Lipid-lowering and antioxidant effects of hydroxytyrosol and its triacetylated derivative recovered from olive tree leaves in cholesterol-fed rats

This study was designed to test the lipid-lowering and antioxidative activities of triacetylated hydroxytyrosol compared with its native compound, hydroxytyrosol, purified from olive tree leaves. Wistar rats fed a standard laboratory diet or a cholesterol-rich diet for 16 weeks were used. The serum lipid levels, the thiobarbituric acid-reactive substances (TBARS) level, as an indicator of lipid peroxidation, and the activity of superoxide dismutase (SOD) as well as that of catalase (CAT) were examined. The cholesterol-rich diet induced hypercholesterolemia that was manifested in the elevation of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C). Administration of hydroxytyrosol and triacetylated hydroxytyrosol (3 mg/kg of body weight) decreased the serum levels of TC, TG, and LDL-C significantly and increased the serum level of high-density lipoprotein cholesterol (HDL-C). Furthermore, the content of TBARS in liver, heart, kidney, and aorta decreased significantly when hydroxytyrosol and its triacetylated derivatives were orally administered to rats compared with those fed a cholesterol-rich diet. In addition, triacetylated hydroxytyrosol and hydroxytyrosol increased CAT and SOD activities in the liver. These results suggested that the hypolipidemic effect of triacetylated hydroxytyrosol and hydroxytyrosol might be due to their abilities to lower serum TC, TG, and LDL-C levels as well as to their antioxidant activities preventing the lipid peroxidation process.     

Structural characterization of the metabolites of hydroxytyrosol, the principal phenolic component in olive oil, in rats

Hydroxytyrosol is quantitatively and qualitatively the principal phenolic antioxidant in olive oil. Recently it was shown that hydroxytyrosol and five metabolites were excreted in urine when hydroxytyrosol was dosed intravenously or orally in an olive oil solution to rats. The conclusive identification of three metabolites of hydroxytyrosol by MS/MS as a monosulfate conjugate, a 3-O-glucuronide conjugate, and 4-hydroxy-3-methoxyphenylacetic acid (homovanillic acid) has been established in this investigation. The structural configurations of the glucuronide conjugate and 4-hydroxy-3-methoxyphenylacetic acid were confirmed by (1)H NMR. The radical scavenging potencies of homovanillic acid, homovanillic alcohol, hydroxytyrosol, and the metabolites were examined with the radical 2,2-diphenyl-1-picrylhydrazyl. These studies showed them to be potent antioxidants with SC(50) values of 14.8 and 11.4 microM for homovanillic acid and homovanillic alcohol, respectively. The 3-O-glucuronide conjugate was more potent than hydroxytyrosol, with an SC(50) of 2.3 in comparison to 11.0 microM, and the monosulfate conjugate was almost devoid of radical scavenging activity.     

Production in large quantities of highly purified hydroxytyrosol from liquid-solid waste of two-phase olive oil processing or "Alperujo"

The effect of hydrothermal treatment of two-phase olive waste (alperujo) on the solubilization of hydroxytyrosol was studied. Different conditions of saturated steam were assayed. A high amount of hydroxytyrosol was solubilized and increased with increasing steaming temperature and time, reaching 1.4-1.7 g/100 g of dry alperujo. The effect of acidic (H(2)SO(4)) and basic (NaOH) catalysts was also evaluated. Acid-catalyzed treatment was more effective at milder conditions, whereas the alkali-catalyzed conditions were not very suitable. In the present study, the extracted hydroxytyrosol was purified by means of a new, simple, and inexpensive chromatographic system, under international patent application (PCT/ES02/00058). From 1000 kg of alperujo, with 70% humidity, can be obtained approximately 4.5-5 kg of hydroxytyrosol. After a purification process, at least 3 kg of hydroxytyrosol, at 90-95% purity, would be obtained. The purified compound was identified by HPLC/UV and (1)H and (13)C NMR analyses, and its antioxidant activity was tested on refined olive oil without antioxidants by Rancimat method. The oxidative stability of refined olive oil was increased by a factor of 1.71 in the presence of 100 ppm of hydroxytyrosol.     

Analysis of total contents of hydroxytyrosol and tyrosol in olive oils

The most abundant phenolic compounds in olive oils are the phenethyl alcohols hydroxytyrosol and tyrosol. An optimized method to quantify the total concentration of these substances in olive oils has been described. It consists of the acid hydrolysis of the aglycons and the extraction of phenethyl alcohols with a 2 M HCl solution. Recovery of the phenethyl alcohols from oils was very high (<1% remained in the extracted oils), and the limits of quantification (LOQ) were 0.8 and 1.4 mg/kg for hydroxytyrosol and tyrosol, respectively. Precision values, both intraday and interday, remained below 3% for both compounds. The final optimized method allowed for the analysis of several types of commercial olive oils to evaluate their hydroxytyrosol and tyrosol contents. The results show that this method is simple, robust, and reliable for a routine analysis of the total concentration of these substances in olive oils.     

Hypocholesterolemic effects of phenolic extracts and purified hydroxytyrosol recovered from olive mill wastewater in rats fed a cholesterol-rich diet

In our previous studies, a phenolic-rich extract of olive mill wastewaters (OMW) was prepared under optimal conditions, using a continuous countercurrent extraction unit, and hydroxytyrosol was purified from the obtained OMW extract. The antioxidant activity of OMW extract and hydroxytyrosol was determined by a series of models in vitro. In this study, the hypocholesterolemic effects of hydroxytyrosol and OMW extract in rats fed a cholesterol-rich diet were tested. Wistar rats, fed a standard laboratory diet or a cholesterol-rich diet for 16 weeks, were used. Serum lipid levels, as well as thiobarbituric acid reactive substances (TBARS) and superoxide dismutase and catalase activities in liver were examined. Cholesterol-rich diet-induced hypercholesterolemia was manifested in the elevation of serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C). Administration of a low-dose (2.5 mg/kg of body weight) of hydroxytyrosol and a high-dose (10 mg/kg of body weight) of OMW extract significantly lowered the serum levels of TC and LDL-C while increasing the serum levels of high-density lipoprotein cholesterol (HDL-C). Furthermore, the TBARS contents in liver, heart, kidney, and aorta decreased significantly after oral administration of hydroxytyrosol and OMW extract as compared with those of rats fed a cholesterol-rich diet. In addition, OMW phenolics increased CAT and SOD activities in liver. These results suggested that the hypocholesterolemic effect of hydroxytyrosol and OMW extract might be due to their abilities to lower serum TC and LDL-C levels as well as slowing the lipid peroxidation process and enhancing antioxidant enzyme activity.     

Toward a high yield recovery of antioxidants and purified hydroxytyrosol from olive mill wastewaters

We investigated to develop effective procedures to recover the potentially high-added-value phenolic compounds contained in the discontinuous three-phase olive processing wastewaters (OMW). Particular emphasis was made to extract and purify hydroxytyrosol, one of the major compounds occurring in OMW. Batch optimization experiments showed that ethyl acetate is the most efficient solvent for the recovery of phenolic monomers from OMW. The latter was used with an optimal pH equal to 2. Furthermore, the percentage of each monomer, and particularly hydroxytyrosol, in the extract was maximum for a solvent ratio and a theoretical extraction stage number equal to 2 and 3, respectively. High yield (85.46%) recovery of hydroxytyrosol was achieved from OMW using a three-staged continuous counter-current liquid-liquid extraction unit. Hydroxytyrosol (1.225 g) were extracted per liter of OMW. One gram of hydroxytyrosol per liter of OMW was then purified by means of a chromatographic system which could be adapted to a large scale production process.     

Antioxidative and antiplatelet effects of aqueous inflorescence Piper betle extract

Piper betle, belonging to the Piperaceae family, is a tropical plant, and its leaf and inflorescence are popularly consumed by betel quid (BQ) chewers in Taiwan and many other South and Southeast Asian countries. However, little is known about the biochemical properties of inflorescence Piper betle (IPB) toward reactive oxygen species (ROS) and platelet functions. In the present work, aqueous IPB extract was shown to be a scavenger of H(2)O(2), superoxide radical, and hydroxyl radical with a 50% inhibitory concentration (IC(50)) of about 80, 28, and 73 microg/mL, respectively. IPB extract also prevented the hydroxyl radical induced PUC18 plasmid DNA breaks at concentrations higher than 40 microg/mL. Since ROS are crucial for platelet aggregation, we further found that IPB extract also inhibited the arachidonic acid (AA) induced and collagen-induced platelet aggregation, with an IC(50) of 207 and 335 microg/mL, respectively. IPB extract also inhibited the AA-, collagen- (>100 microg/mL of IPB), and thrombin (>250 microg/mL of IPB)-induced thromboxane B(2) (TXB(2)) production by more than 90%. However, IPB extract showed little effect on thrombin-induced aggregation. These results indicated that aqueous components of IPB are potential ROS scavengers and may prevent the platelet aggregation possibly via scavenging ROS or inhibition of TXB(2) production.     

Cytoprotective effect of hydroxytyrosyl alkyl ether derivatives after oral administration to rats in a model of glucose-oxygen deprivation in brain slices

This study was designed to determine whether the oral administration of hydroxytyrosol (HT) alkyl ether derivatives has a neuroprotective effect in rats. The animals were treated for 7 days with HT or ethyl, butyl, hexyl, octyl, and dodecyl HT ether. A method of in vitro hypoxia-reoxygenation in brain slices was used. Hexyl, octyl, and dodecyl HT derivatives reduced brain cell death (LDH efflux). Lipid peroxidation and nitrite concentrations were inhibited most by hexyl, octyl, and dodecyl derivatives. Concentrations of 3-nitrotyrosine were reduced by HT butyl, hexyl, octyl, and dodecyl ether derivatives. Interleukin-1β was significantly reduced in brain slices from rats treated with all HT ether derivatives. LDH efflux showed a linear correlation with brain concentrations of lipid peroxides, nitrites plus nitrates, and interleukin 1β. The reduction in oxidative and nitrosative stress and decreased production of pro-inflammatory interleukins may be the basis for the observed neuroprotective effects.     

Static-dynamic superheated liquid extraction of hydroxytyrosol and other biophenols from alperujo (a semisolid residue of the olive oil industry)

Hydroxytyrosol and other olive biophenols (OBPs) such as tyrosol, verbascoside, apigenin-7-glucoside, and alpha-taxifolin have been extracted from alperujo by using static-dynamic superheated liquids. Multivariate methodology has been used to carry out a detailed optimization of the extraction. Under the optimal working conditions no further extraction of the target analytes was achieved after 27 min (up to 2800 and 1500 mg/kg of hydroxytyrosol and tyrosol, respectively), so complete removal of them within this interval was assumed. The extract was injected into a chromatograph-photodiode array detector assembly for individual separation-quantification. The efficacy of ethanol/water mixtures to extract OBPs from alperujo has been demonstrated and compared with that of a conventional stirring-based method. These less toxic extractant mixtures are of interest with a view to future human uses of OBPs.     

NO合酶抑制剂 L-NAME和NO前体L-Arg对小鼠体内卵母细胞成熟的影响

摘要: 采用腹腔单独注射L-NAME(左旋硝基精氨酸甲基酯)或联合注射L-NAME和L-Arg, 取出卵母细胞体外观察，研究了NO对小鼠卵母细胞体内成熟的影响。结果表明,（1）单独注射5、10、20 mg / kg体重L-NAME均能显著抑制卵母细胞第一极体的释放(P <0.01)，尤以10 mg / kg 体重L-NAME组作用最为明显, 各剂量组对生发泡的破裂均无影响(P >0.05)；（2）10 mg / kg 体重L-NAME对卵母细胞成熟的抑制作用能被同时注射的100 mg / kg 或200 mg /kg 体重L-Arg逆转，其中以200 mg / kg体重作用最为明显;（3）L-NAME对卵母细胞畸形率的影响和培养时间有关，而对卵母细胞的存活率没有统计学影响。实验结果证明NO参与了小鼠卵母细胞的体内成熟调控。     

Inhibition of reactive nitrogen species effects in vitro and in vivo by isoflavones and soy-based food extracts

Recent studies have shown that soy isoflavone inhibits inducible nitric oxide (NO) synthase activities and is reported to have peroxynitrite scavenging ability. Consequently, we investigated whether isoflavones (daidzein and genistein) and extracts from soy-based products (miso, soymilk, tofu, soy sprout, black soybean, soybean, and yuba) would inhibit the reactive nitrogen species (RNS) effect in vitro and in vivo. In the in vitro experiments [including the protection of cellular DNA from peroxynitrite or sodium nitroprusside damage, an inhibitory effect on nitric oxide production from lipopolysaccharide (LPS)-induced RAW 264.7 cells, and nitric oxide scavenging ability], extracts from soy-based foods showed a potent antioxidant activity and an inhibiting effect on RNS activity. These effects were correlated with total isoflavone content. In the in vivo experiments, rats were given isoflavones (4.0 mg/kg bw) or soy-based product extracts (1.0 g/kg bw) orally for 1 week and were injected with vehicle H(2)O (1 mL/kg bw) or LPS (10 mg/kg bw) on the day 7. Twelve hours after treatment, the rats were killed, and blood serum was collected for analysis. The intraperitoneal administration of LPS resulted in an increase in serum nitrite, nitrate, and nitrotyrosine concentrations. These are stable metabolite end products of nitric oxide, to 4-, 16-, and 5-fold levels, (4, 10 microM and 58 +/- 14 pmol/mL), of the placebo control, respectively. Results showed that oral administration of isoflavones and extracts from soy-based products significantly decreased serum nitrite, nitrate, and nitrotyrosine levels in LPS-induced rats. This study demonstrates that soy isoflavone supplementation may inhibit RNS-induced oxidation both in vitro and in vivo.     

Inhibitory effect of caffeic acid phenethyl ester on angiogenesis, tumor invasion, and metastasis

Caffeic acid phenethyl ester (CAPE) derived from honeybee propolis has been used as a folk medicine and has several proven biological activities. The present study investigated the effect of CAPE on angiogenesis, tumor invasion, and metastasis. A cytotoxicity assay of CAPE in CT26 colon adenocarcinoma cells showed a dose-dependent decrease in cell viability but no significant influence on the growth of human umbilical vein epithelial cells (HUVEC). A low concentration of CAPE (1.5 microg/mL) inhibited 52.7% of capillary-like tube formation in HUVEC culture on Matrigel. CAPE (6 microg/mL)-treated CT26 cells showed not only inhibited cell invasion by 47.8% but also decreased expression of matrix metalloproteinase (MMP)-2 and -9. Vascular endothelial growth factor (VEGF) production from CT26 cells was also inhibited by treatment with CAPE (6 microg/mL). Intraperitoneal injection of CAPE (10 mg/kg/day) in BALB/c mice reduced the pulmonary metastatic capacity of CT26 cells accompanied with a decreased plasma VEGF level. CAPE treatment also prolonged the survival of mice implanted with CT26 cells. These results indicate that CAPE has potential as an antimetastatic agent.     

Suppression of lipopolysaccharide and galactosamine-induced hepatic inflammation by red grape pomace

Grape pomace is generated in the production process of wine and grape juices and is an industrial waste. This study investigated whether an intake of grape pomace was able to suppress chronic inflammation induced by lipopolysaccharide (LPS) and galactosamine (GalN) in vivo. When Sprague-Dawley rats were orally given methanolic extracts from red and white grape pomace, the extracts inhibited the LPS/GalN-evoked activation of nuclear factor-κB (NF-κB) dose-dependently, and red grape pomace exerted a stronger effect than white grape one. Next, rats were fed an AIN93 M-based diet containing 5% red grape pomace for 7 days, followed by the intraperitoneal injection of LPS and GalN. The intake of the red grape pomace-supplemented diet was found to suppress the LPS/GalN-induced activation of NF-κB and expression of inducible nitric oxide synthase and cyclooxygenase-2 proteins. These results suggest that red grape pomace may contain an abundance of effective compound(s) for anti-inflammatory action.     

Mechanisms involved in the antiplatelet activity of rutin, a glycoside of the flavonol quercetin, in human platelets

The aim of this study was to systematically examine the inhibitory mechanisms of rutin, a well-known flavonoid in platelet aggregation. In this study, rutin concentration-dependently (250 and 290 microM) inhibited platelet aggregation in human platelets stimulated by agonists (i.e., collagen). Rutin (250 and 290 microM) did not significantly interfere with the binding of FITC-triflavin to the glycoprotein IIb/IIIa complex in human platelets. Rutin (250 and 290 microM) markedly inhibited intracellular Ca(2+) mobilization and thromboxane A(2) formation in human platelets stimulated by collagen. Rapid phosphorylation of a platelet protein of M(r) 47000 (P47), a marker of protein kinase C activation, was triggered by collagen (1 microg/mL). This phosphorylation was markedly inhibited by rutin (250 and 290 microM). On the other hand, rutin (250 and 290 microM) did not significantly increase the formations of cyclic AMP and nitric oxide/cyclic GMP in platelets. In conclusion, these results indicate that the antiplatelet activity of rutin may involve the following pathways: rutin inhibited the activation of phospholipase C, followed by inhibition of protein kinase C activity and thromboxane A(2) formation, thereby leading to inhibition of the phosphorylation of P47 and intracellular Ca(2+) mobilization, finally resulting in inhibition of platelet aggregation.     

Minor components of olive oil modulate proatherogenic adhesion molecules involved in endothelial activation

The Mediterranean diet reduces the risk of coronary artery disease as a consequence of its high content of antioxidants, namely, hydroxytyrosol (HT) and oleuropein aglycone (OleA), typical of virgin olive oil. Because intercellular and vascular cell adhesion molecules (ICAM-1 and VCAM-1) and E-selectin are crucial for endothelial activation, the role of the phenolic extract from extra virgin olive oil (OPE), OleA, HT, and homovanillyl alcohol (HVA) on cell surface and mRNA expression in human umbilical vascular endothelial cells (HUVEC) was evaluated. OPE strongly reduced cell surface expression of ICAM-1 and VCAM-1 at concentrations physiologically relevant (IC50 < 1 microM), linked to a reduction in mRNA levels. OleA and HT were the main components responsible for these effects. HVA inhibited cell surface expression of all the adhesion molecules, whereas the effect on mRNA expression was weaker. These results supply new insights on the protective role of olive oil against vascular risk through the down-regulation of adhesion molecules involved in early atherogenesis.     

Antioxidant and cellular activities of anthocyanins and their corresponding vitisins A--studies in platelets, monocytes, and human endothelial cells

During red wine aging, there is a loss of anthocyanins and the formation of various other pigments, so-called vitisins A, which are formed through the chemical interaction of the original anthocyanins with pyruvic acid. The objective of this study was to investigate the antioxidant activities of the most abundant anthocyanins present in red wine (glycosides of delphinidin, petunidin, and malvidin) and their corresponding vitisins A. Anthocyanins exhibited a higher iron reducing as well as 2,2'-azinobis (3-ethyl-benzothiazoline-6-sulfonate) and peroxyl radical scavenging activity than their corresponding vitisins A. Delphinidin showed the highest antioxidant effect of the tested compounds in all of the assays used. Furthermore, we studied the effect of anthocyanins and vitisins A on platelet aggregation and monocyte and endothelial function. Anthocyanins and vitisins did not affect nitric oxide production and tumor necrosis factor-alpha (TNF-alpha) secretion in lipopolysaccharide plus interferon-gamma-activated macrophages. Furthermore, anthocyanins and vitisins did not change collagen-induced platelet aggregation in vitro. However, anthocyanins and to a lesser extent vitisins exhibited protective effects against TNF-alpha-induced monocyte chemoattractant protein production in primary human endothelial cells.     

Hydroxytyrosol-rich olive mill wastewater extract protects brain cells in vitro and ex vivo

Elevated oxidative and nitrosative stress both impair the integrity and functioning of brain tissue, especially in aging. As long-term intake of plant foods rich in antioxidant phenolics, such as extra virgin olive oil, positively modulates surrogate markers of many human pathological alterations, the interest in cheap and abundant sources of such phenolics is rapidly growing. Olive mill wastewater is particularly rich in hydroxytyrosol, an o-diphenol with powerful antioxidant, anti-inflammatory, and antithrombotic activities. Due to the deleterious effect of oxidative stress on brain cell survival, the efficacy of a hydroxytyrosol-rich extract to attenuate Fe2+- and nitric oxide (NO)-induced cytotoxicity in murine-dissociated brain cells was investigated. The addition of either Fe2+ or SNP (an NO donor) caused both a severe loss of cellular ATP and a markedly depolarized mitochondrial membrane potential. Preincubation with hydroxytyrosol significantly attenuated the cytotoxic effect of both stressors, although with different efficiencies. Mice feeding studies were performed to assess the brain bioactivity of hydroxytyrosol ex vivo. Subchronic, but not acute, administration of 100 mg of hydroxytyrosol per kilogram body weight for 12 days enhanced resistance of dissociated brain cells to oxidative stress, as shown by reduced basal and stress-induced lipid peroxidation. Also, basal mitochondrial membrane potential was moderately hyperpolarized (P < 0.05), an effect suggestive of cytoprotection. In synthesis, the ex vivo data provide the first evidence of neuroprotective effects of oral hydroxytyrosol intake. Keywords: Hydroxytyrosol; olive mill wastewater; dissociated brain cells; oxidative stress; brain; Mediterranean diet.     

A highly convenient synthesis of hydroxytyrosol and its recovery from agricultural waste waters

Hydroxytyrosol, a polyphenol with very interesting antioxidant properties, which naturally occurs in virgin olive oil and mainly in olive oil mill waste waters, was synthesized by reducing 3, 4-dihydroxyphenylacetic acid with LiAlH(4) in tetrahydrofuran under refluxing for 2 h. The yield of reaction was 82.8%. The spectroscopic and HPLC data of the synthesized compound proved to coincide fully with those of a pure sample obtained by the chromatographic recovery from olive oil mill waste waters (yield = 91 mg/L). This synthetic method appears to be the most convenient compared with those reported in the literature and is more convenient than the chromatographic recovery. The tri- and diacetyl derivatives of the synthetic compound were also prepared for structure-bioactivity relationship studies. A brief discussion is given on the economical and ecological aspects regarding the production of hydroxytyrosol.     

High-level expression of milk-derived antihypertensive peptide in Escherichia coli and its bioactivity

An optimal antihypertensive peptide (AHP), KVLPVP, was linked to form a six-copy of tandem dotetracontapeptide with the specific cleavage site (Arg-X) of clostripain. The gene of the dotetracontapeptide was synthesized and expressed in Escherichia coli BL21. After a 5 h induction with 1.2 mM isopropyl-beta-D-thiogalactopyranoside the recombinant AHP fused with glutathione-S-transferase tag reached the maximal production, 24.6% of total intracellular protein. Following digestion with clostripain and carboxypeptidase B, the product was separated with ultrafiltration and reversed-phase HPLC, and 170 mg of pure recombinant AHP was obtained from 1 L of E. coli culture. The IC50 of the recombinant AHP was 4.6 microM. The systolic blood pressure of spontaneously hypertensive rats could be decreased dramatically by the recombinant AHP in a dose-dependent manner after delivering 0.3 mg of AHP/kg of body weight (BW) or 0.6 mg of AHP/kg of BW orally. The strong antihypertensive effect was reached 4-24 h after oral administration of 0.3 mg of AHP/kg of BW, and the peak point was at the fourth hour (-21.4 +/- 7.2 mm of Hg). This study overcame traditional enzymatic digestion problems in preparing AHP and established a novel approach for industrial production of AHP.