenciadas control y sometidas a H2 O2 y con extracto que tienen la misma relación de 1,02 (SD 0,01-0,03). Conclusión: el extracto de huesos de aceitunas presenta una actividad antiapoptótica frente a la provocación de la muerte celular por peróxido de hidrógeno, preservando a las células de neuroblastoma humano SH-SY5Y en su estado de normalidad, al defenderlas del estrés oxidativo que produce un significativo aumento de la relación de genes apoptóticos frente a antiapoptóticos (Bax/Bcl2) (AU)

Anti-adipogenic activity of an olive seed extract in mouse fibroblasts.

UNLABELLED: The administration of different polyphenols protects against increased body weight and fat accumulation. The aim of the study was to determine the anti-adipogenic activity of an olive-seed polyphenolic extract, by means of mouse fibroblast cell line 3T3-L1 adipocyte differentiation. MATERIAL AND METHODS: Cells were incubated and differentiated (6000 cells/cup) in the presence of olive-seed extract at 10 and 50 mg/l biosecure concentrations of polyphenols, and with no extract in the control sample. After 5 to 7 days mature adipocytes are formed. The fat clusters are quantified by means of red-oil staining, 490 nm absorbance, and the expression of the leptin and PPARg genes, and then compared to the values obtained in the cultures before and after adipocyte differentiation. RESULTS: The control samples, with no extract, presented an accumulation of fat of 100%. By contrast, the addition of 50 mg/l of olive-seed extract polyphenols resulted in a 50% accumulation of fat, similar to that of the non-differentiated cells. A 10 mg/l extract concentration had no effect. Anti-adipogenic activity is thus confirmed, as the expression of the PPARg and leptin genes is reduced in adipocyte differentiation in the presence of extract at 50 mg/l. In conclusion, both the formation of fatty substances characteristic of adipogenesis, and the expression of the adipogenic PPARg and leptin genes are found to be inhibited by the prior addition of olive-seed extract polyphenols at a 50 mg/l concentration.

La administración de diferentes polifenoles protege contra el incremento de peso y la acumulación de grasa. Objetivo: comprobar la actividad anti-adipogénica de un extracto polifenólico de huesos de aceituna, utilizando la diferenciación a adipocitos de la línea celular 3T3-L1 de fibroblastos de ratón. Material y métodos: se cultivan y diferencian las células (6.000 células/pocillo) en presencia del extracto de huesos de aceitunas a 10 y 50 mg/l de polifenoles, concentraciones bioseguras, y sin extracto como control. A los 5-7 días se forman los adipocitos maduros. Se cuantifican los cúmulos de grasa formados mediante tinción con Oil- Red y medida de la absorbancia a 490 nm y la expresión de los genes de leptina y PPARg, relacionándolos con los valores en los cultivos antes y después de diferenciarse a adipocitos. Resultados: las muestras control, sin extracto, se consideran el 100% de acumulación de grasas. En contraste, la adición de 50 mg/l de extracto de polifenoles de huesos de aceituna muestra un cúmulo de grasa de alrededor del 50%, semejante a las células no diferenciadas. Con 10 mg/l de extracto no se muestra efecto. Se confirma la actividad antiadipogénica, observándose disminución en la expresión de los genes PPARg y de leptina en la diferenciación a adipocitos en presencia del extracto a 50 mg/l. En conclusión, la formación de los cuerpos grasos característicos de la adipogénesis queda inhibida previa adición de 50 mg/l de polifenoles de extracto de huesos de aceituna, así como la expresión de los genes adipogénicos PPARg y de leptina.

Anti-adipogenic activity of an olive seed extract in mouse fibroblasts / Actividad anti-adipogénica de un extracto de huesos de aceituna en fibroblastos de ratón

The administration of different polyphenols protects against increased body weight and fat accumulation. The aim of the study was to determine the anti-adipogenic activity of an olive-seed polyphenolic extract, by means of mouse fibroblast cell line 3T3-L1 adipocyte differentiation. Material and methods: cells were incubated and differentiated (6000 cells/cup) in the presence of olive-seed extract at 10 and 50 mg/l biosecure concentrations of polyphenols, and with no extract in the control sample. After 5 to 7 days mature adipocytes are formed. The fat clusters are quantified by means of red-oil staining, 490 nm absorbance, and the expression of the leptin and PPARg genes, and then compared to the values obtained in the cultures before and after adipocyte differentiation. Results: the control samples, with no extract, presented an accumulation of fat of 100%. By contrast, the addition of 50 mg/l of olive-seed extract polyphenols resulted in a 50% accumulation of fat, similar to that of the non-differentiated cells. A 10 mg/l extract concentration had no effect. Anti-adipogenic activity is thus confirmed, as the expression of the PPARg and leptin genes is reduced in adipocyte differentiation in the presence of extract at 50 mg/l. In conclusion, both the formation of fatty substances characteristic of adipogenesis, and the expression of the adipogenic PPARg and leptin genes are found to be inhibited by the prior addition of olive-seed extract polyphenols at a 50 mg/l concentration (AU)

La administración de diferentes polifenoles protege contra el incremento de peso y la acumulación de grasa. Objetivo: comprobar la actividad anti-adipogénica de un extracto polifenólico de huesos de aceituna, utilizando la diferenciación a adipocitos de la línea celular 3T3-L1 de fibroblastos de ratón. Material y métodos: se cultivan y diferencian las células (6.000 células/pocillo) en presencia del extracto de huesos de aceitunas a 10 y 50 mg/l de polifenoles, concentraciones bioseguras, y sin extracto como control. A los 5-7 días se forman los adipocitos maduros. Se cuantifican los cúmulos de grasa formados mediante tinción con OilRed y medida de la absorbancia a 490 nm y la expresión de los genes de leptina y PPARg, relacionándolos con los valores en los cultivos antes y después de diferenciarse a adipocitos. Resultados: las muestras control, sin extracto, se consideran el 100% de acumulación de grasas. En contraste, la adición de 50 mg/l de extracto de polifenoles de huesos de aceituna muestra un cúmulo de grasa de alrededor del 50%, semejante a las células no diferenciadas. Con 10 mg/l de extracto no se muestra efecto. Se confirma la actividad antiadipogénica, observándose disminución en la expresión de los genes PPARg y de leptina en la diferenciación a adipocitos en presencia del extracto a 50 mg/l. En conclusión, la formación de los cuerpos grasos característicos de la adipogénesis queda inhibida previa adición de 50 mg/l de polifenoles de extracto de huesos de aceituna, así como la expresión de los genes adipogénicos PPARg y de leptina (AU)