Olea europaea leaf (Ph.Eur.) extract as well as several of its isolated phenolics inhibit the gout-related enzyme xanthine oxidase

In Mediterranean folk medicine Olea europaea L. leaf (Ph.Eur.) preparations are used as a common remedy for gout. In this in vitro study kinetic measurements were performed on both an 80% ethanolic (v/v) Olea europaea leaf dry extract (OLE) as well as on nine of its typical phenolic constituents in order to investigate its possible inhibitory effects on xanthine oxidase (XO), an enzyme well known to contribute significantly to this pathological process. Dixon and Lineweaver-Burk plot analysis were used to determine K_i values and the inhibition mode for the isolated phenolics, which were analysed by RP-HPLC for standardisation of OLE. The standardised OLE as well as some of the tested phenolics significantly inhibited the activity of XO. Among these, the flavone aglycone apigenin exhibited by far the strongest effect on XO with a K_i value of 0.52 μM. In comparison, the known synthetic XO inhibitor allopurinol, used as a reference standard, showed a K_i of 7.3 μM. Although the phenolic secoiridoid oleuropein, the main ingredient of the extract (24.8%), had a considerable higher K_i value of 53.0 μM, it still displayed a significant inhibition of XO. Furthermore, caffeic acid (K_i of 11.5 μM; 1.89% of the extract), luteolin-7-O-β-d-glucoside (K_i of 15.0 μM; 0.86%) and luteolin (K_i of 2.9 μM; 0.086%) also contributed significantly to the XO inhibiting effect of OLE. For oleuropein, a competitive mode of inhibition was found, while all other active substances displayed a mixed mode of inhibition. Tyrosol, hydroxytyrosol, verbascoside, and apigenin-7-O-β-d-glucoside, which makes up for 0.3% of the extract, were inactive in all tested concentrations. Regarding the pharmacological in vitro effect of apigenin-7-O-β-d-glucoside, it has to be considered that it is transformed into the active apigenin aglycone in the mammalian body, thus also contributing substantially to the anti-gout activity of olive leaves. For the first time, this study provides a rational basis for the traditional use of olive leaves against gout in Mediterranean folk medicine.     

Olive (Olea europaea) leaf extract effective in patients with stage-1 hypertension: Comparison with Captopril

A double-blind, randomized, parallel and active-controlled clinical study was conducted to evaluate the anti-hypertensive effect as well as the tolerability of Olive leaf extract in comparison with Captopril in patients with stage-1 hypertension. Additionally, this study also investigated the hypolipidemic effects of Olive leaf extract in such patients. It consisted of a run-in period of 4 weeks continued subsequently by an 8-week treatment period. Olive (Olea europaea L.) leaf extract (EFLA^®943) was given orally at the dose of 500 mg twice daily in a flat-dose manner throughout the 8 weeks. Captopril was given at the dosage regimen of 12.5 mg twice daily at start. After 2 weeks, if necessary, the dose of Captopril would be titrated to 25 mg twice daily, based on subject's response to treatment. The primary efficacy endpoint was reduction in systolic blood pressure (SBP) from baseline to week-8 of treatment. The secondary efficacy endpoints were SBP as well as diastolic blood pressure (DBP) changes at every time-point evaluation and lipid profile improvement. Evaluation of BP was performed every week for 8 weeks of treatment; while of lipid profile at a 4-week interval. Mean SBP at baseline was 149.3 ± 5.58 mm Hg in Olive group and 148.4 ± 5.56 mm Hg in Captopril group; and mean DBPs were 93.9 ± 4.51 and 93.8 ± 4.88 mm Hg, respectively. After 8 weeks of treatment, both groups experienced a significant reduction of SBP as well as DBP from baseline; while such reductions were not significantly different between groups. Means of SBP reduction from baseline to the end of study were −11.5 ± 8.5 and −13.7 ± 7.6 mm Hg in Olive and Captopril groups, respectively; and those of DBP were −4.8 ± 5.5 and −6.4 ± 5.2 mm Hg, respectively. A significant reduction of triglyceride level was observed in Olive group, but not in Captopril group. In conclusion, Olive (Olea europaea) leaf extract, at the dosage regimen of 500 mg twice daily, was similarly effective in lowering systolic and diastolic blood pressures in subjects with stage-1 hypertension as Captopril, given at its effective dose of 12.5-25 mg twice daily.     

Technique for cryopreservation of intestinal smooth muscle cells

Attempts were made to develop a simplified procedure for long-term cryopreservation of intestinal smooth muscle cells (ISMC). ISMC were collected from the ileum of Sprague-Dawley neonatal rats through cellular dissociation in trypsin. Cryopreservation method comprised of a rapid 1-step (protocol 1) and a slow 3-step (protocol 2) freezing of ISMC for 1 week. Preparations were thawed and single ISMC were assessed via the comet assay and damaged DNA was quantified through comet tail moment. The control unfrozen ISMC exhibited DNA damage of 2.34 ± 0.35 compared to ISMC cooled via protocol 2 (2.62 ± 0.36) and protocol 1 (10.15 ± 0.72). Thereafter, protocol 2 freezing method was adopted and ISMC were cryopreserved for 1-week, 1-month, and 4-months to analyse the temporal and long-term cryopreservation of ISMC. This revealed a DNA damage of 2.62 ± 0.36 (1-week), 3.81 ± 0.72 (1-month), and 5.1 ± 0.9 (4-months). Gradual cooling is suitable for continuing storage of ISMC and although fluctuation in cryoinjury is observed with time this is considered to reflect cell-to-cell variability.     

Production of added‐value metabolites by Yarrowia lipolytica growing in olive mill wastewater‐based media under aseptic and non‐aseptic conditions

Yarrowia lipolytica ACA‐YC 5033 was grown on glucose‐based media in which high amounts of olive mill wastewaters (OMWs) had been added. Besides shake‐flask aseptic cultures, trials were also performed in previously pasteurized media while batch bioreactor experiments were also done. Significant decolorization (～58%) and remarkable removal of phenolic compounds (～51% w/w) occurred, with the latter being amongst the highest ones reported in the international literature, as far as yeasts were concerned during their growth on phenol‐containing media. In nitrogen‐limited flask fermentations the microorganism produced maximum citric acid quantity ≈19.0 g/L [simultaneous yield of citric acid produced per unit of glucose consumed (Y_Cit/Glc)≈0.74 g/g]. Dry cell weight (DCW) values decreased at high phenol‐containing media, but, on the other hand, the addition of OMWs induced reserve lipid accumulation. Maximum citric acid concentration achieved (≈52.0 g/L; Y_Cit/Glc≈0.64 g/g) occurred in OMW‐based high sugar content media (initial glucose added at ≈80.0 g/L). The bioprocess was successfully simulated by a modified logistic growth equation. A satisfactory fitting on the experimental data occurred while the optimized parameter values were found to be similar to those experimentally measured. Finally, a non‐aseptic (previously pasteurized) trial was performed and its comparison with the equivalent aseptic experiment revealed no significant differences. Yarrowia lipolytica hence can be considered as a satisfactory candidate for simultaneous OMWs bioremediation and the production of added‐value compounds useful for the food industry.     

Cloning and characterization of phospholipase D from olive flounder (Paralichthys olivaceus)

The phospholipase D1 (PLD1) cDNA, designated PoPLD, encoding a predicted protein of 1053 amino acids in olive flounder (Paralichthys olivaceus) has been cloned. The deduced amino acid sequence shares high identity with that of PLD1s and PLD2 in human, rat and mouse. The phylogenic analysis and sequence comparison of PoPLD with other PLD isozymes were found to be closely related to the PLD1 isozyme in primary structure. The tissue expression analysis of PoPLD showed that the mRNA of PoPLD was predominantly expressed in the brain, gullet, muscle, stomach, head kidney, pyloric caeca, intestine and gill. The expression of the PoPLD gene was examined in various tissues of flounder by RT-PCR following stimulation with LPS and compared also with that of the inflammatory cytokines IL-1beta and IL-8 in various tissues of the stimulated flounder. This provides indirect evidence that PLD1 might have a relevant role in immune responses against pathogens and in inflammation. In addition, the recombinant protein of PoPLD (GFP-PoPLD), which demonstrated a phosphatidylcholine (PC)-hydrolyzing activity, was partially localized as a distinct ring-shaped form surrounding the rim of the nucleus in EPC cells. Together, our results suggest that PoPLD is similar to the mammalian PLD1 isoform, is generally widespread within olive flounder tissue, might have a relevant role in the fish immune system against pathogens and specifically may be localized in the subcellular membranes of the nuclear rim in EPC cells.     

Development of a stochastic dynamic model for ecological indicators’ prediction in changed Mediterranean agroecosystems of north-eastern Portugal

A holistic stochastic dynamic model was developed by focusing on the interactions between conceptually isolated key-components, such as local passerine guilds and changes in habitat conditions, in Mediterranean agroecosystems of the “Terra Quente Transmontana region” (north-eastern Portugal). The ecological integrity of the typical patchwork of this region, with respect to land use, can be partly assessed by the observation of the occurrence of passerine guilds. These important indicators and state variables are the underlying database of our model. This model aimed the prediction of the ecological changes which can be expected when olive orchards are being intensified. The model proposed was preceded by a conventional multivariate statistical procedure (stepwise multiple regression analysis) performed to discriminate the significant relationships between guild richness and environmental variables. Since this statistical analysis is static, the dataset recorded from the field included true gradients of habitat changes. The model parameters were estimated from the results of the stochastic treatment and from regional data regarding tendencies within the use of land. A period of 50 years was considered. The final model provided some basis to analyse the responses of passerine guilds to the environmental scenarios that will characterize the new agroecosystems of the region. The model simulations were incorporated into a Geographical Information System (GIS) approach. The results of the simulation revealed a structural drift within the different guild richness in response to the expected gradient of habitat changes. The possible local extinction of several species within the less well-represented guilds, such as the steppe passerine species, may be associated with a predictable reduction in ecological integrity of the typical agroecosystems. Therefore, a new structure of the passerine communities indicates that future agroecosystems will diverge from the initial or actual ecological state.     

Effect of different drying procedures on the nutritive value of olive (Olea europaea var. europaea) leaves for ruminants

Fresh, freeze-, air- and oven-dried at 60 °C and 100 °C olive leaves (OL) were studied in order to determine the effect of different drying procedures on OL chemical composition, in vitro digestibility, ruminal degradability, and intestinal digestibility. The drying procedure affected all the parameters measured except for gross energy (GE; P=0.194). Protein-bound condensed tannins (CT) decreased (P=0.001) with freeze-, air- and 60 °C drying (from 1.25 up to 0.82 g/kg dry matter, DM). Total CT were only decreased (P=0.001) by drying at 60 °C (from 10.0 to 6.24 g/kg DM). The in vitro crude protein (CP) digestibility increased (P<0.001) with drying except for oven-drying at 100 °C up to 58%. Values for CP digestibility found in freeze- and air-dried OL were not different (P>0.05). No differences (P>0.05) were observed between CP digestibility in air- and oven-dried at 60 °C OL. Effective degradability of DM and CP increased from 0.53 to 0.62 (P=0.005) and from 0.46 to 0.64 (P=0.002), respectively after treatment. The apparent intestinal digestibility of undegraded CP in the rumen was only affected (P=0.046) by oven-drying, which increased it from 0.33 to 0.39. As air-drying did not have detrimental effects on the OL nutritive value it could be an appropriate, simple and low-cost procedure for olive-leaves preservation.     

Olive mill wastewater stabilization in open-air ponds: impact on clay-sandy soil

The aim of this work was to study the natural biodegradation of the stored olive mill wastewater (OMW) in ponds and the infiltration as well as the impact on soil of the effluent in the evaporation pond used for the storage over the past eight years. For this, two approaches were considered. First, a laboratory-scale column was used for the infiltration of OMW through soil (clay and sand) to predict the effect of the clayey soil in reducing OMW pollution. Second, the ponds including the effluent annually stored and having this clayey structure were investigated. At the laboratory-scale, a modification of OMW contents was noticed, with the elimination of 95% of total suspended solids (TSS), 60% of chemical oxygen demand (COD), 40% of total organic carbon (TOC), 50% of total P, 50% of phenols and 40% of minerals (K+, Mg++ and Na+). The experimented soil was able to restrain the considerable effects of OMW pollution. In the ponds, the granulometric characteristics, the physico-chemical and the biological parameters of the soil profile from the contaminated pond were compared to those of a control soil, located near the contaminated pond. Property modifications of the contaminated soil were noted, especially pH, electrical conductivity, COD and microflora. These changes can be explained by the infiltration of OMW constituents, which were noticed in the soil layers, especially phenolic compounds that have a negative effect on the ground water.     

Effect of dietary coenzyme Q and fatty acids on the antioxidant status of rat tissues

Summary.  Wistar rats were fed with different diets with or without supplement coenzyme Q₁₀ (CoQ₁₀) and with oil of different sources (sunflower or virgin olive oil) for six or twelve months. Ubiquinone contents (CoQ₉ and CoQ₁₀) were quantified in homogenates of livers and brains from rats fed with the four diets. In the brain, younger rats showed a 3-fold higher amount of ubiquinone than older ones for all diets. In the liver, however, CoQ₁₀ supplementation increased the amount of CoQ₉ and CoQ₁₀ in both total homogenates and plasma membranes. Rats fed with sunflower oil as fat source showed higher amounts of ubiquinone content than those fed with olive oil, in total liver homogenates, but the total ubiquinone content in plasma membranes was similar with both fat sources. Older rats showed a higher amount of ubiquinone after diets supplemented with CoQ₁₀. Two ubiquinone-dependent antioxidant enzyme activities were measured. NADH-ferricyanide reductase activity in hepatocyte plasma membranes was unaltered by ubiquinone accumulation, but this activity increased slightly with age. Both cytosolic and membrane-bound dicumarol-sensitive NAD(P)H:(quinone acceptor) oxidoreductase (DT-diaphorase, EC 1.6.99.2) activities were decreased by diets supplemented with CoQ₁₀. Animals fed with olive oil presented lower DT-diaphorase activity than those fed with sunflower oil, suggesting that the CoQ₁₀ antioxidant protection is strengthened by olive oil as fat source. Received May 22, 2002; accepted September 20, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Departamento de Biología Celular, Fisiología e Inmunología, Universidad de Córdoba Edificio Severo Ochoa, Campus de Rabanales, 14071 Córdoba, Spain.     

Potential of agroindustrial waste from olive oil industry for fuel ethanol production

Olive pulp (OP) is a highly polluting semi-solid residue generated from the two-stage extraction processing of olives and is a major environmental issue in Southern Europe, where 80% of the world olive oil is produced. At present, OP is either discarded to the environment or combusted with low calorific value. In this work, utilization of OP as a potential substrate for production of bioethanol was studied. Enzymatic hydrolysis and subsequent glucose fermentation by baker's yeast were evaluated for OP from 10% to 30% dry matter (i.e., undiluted). Enzymatic hydrolysis resulted in an increase in glucose concentration by 75%, giving final glucose yields near 70%. Fermentation of undiluted OP hydrolysate (OPH) resulted in the maximum ethanol produced (11.2 g/L) with productivity of 2.1 g/L/h. Ethanol yields were similar for all tested OPH concentrations and were in the range of 0.49-0.51 g/g. Results showed that yeast could effectively ferment OPH even without nutrient addition, revealing the tolerance of yeast to OP toxicity. Because of low xylan (12.4%) and glucan (16%) content in OP, this specific type of OP is not a suitable material for producing only ethanol and thus, bioethanol production should be integrated with production of other value-added products.