Protective effect against oxidation of human low-density lipoprotein and plasmid DNA strand scission of Tamarind seed coat extract in vitro

Methanolic extract from seed coat of Thai Tamarindus indica L. containing polyphenol; procyanidins, (−)-epicatechin was studied in vitro for protective effect against Cu²⁺-induced human low-density lipoprotein (hLDL) oxidation and oxidative damage of plasmid DNA induced by Fenton reactant. LDL oxidation was monitored by formation of conjugated dienes, fluorescent products, protein carbonylation and thiobarbituric acid reactive substances (TBARS). Supercoiled DNA strand scission was evaluated by gel electrophoresis. The extract inhibited formation of all four products of oxidized LDL in a dose-dependent manner over 25-1000 ng/ml extracts. Furthermore, 1 mg/ml extract also protected supercoiled DNA strand in plasmid pBR322 against scission induced by Fenton-mediated hydroxyl radical similar to that found with reference antioxidant, 0.5 μg/ml Trolox. Thus, seed coat extract from Thai Tamarind shows antioxidant effects against in vitro Cu²⁺-mediated LDL oxidation and hydroxyl radical-induced supercoiled DNA damage. These results suggest that seed coat extract from Thai Tamarind may be useful for preventing LDL oxidation and DNA damage.     

Dihydrocaffeic acid,a major microbial metabolite of chlorogenic acids,shows similar protective effect than a yerba mate phenolic extract against oxidative stress in HepG2 cells

The hepatoprotective effect of a yerba mate phenolic extract (YMPE), rich in chlorogenic acids, and its main circulating metabolites dihydrocaffeic (DHCA) and dihydroferulic (DHFA) acids were assessed in human hepatoma HepG2 cells subjected to oxidative damage induced by tert-butylhydroperoxide (t-BOOH). Direct treatment of HepG2 cells with realistic concentrations of YMPE (1, 10 and 50 μg/mL), DHCA or DHFA (0.2, 1, 10 μM) for 20 h was not cytotoxic and significantly decreased ROS generation. Pre-treatment with YMPE and DHCA prevented the cytotoxicity and macromolecular damage induced by t-BOOH. Moreover, decreased levels of reduced glutathione (GSH), and increased ROS levels and antioxidant enzyme activity induced by t-BOOH were dose-dependently recovered. DHFA only showed a slight protection against cell cytotoxicity, lipid oxidation and GSH depletion. In conclusion, YMPE and one of its major microbial metabolites, DHCA, confer significant protection against oxidative damage, adding evidences to the beneficial health effects associated with mate intake.     

Study of the inhibitory activity of phenolic compounds found in olive products and their degradation by Lactobacillus plantarum strains

Lactobacillus plantarum is the main species responsible for the spontaneous fermentation of Spanish-style green olives. Olives and virgin oil provide a rich source of phenolic compounds. This study was designed to evaluate inhibitory growth activities of nine olive phenolic compounds against four L. plantarum strains isolated from different sources, and to explore the L. plantarum metabolic activities against these phenolic compounds. None of the nine compounds assayed (oleuropein, hydroxytyrosol, tyrosol, as well as vanillic, p-hydroxybenzoic, sinapic, syringic, protocatechuic and cinnamic acids) inhibited L. plantarum growth at the concentration found in olive products. Oleuropein and tyrosol concentrations higher than 100 mM were needed to inhibit L. plantarum growth. On the other hand, sinapic and syringic acid showed the highest inhibitory activity since concentrations ranging from 12.5 to 50 mM inhibited L. plantarum growth in all the strains analyzed. Among the nine compounds assayed, only oleuropein and protocatechuic acid were metabolized by L. plantarum strains grown in the presence of these compounds. Oleuropein was metabolized mainly to hydroxytyrosol, while protocatechuic acid was decarboxylated to catechol. Metabolism of oleuropein was carried out by inducible enzymes since a cell-free extract from a culture grown in the absence of oleuropein was unable to metabolize it. Independent of their isolation source, the four L. plantarum strains analysed showed similar behaviour in relation to the inhibitory activity of phenolic compounds, as well as their ability to metabolize these compounds.     

Health-beneficial qualities of the edible mushroom, Agrocybe aegerita

The black poplar mushroom, Agrocybe aegerita is a popular edible mushroom with reported anti-tumor properties. A bioactivity-guided investigation gave positive results for ceramide (1), methyl-β-d-glucopyranoside and α-d-glucopyranoside, along with already reported linoleic acid and its methyl ester. The structure elucidation of the above was accomplished by NMR and mass spectral methods. The ceramide (1) inhibited cyclooxygenase enzymes, COX-1 and -2, by 43 and 92.3%, respectively at 25 μg/ml (34.4 μM). The 50% inhibition concentration (IC₅₀) of compound 1 against COX-2 was 5.3 μg/ml (7.3 μM). Similarly, its anti-cancer potential was investigated against five human cancer cell lines in vitro and it was found to inhibit the proliferation of stomach, breast and CNS cancer cell lines at 26.9, 23.2 and 39.1%, respectively, at 100 μg/ml (139 μM) concentration. This is the first report of the isolation of ceramide from A. aegerita and its COX and tumor cell proliferation inhibitory activities. This suggested that the consumption of A. aegerita would assist in alleviating inflammatory conditions, as well as reducing the development of the above cancers.     

Antioxidant properties of mashua (Tropaeolum tuberosum) phenolic extracts against oxidative damage using biological in vitro assays

Purified mashua extracts (PME) from four different coloured mashua genotypes were assayed for oxidative damage prevention. Three in vitro assays for oxidative damage to biological structures rich in polyunsaturated fatty acids (PUFA), such as LDL and erythrocytes, were tested: AAPH-induced TBARS assay and Cu²⁺-induced conjugated dienes assay for LDL oxidation and AAPH-induced oxidative hemolysis of erythrocytes. Additionally, ORAC antioxidant capacity, total phenolics (TP), total flavanoids (TFA) and total anthocyanins (TA) were evaluated. In the presence of 5 μM of gallic acid equivalents (GAE), inhibitions of LDL oxidation for the PME ranged from 29.1% to 34.8% and from 51.8% to 58.1% when the TBARS and conjugated dienes assays were performed, respectively. PME inhibited the hemolysis of erythrocytes within the range 20.8–25.1%. Thus, mashua phenolic extracts are capable of scavenging peroxyl radicals, as well as chelating redox metal ions in vitro. ORAC and LDL protection (TBARS and conjugated dienes assays) showed good correlations with the TP and TFA, suggesting that these compounds have a good ability to protect LDL molecules under the employed conditions. In contrast, inhibition of hemolysis did not show any correlation with the evaluated phenolic assays (TP, TA, TFA) or with any of the evaluated oxidative LDL assays, suggesting a specific action of some non-evaluated compounds present in the PME. The results of this study indicate that the mashua polyphenol extracts displayed good antioxidant properties against oxidative damage in biological structures rich in PUFA. The displayed antioxidant properties could be applied in the field of food or cosmetic industry.     

In vitro activity of the essential oils of Origanum vulgare, Satureja montana and their main constituents in peroxynitrite-induced oxidative processes

The essential oil obtained from aerial parts of Satureja montana L. and Origanum vulgare L. (Labiatae) along with four of its main components, p-cymene, carvacrol, thymol and γ-terpinene were tested in models of in vitro peroxynitrite-induced formation of both 3-nitrotyrosine and malondialdehyde, two biomarkers of the oxidative stress of recognised pathological and toxicological significance. The essential oils showed a significant activity, thus decreasing 3-nitrotyrosine formation (IC₅₀ values of 43.9 μg/ml for S. montana and 19.2 μg/ml for O. vulgare), and also inhibited the peroxynitrite induced malondialdehyde formation (IC₅₀ values of 27.2 μg/ml and 17.0 μg/ml respectively). Thymol and carvacrol inhibited 3-nitrotyrosine formation (IC₅₀ values of 81.3 μM and 106.3 μM; ascorbic acid IC₅₀ = 400 μM) and reduced malondialdehyde formation (IC₅₀ values of 43.9 μM and 70.1 μM respectively; trolox IC₅₀ = 240 μM). On the contrary, p-cymene and γ-terpinene were completely inactive in both assays under the concentration of 300 μg/ml. These results support, in particular for origanum, the nutraceutical value of these spices and the potential of thymol and carvacrol in preventing the formation of toxic products by the action of reactive nitrogen species.     

Inhibition of the formation of advanced glycation end products by thymoquinone

The inhibitory activity of thymoquinone, a major quinone from black seeds (Nigella sativa) against the formation of advanced glycation end products was studied using the hemoglobin-δ-gluconolactone, human serum albumin–glucose, and the N-acetyl-glycyl-lysine methyl ester–ribose assays. A comparison was made with the inhibitory activity of aminoguanidine. The cytotoxicity of thymoquinone was studied by the release of lactate dehydrogenase from platelets and the levels of plasma thiols. At 20 μM, thymoquinone inhibited 39% of hemoglobin glycation, 82% of post-Amadori glycation products, reduced methyglyoxal-mediated human serum albumin glycation by 68%, inhibited 78% of late glycation end products. Aminoguanidine at 10 mM was less effective than thymoquinone. The IC₅₀ for thymoquinone and aminoguanidine were 7.2 μM and 1.25 mM, respectively. Thymoquinone at 20–50 μM was not toxic to platelet lactate dehydrogenase and plasma thiols. The potential of thymoquinone in food applications is discussed.     

Curtailing Oxidation‐Induced Loss of Myosin Gelling Potential by Pyrophosphate Through Shielding the S1 Subfragment

In muscle food processing, where oxidation is inevitable, phosphates are usually added to improve water binding. This present study attempted to investigate the interactive roles of protein oxidation and pyrophosphate (PP) during thermal gelation of myosin. Myosin isolated from pork muscle was solubilized in 0.5 M NaCl at pH 6.2 then oxidatively stressed with an iron‐redox cycling system that produces hydroxyl radicals with or without 1 mM PP and 2 mM MgCl₂ at 4 °C for 12 or 24 h then heated to 50 °C at 1.3 °C/min. Protein conformational stability was measured by differential scanning calorimetry, and covalent cross‐linking was examined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis following chymotrypsin digestion. The binding of PP to myosin suppressed disulfide bond formation in myosin subfragments 1 and 2 and partially inhibited oxidation‐initiated cross‐linking of heavy meromyosin during myosin gelation with a lesser effect on light meromyosin. In the presence of PP, myosin exhibited less loss of conformational integrity upon oxidation than myosin without PP. Rheological analysis from 20 to 75 °C indicated up to 32% decreases (P < 0.05) in elastic modulus (G′) of myosin gels due to oxidation. However, the presence of 1 mM PP, which also lowered the gelling capacity of myosin, inhibited the oxidation‐induced G′ by nearly half (P < 0.05). These results suggest that the protection of myosin head from oxidative modification by PP can be a significant factor for the minimization of gelling property losses during cooking of comminuted meats.     

Salvianolic acid B inhibits low‐density lipoprotein oxidation and neointimal hyperplasia in endothelium‐denuded hypercholesterolaemic rabbits

BACKGROUND: Atherosclerosis and restenosis are inflammatory responses involving free radicals and lipid peroxidation and may be prevented/cured by antioxidant‐mediated lipid peroxidation inhibition. Salvianolic acid (Sal B), a water‐soluble antioxidant obtained from a Chinese medicinal herb, is believed to have multiple preventive and therapeutic effects against human vascular diseases. In this study the in vitro and in vivo inhibitory effects of Sal B on oxidative stress were determined. RESULTS: In human aortic endothelial cells (HAECs), Sal B reduced oxidative stress, inhibited low‐density lipoprotein (LDL) oxidation and reduced oxidised LDL‐induced cytotoxicity. Sal B inhibited Cu²⁺‐induced LDL oxidation in vitro (with a potency 16.3 times that of probucol) and attenuated HAEC‐mediated LDL oxidation as well as reactive oxygen species (ROS) production. In cholesterol‐fed New Zealand White rabbits (with probucol as positive control), Sal B intake reduced Cu²⁺‐induced LDL oxidation, lipid deposition in the thoracic aorta, intimal thickness of the aortic arch and thoracic aorta and neointimal formation in the abdominal aorta. CONCLUSION: The data obtained in this study suggest that Sal B protects HAECs from oxidative injury‐mediated cell death via inhibition of ROS production. The antioxidant activity of Sal B may help explain its efficacy in the treatment of vascular diseases. Copyright © 2010 Society of Chemical Industry     

In vitro inhibition of α-glucosidases and glycogen phosphorylase by catechin gallates in green tea

We investigated in vitro inhibition of mammalian carbohydrate-degrading enzymes by green tea extract and the component catechins, and further evaluated their inhibitory activities in cell cultures. The extract showed good inhibition toward rat intestinal maltase and rabbit glycogen phosphorylase (GP) b, with IC₅₀ values of 45 and 7.4 μg/ml, respectively. The polyphenol components, catechin 3-gallate (CG), gallocatechin 3-gallate (GCG), epicatechin 3-gallate (ECG), and epigallocatechin 3-gallate (EGCG), were good inhibitors of maltase, with IC₅₀ values of 62, 67, 40, and 16 μM, respectively, and EGCG also showed good inhibition toward maltase expressed on Caco-2 cells, with an IC₅₀ value of 27 μM. The ungallated catechins, such as catechin, gallocatechin (GC), epicatechin (EC), and epigallocatechin (EGC), showed no significant inhibition toward GP b, whereas the gallated catechins CG, GCG, ECG, and EGCG inhibited the enzyme, with IC₅₀ values of 35, 6.3, 27, and 34 μM. From multiple inhibition studies by Dixon plots, GCG appears to bind a new allostelic site, the indole inhibitor site. These gallated catechins also inhibited glucagon-stimulated glucose production dose-dependently, with IC₅₀ values ranging from 33 to 55 μM. Dietary supplementation with these gallated catechins or the green tea extract containing them, which inhibits both α-glucosidases and GP in vitro and in cell culture, would contribute to the protection or improvement of type 2 diabetes.     

Electrochemical behaviour of Sudan I at Fe3O4 nanoparticles modified glassy carbon electrode and its determination in food samples

In this work, a simple and sensitive electrochemical method was developed to determine Sudan I based on magnetic Fe₃O₄ nanoparticles modified glassy carbon electrode using cyclic voltammetry and differential pulse voltammetry. The sensor exhibited an obviously electrocatalytic activity towards the oxidation of Sudan I, which can be confirmed by the increased oxidation peak current and the decreased oxidation peak potential when compared with the bare GCE. The determination conditions, such as pH, modifier amount, accumulation time and accumulation potential, were optimised. And some kinetic parameters were calculated. Under the optimum experimental conditions, the oxidation current of Sudan I was proportional to its concentration from 0.01 to 1 μM and 1 to 20 μM. The detection limit was estimated to be 0.001 μM (S/N = 3). The developed method was successfully applied to determine Sudan I content in food samples with satisfactory results.     

Antioxidant and hepatoprotective activity of ethanolic extract of leaves of Solidago microglossa containing polyphenolic compounds

The antioxidative and hepatoprotective potential of Solidago microglossa D.C, a widely used medicinal plant from Brazil was investigated. The leaf extract showed inhibition against thiobarbituric acid reactive species (TBARS) induced by different prooxidants (10 μM FeSO₄ and 5 μM sodium nitroprusside SNP) in rat liver, brain and phospholipid homogenates from egg yolk. Moreover, the free radical scavenging activities of the extract was evaluated by the scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) (IC_50, 3.8 ± 0.5 μg/ml) and hydroxyl radical on benzoic acid hydroxylation (IC_50, 32.3 ± 1.3 μg/ml) and deoxyribose (IC_50, 39.1 ± 2.4 μg/ml) assays. The ethanolic extract showed significant hepatoprotective activity against paracetamol (250 mg/kg) induced liver damage in mice in a dose dependent manner. The phenolic composition and their quantification by high performance liquid chromatography (HPLC) resulted in the identification of gallic acid and flavonoids: quercetrin (quercetin-3-O-rhamnoside), rutin (quercetin-3-O-rutinoside) and quercetin.     

Palm-tocotrienol rich fraction (TRF) is a more effective inhibitor of LDL oxidation and endothelial cell lipid peroxidation than α-tocopherol in vitro

Comparative protective effects of palm tocotrienol rich fractions (TRF) and α-tocopherol on the copper-induced oxidation of plasma low density lipoprotein (LDL) and indices of lipid peroxidation in human umbilical vein endothelial cells (HUVEC) were investigated. LDL (100 μg protein/ml) was incubated at 37 °C with 1 μM CuSO₄ with or without the presence of 50 μM (TRF) or 50 μM α-tocopherol. Aliquots were collected at 0,1,2,3,6,9,12 and 24 h and thiobarbituric acid reactive substances (TBARS) determined. In the absence of antioxidant, TBARS increased significantly in LDL reaching a maximum after 6 h. α-Tocopherol and TRF delayed TBARs formation by 6 h with a maximum effect after 12 h. The rate of TBARs formation with TRF was significantly slower than with α-tocopherol, indicating a higher antioxidant efficacy for TRF. Low density lipoprotein isolated from volunteers consuming low or high polyunsaturated fatty acid (PUFA) diets were similarly incubated with 1 μM CuSO₄ with or without 50 μM TRF or 50 μM α-tocopherol. LDL oxidation was not significantly different between low and high PUFA groups. However, LDL treated with TRF was better protected against copper-induced oxidation than that treated with α-tocopherol. In HUVEC pre-incubated with 100 μM arachidonic acid, 25 μM TRF treatment decreased TBARS by ∼73% compared to ∼50% with 25 μM of α-tocopherol. Higher concentrations of TRF did not further decrease TBARS formation in the medium. However, treatment with higher concentrations of α-tocopherol again increased TBARS formation. Formation of conjugated diene in HUVEC subjected to arachidonic acid (ARA)-induced oxidative stress was decreased equally with either 10 μM TRF or 25 μM α-tocopherol. Results suggest that TRF is a more potent antioxidant than α-tocopherol, at least in vitro.     

Metal-Catalyzed Oxidation of Ascorbate, Deoxyribose and Linoleic Acid as Affected by Phytic Acid in a Model System

Antioxidant activity of phytate was investigated in metal-catalyzed model systems. In a dose-dependent manner, phytate facilitated oxidation of Fe (II) to Fe (III) and inhibited formation of thiobarbituric acid-reactive substances (TBARS) from Fe (II)- or hemeprotein-catalyzed deoxyribose degradation. In the presence of 100 μM Fe (III), phytate inhibited reduction of Fe (III) to Fe (II) by 100 μM ascorbic acid and it consequently inhibited ascorbate oxidation. Phytate inhibited hemeprotein- and H₂O₂-catalyzed TBARS formation from linoleic acid micelles. Inhibition by phytate of iron + ascorbate-dependent lipid peroxidation depended on the concentration of ascorbate. These results indicate that phytate may be a useful antioxidant in the protection against oxidative deterioration of foods.     

Antifungal,antiaflatoxin and antioxidant potential of chemically characterized Boswellia carterii Birdw essential oil and its in vivo practical applicability in preservation of Piper nigrum L. fruits

The study explores the chemical profile, antimicrobial and antioxidant activity of Boswellia carterii essential oil (EO). The EO significantly inhibited growth and aflatoxin production by the food borne toxigenic strain of Aspergillus flavus at 1.75 μl/ml and 1.25 μl/ml respectively. It exhibited broad fungitoxic spectrum against 12 food borne moulds and also showed strong antioxidant activity, IC₅₀ value and % inhibition of linoleic acid peroxidation being 0.64 μl/ml and 51.68% respectively. The antifungal action of EO was observed in terms of reduction in ergosterol content of plasma membrane of A. flavus. As fumigant in food system in storage containers, the EO provided 65.38% protection against fungal deterioration of Piper nigrum. GC–MS results revealed 31 components of EO. The chemically characterized B. carterii EO may thus be recommended as plant based preservative in view of its antifungal, antiaflatoxigenic, antioxidant activity and efficacy in food system.     

Flavonoids isolated from Syzygium aqueum leaf extract as potential antihyperglycaemic agents

Syzygium aqueum is a medicinal plant which is grown in tropical regions. In this study, the ethanolic extracts of S. aqueum leaf were investigated for its antihyperglycaemic activity. Our investigation revealed its effectiveness in inhibiting the carbohydrate hydrolysing enzymes, α-glucosidase (EC₅₀ = 11 μg/ml) and α-amylase (EC₅₀ = 8 μg/ml), at significant level than the commercial drug acarbose (EC₅₀ = 28 μg/ml, α-glucosidase; EC₅₀ = 12 μg/ml, α-amylase). In addition, the ethanolic leaf extracts were able to inhibit the key enzyme in the polyol pathway, aldose reductase (EC₅₀ = 0.03 μg/ml) and prevent the AGEs formation by 89%. Six flavonoid compounds, 4-hydroxybenzaldehyde (1), myricetin-3-O-rhamnoside (2), europetin-3-O-rhamnoside (3), phloretin (4), myrigalone-G (5) and myrigalone-B (6), were isolated from the ethanolic leaf extracts. Compounds (2) and (3) showed high inhibitory activities, with EC₅₀ values of 1.1 μM and 1.9 μM against α-glucosidase and EC₅₀ values of 1.9 μM and 2.3 μM against α-amylase, respectively. These findings provide a strong rationale to establish S. aqueum’s capability as an antihyperglycaemic agent.     

Coumestrol induces senescence through protein kinase CKII inhibition-mediated reactive oxygen species production in human breast cancer and colon cancer cells

An inhibitor of the protein kinase CKII (CKII) was purified from leaves of Glycine max (L.) Merrill and was identified as coumestrol by structural analysis. Coumestrol inhibited the phosphotransferase activity of CKII toward β-casein, with an IC₅₀ of about 5 μM. It acted as a competitive inhibitor with respect to ATP as a substrate, with an apparent K_i value of 7.67 μM. Coumestrol at 50 μM resulted in 50% and 30% growth inhibition of human breast cancer MCF-7 and colorectal cancer HCT116 cells, respectively. Coumestrol promoted senescence through the p53-p21^Cip1/WAF1 pathway by inducing reactive oxygen species (ROS) production in MCF-7 and HCT116 cells. The ROS scavenger N-acetyl-l-cysteine (NAC), NADPH oxidase inhibitor apocynin and p22^phox siRNA almost completely abolished this event. Overexpression of CKIIα antagonised cellular senescence mediated by coumestrol, indicating that this compound induced senescence via a CKII-dependent pathway. Since senescence is an important tumour suppression process in vivo, these results suggest that coumestrol can function by inhibiting oncogenic disease, at least in part, through CKII inhibition-mediated cellular senescence.     

Acute ingestion of yerba mate infusion (Ilex paraguariensis) inhibits plasma and lipoprotein oxidation

Yerba mate extract (Ilex paraguariensis) is a source of phenolic compounds that possesses in vitro antioxidant activities and may contribute to a reduction in the risk of cardiovascular disease. In this study we examined the acute effects of the consumption of mate infusion on ex vivo plasma and low-density lipoprotein (LDL) oxidation, plasma antioxidant capacity, and platelet aggregation. Twelve healthy fasted subjects ingested 500 mL of mate infusion and blood samples were collected before and 1 h after mate intake. Lipid peroxidation of plasma and LDL was monitored by the measurement of cholesteryl-ester hydroperoxides (CE-OOH) and cholesterol oxides. The plasma antioxidant capacity was measured as ferric-reducing antioxidant potential (FRAP). Platelet aggregation was evaluated in platelet-rich plasma stimulated with adenosine diphosphate and coagulation was tested in platelet-poor plasma. Ingestion of mate infusion diminished the ex vivo oxidizability of both plasma and LDL particles. After mate intake, the CE-OOH levels were around 50% lower in plasma oxidized with copper or 2,2′-azobis[2-amidine-propane-hydrochloride] (AAPH) and the lag time to plasma oxidation increased 2-fold (P < 0.05). Copper- and AAPH-induced LDL peroxidation were also inhibited by around 50% and 20%, respectively, after mate consumption (P < 0.05). The levels of various oxysterols were significantly reduced in oxidized-plasma and LDL (P < 0.05) and FRAP increased by 7.7% after mate intake (P < 0.01). However, mate consumption did not inhibit platelet aggregation or blood coagulation. In summary, intake of yerba mate infusion improved the antioxidant capacity and the resistance of plasma and LDL particles to ex vivo lipid peroxidation.