David D. Kitts

Antioxidant activity of the flaxseed lignan secoisolariciresinol diglycoside and its mammalian lignan metabolites enterodiol and enterolactone.

The antioxidant activities of the flaxseed lignan secoisolariciresinol diglycoside (SDG) and its mammalian lignan metabolites, enterodiol (ED) and enterolactone (EL), were evaluated in both lipid and aqueous in vitro model systems. All three lignans significantly (p ≤ 0.05) inhibited the linoleic acid peroxidation at both 10 and 100 μM over a 24-48 h of incubation at 40°C. In a deoxyribose assay, which evaluates the non site-specific and site-specific Fenton reactant-induced ·OH scavenging activity, SDG demonstrated the weakest activity compared to ED and EL at both 10 and 100 μM; the greatest ·OH scavenging for ED and EL was observed at 100 μM in both assays. The incubation of pBR322 plasmid DNA with Fenton reagents together with SDG, ED or EL showed that the inhibition of DNA scissions was concentration dependent. The greatest non site-specific activity of lignans was at 100 μM, thus, confirming the results of the deoxyribose test. In contrast, the protective effect of SDG and EL in the site-specific assay was lost and that of ED was minimal. Therefore, the results indicate a structure-activity difference among the three lignans with respect to specific antioxidant efficacy. All three lignans did not exhibit reducing activity compared to ascorbic acid, therefore, did not possess indirect prooxidant activity related to potential changes in redox state of transition metals. The efficacy of SDG and particularly the mammalian lignans ED and EL to act as antioxidants in lipid and aqueous in vitro model systems, at relatively low concentrations (i.e. 100 μM), potentially achievable in vivo, is an evidence of a potential anticarcinogenic mechanism of flaxseed lignan SDG and its mammalian metabolites ED and EL.

Antioxidant properties of a North American ginseng extract

A North American ginseng extract (NAGE) containing known principle ginsenosides for Panax quinquefolius was assayed for metal chelation, affinity to scavenge DPPH-stable free radical, and peroxyl (LOO·) and hydroxyl (·OH) free radicals for the purpose of characterizing mechanisms of antioxidant activity. Dissociation constants (Kd) for NAGE to bind transition metals were in the order of Fe2+ > Cu2+ > Fe3+ and corresponded to the affinity to inhibit metal induced lipid peroxidation. In a metal-free linoleic acid emulsion, NAGE exhibited a significant (p ≤ 0.05) concentration (0.01-10 mg/mL) dependent mitigation of lipid oxidation as assessed by the ammonium thiocyanate method. Similar results were obtained when NAGE was incubated in a methyl linoleate emulsion containing haemoglobin catalyst and assessed by an oxygen electrode. NAGE also showed strong DPPH radical scavenging activity up to a concentration of 1.6 mg/mL (r2 = 0.996). Similar results were obtained for scavenging of both site-specific and non site-specific ·OH, using the deoxyribose assay method. Moreover, NAGE effectively inhibited the non site-specific DNA strand breakage caused by Fenton agents, and suppressed the Fenton induced oxidation of a 66 Kd soluble protein obtained from mouse brain over a concentration range of 2-40 mg/mL. These results indicate that NAGE exhibits effective antioxidant activity in both lipid and aqueous mediums by both chelation of metal ions and scavenging of free radicals.

Structure-function relationship exists for ginsenosides in reducing cell proliferation and inducing apoptosis in the human leukemia (THP-1) cell line

Ginsenosides of the 20(S)-protopanaxadiol and 20(S)-protopanaxatriol classifications including the aglycones, protopanaxadiol (PD), protopanaxatriol (PT), and ginsenosides Rh2 and Rh1 were shown to posses characteristic effects on the proliferation of human leukemia cells (THP-1). A similar efficacy was not apparent for ginsenoside Rg3. The concentrations to inhibit 50% of cells (LC50) for PD, Rh2, PT, and Rh1 were 13, 15, 19, and 210 microg/mL, respectively. PD and PT induced DNA fragmentation at the LC50 after 72 h of treatment, compared to Rh2, Rh1, dexamethasone, and untreated cells. Cell-cycle analysis confirmed apoptosis with PD and PT treatment of THP-1 cells resulting in a buildup of sub-G1 cells after 24, 48, and 72 h of treatment. Rh2 and dexamethasone treatments also increased apoptotic cells after 24 h, whereas Rh1 did not. After 48 and 72 h, Rh2, Rh1, and dexamethasone similarly increased apoptosis, but these effects were significantly (P<0.05) lower than those observed for both PD and PT treatments. Furthermore, treatments that produced the largest buildup of apoptotic cells were also found to have the largest release of lactate dehydrogenase. It can be concluded from these studies that the presence of sugars in PD and PT aglycone structures reduces the potency to induce apoptosis, and alternately alter membrane integrity. These cytotoxic effects were different to THP-1 cells than dexamethasone.

Luteolin and luteolin-7-O-glucoside from dandelion flower suppress iNOS and COX-2 in RAW264.7 cells

Both reactive oxygen- and nitrogen-derived reactive species play important roles in physiological and pathophysiological conditions. Flavones, luteolin and luteolin-7-O-glucoside along with a rich plant source of both flavones, namely dandelion (Taraxacum officinale) flower extract were studied for antioxidant activity in different in vitro model systems. In this current study, luteolin and luteolin-7-O-glucoside at concentrations lower than 20 μM, significantly (p < 0.05) suppressed the productions of nitric oxide and prostaglandin E2 (PGE2) in bacterial lipopolysaccharide activated-mouse macrophage RAW264.7 cells without introducing cytotoxicity. The inhibitory effects were further attributed to the suppression of both inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression, and not reduced enzymatic activity. Similar suppression for both inducible enzymes was also found with the presence of dandelion flower extract, specifically, the ethyl acetate fraction of dandelion flower extract which contained 10% luteolin and luteolin-7-O-glucoside.

Antioxidant activity of a Rhus verniciflua Stokes ethanol extract.

A fractionated ethanol extract derived from Rhus Verniciflua Stokes (RVS) was assessed in both organic and aqueous media for the purpose of characterizing the mechanisms of antioxidant activity. RVS, an indigenous plant to Korea, was initially extracted with ethanol and characterized to contain a 90 KDa-ABTS reactive protein possessing 0.662 ng/mg copper. This characterization suggested that a primary component of RVS was Laccase, an oxidase enzyme complex. RVS exhibited a significant (P < 0.01) concentration-dependent inhibition of linoleic acid oxidation in an emulsion system up to 48 hours of incubation. Free radical scavenging activity of both a stable radical (e.g DPPH) and hydroxyl (e.g. *OH) radical followed a concentration-dependent pattern in different model systems. Using a liposome model with peroxyl radicals generated by AAPH, a significant extension of both the lag phase and a reduction of peak propagation of peroxyl radicals by RVS over a concentration range of 1 to 10 microg/ml was observed. RVS ethanol extract was also found to protect human low-density lipoprotein (LDL) from oxidative modification, mediated by cupric ion at 37 degrees C. Finally, RVS was found to be effective at protecting against plasmid DNA strand breakage induced by peroxyl free radicals in an aqueous medium. Our findings show that the ethanol fraction derived from RVS contained significant antioxidant activity in both polar and non-polar mediums.

In vitro and in vivo inhibition of muscle lipid and protein oxidation by carnosine.

Carnosine, a β‐alanyl‐L‐histidine dipeptide with antioxidant properties is present at high concentrations in skeletal muscle tissue. In this study, we report on the antioxidant activity of carnosine on muscle lipid and protein stability from both in vitro and in vivo experiments. Carnosine inhibited lipid peroxidation and oxidative modification of protein in muscle tissue prepared from rat hind limb homogenates exposed to in vitro Fenton reactant (Fe2+, H2O2)‐generated free radicals. The minimum effective concentrations of carnosine for lipid and protein oxidation were 2.5 and 1 mM, respectively. Histidine and β‐alanine, active components of carnosine, showed no individual effect towards inhibiting either lipid or protein oxidation. Skeletal muscle of rats fed a histidine supplemented diet for 13 days exhibited a marked increase in carnosine content with a concomitant reduction in muscle lipid peroxidation and protein carbonyl content in skeletal muscle caused by subjecting rats to a Fe‐nitrilotriacetate administration treatment. This significant in vitro result confirms the in vivo antioxidant activity of carnosine for both lipid and protein constituents of muscle under physiological conditions.

Stability of bovine immunoglobulins to thermal treatment and processing

Abstract Thermal stability of bovine immunoglobulin (IgG) in model systems and in commercially processed milk products was investigated. Bovine serum IgG dissolved in either phosphate buffered saline (PBS), boiled milk or ultra high temperature (UHT) sterilized milk was heated at temperatures ranging from 62.7 °C to 80 °C. The D -values ranged from 90, 200 and 170 s at 80 °C to 25.5, 27.2 and 32.8 min at 72 °C for IgG in PBS, boiled milk and UHT milk, respectively. These results suggest slightly greater thermal stability of IgG in milk than in phosphate buffer. IgG content was not changed after 30 min at 62.7 °C, nor by holding for 24 h at either ambient temperature or 4 °C. Over the temperature range from 72 ° to 80 °C, z -values were 6.7, 8.9 and 8.5 °C, and energies of activation were 353.5, 258.2 and 298.5 kJ mol −1 for thermal destruction of bovine serum IgG in PBS, boiled milk and UHT milk, respectively. These findings in model systems suggest that commercial pasteurization processes should not result in complete destruction of IgG. Comparison of IgG content in raw milks and corresponding HTST-pasteurized milks of varying fat content indicated 59–76% retention after pasteurization, with no apparent effect by either homogenization, skimming or standardization to 1 or 2% fat levels. The IgG contents and specific antibody activity against lipopolysaccharide fractions of five bacteria were determined for several commercially processed milk products. HTST-pasteurized milks, reconstituted skim milk powder and whey from cheddar cheese production all showed high levels of IgG and specific antibody activity. However, canned evaporated milk and ultra high temperature (UHT) sterilized milk had little or no IgG. This study demonstrates the dependence of bovine IgG stability in milk products on severity of thermal treatment used in various commercial processes.

Role of Chlorogenic Acids in Controlling Oxidative and Inflammatory Stress Conditions

Chlorogenic acids (CGAs) are esters formed between caffeic and quinic acids, and represent an abundant group of plant polyphenols present in the human diet. CGAs have different subgroups that include caffeoylquinic, p-coumaroylquinic, and feruloyquinic acids. Results of epidemiological studies suggest that the consumption of beverages such as coffee, tea, wine, different herbal infusions, and also some fruit juices are linked to reduced risks of developing different chronic diseases. These beverages contain CGAs present in different concentrations and isomeric mixtures. The underlying mechanism(s) for specific health benefits attributed to CGAs involves mitigating oxidative stress, and hence the related adverse effects associated with an unbalanced intracellular redox state. There is also evidence to show that CGAs exhibit anti-inflammatory activities by modulating a number of important metabolic pathways. This review will focus on three specific aspects of the relevance of CGAs in coffee beverages; namely: (1) the relative composition of different CGA isomers present in coffee beverages; (2) analysis of in vitro and in vivo evidence that CGAs and individual isomers can mitigate oxidative and inflammatory stresses; and (3) description of the molecular mechanisms that have a key role in the cell signaling activity that underlines important functions.

Evaluation of antioxidant activity of epigallocatechin gallate in biphasic model systems in vitro.

The antioxidant activity of epigallocatechin gallate (EGCG) was studied in different in vitro model systems, which enabled evaluation of both chemical and physical factors involved in assessing the role of EGCG in oxidative reactions. EGCG suppressed the initiation rate and prolonged the lag phase duration of peroxyl radical-induced oxidation in a phospholipid liposome model to a greater extent (p < 0.01) compared to both Trolox and α-tocopherol. Effectiveness of these antioxidants to prolong the peroxyl radical-induced lag phase was inversely related to lipophilic character. EGCG also protected against both peroxyl radical and hydroxyl radical-induced supercoiled DNA nicking. The rate constant describing EGCG reaction against hydroxyl radical was 4.22 ± 0.07 × 1010 M–1·sec–1, which was comparable to those of Trolox and α-tocopherol, respectively. EGCG exhibited a synergistic effect with α-tocopherol in scavenging 1,1-diphenyl-2-picylhydrazyl (DPPH) radical, thus displaying a direct free radical scavenging capacity. In vitro Cu2+-induced-human LDL oxidation was accelerated in the presence of EGCG and attributed to the conversion of Cu2+ to Cu+. We conclude that the particularly effective antioxidant properties of EGCG noted in both chemical and biological biphasic systems were related to a unique hydrophilic and lipophilic balance which enabled effective free radical scavenging. The same chemical-physical properties of EGCG also enabled prooxidant activity, only when in contact with unbound transition metal ions in a multiphasic system.

Effect of casein, casein phosphopeptides and calcium intake on ileal 45Ca disappearance and temporal systolic blood pressure in spontaneously hypertensive rats

Paracellular 45Ca absorption and temporal systolic blood pressure (SBP) measurements were recorded in spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats fed on casein (C) and soya-bean-protein isolate (S) diets, containing 20 (H), 5 (H) and 0.5 (L) g Ca/kg. Similar measurements were also taken in SHR rats only fed on C-M and S-M diets supplemented with 30 g caseinophosphopeptides (CPP)/kg. Absorption of 45Ca from the ileal loop was equivalent in both SHR and WKY animals and largely affected by the level of dietary Ca. In addition, animals fed on C diets exhibited significantly (P < 0.05) greater ileal absorption of 45Ca compared with S-fed animals. This result was attributed to the presence of CPP and a greater (P < 0.05) proportion of soluble 45Ca in the contents of the ileum. Animals fed on S diets supplemented with CPP confirmed this finding. The SBP of SHR rats was higher (P < 0.01) than WKY controls after 9-10 weeks of age. The temporal pattern of observed hypertension was independent of dietary influence in the SHR. The severity of hypertension in SHR rats was affected only by dietary Ca deficiency, and not by Ca supplementation or CPP enhancement of Ca bioavailability. These findings suggest that tryptic digestion products of casein in milk can enhance Ca bioavailability by increasing Ca solubility; however, this action had no effect in reducing hypertension in SHR.