Jack N. Losso

Evolution of Phenolic Compounds from Color and Flavor Problems to Health Benefits

Early studies focused on the negative effects on color and flavor of foods, followed by exploration of the antioxidant properties and the associated health benefits. The growing body of evidence suggests that plant-based polyphenols may help prevent or delay the onset of a multiplicity of diseases. Newer work suggests that a variety of polyphenols can alter the expression of genes in the inflammatory pathway. Data also show that the absorption of the polyphenols is very limited. Insulin resistance and endothelial and mitochondrial dysfunction are hallmarks of the metabolic syndrome and aging and occur at the early stages of the disease. There is limited clinical evidence that certain polyphenolic metabolites by virtue of their anti-inflammatory activities can improve insulin sensitivity and endothelial and mitochondrial function, suggesting that polyphenols are good for disease prevention. The goal of this review is to summarize the evolution and emphasize the potential benefits of polyphenols.

Determination of pharmacologically active compounds in root extracts of Cassia alata L. by use of high performance liquid chromatography

A simple high performance liquid chromatography (HPLC) method was developed and validated for the determination of six phenolic compounds, five anthraquinones (rhein, aloe-emodin, emodin, chrysophanol and physcion) and a flavonoid (kaempferol), in root extracts from Cassia alata L. Solid-phase extraction, using C(18) cartridges, was used to remove interfering substances from the root extracts. The extracts were analyzed on a C(18) column using an isocratic mobile phase which consisted of acetonitrile, methanol, and 10mM aqueous ammonium acetate (25:55:20, v/v). Identification of the analytes was performed by use of standards and on-line mass spectrometric detection using atmospheric pressure chemical ionization. The concentration of the phenolic compounds in the root extracts was determined using HPLC with ultraviolet detection at 260nm. The limits of detection obtained for the anlytes were in the range of 0.23-4.61ppm. The overall R.S.D. precision values (intra- and inter-day) for the retention times and peak-areas were lower than 0.16 and 2.10%, respectively. In addition, the recovery of the developed method for the analysis of these phenolic compounds was determined, and ranged from 81.2+/-4.3 to 106+/-2%.

Rhein inhibits angiogenesis and the viability of hormone-dependent and -independent cancer cells under normoxic or hypoxic conditions in vitro

Hypoxia is a hallmark of solid tumors, including breast cancer, and the extent of tumor hypoxia is associated with treatment resistance and poor prognosis. Considering the limited treatment of hypoxic tumor cells and hence a poor prognosis of breast cancer, the investigation of natural products as potential chemopreventive anti-angiogenic agents is of paramount interest. Rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid), the primary anthraquinone in the roots of Cassia alata L., is a naturally occurring quinone which exhibits a variety of biologic activities including anti-cancer activity. However, the effect of rhein on endothelial or cancer cells under hypoxic conditions has never been delineated. Therefore, the aim of this study was to investigate whether rhein inhibits angiogenesis and the viability of hormone-dependent (MCF-7) or -independent (MDA-MB-435s) breast cancer cells in vitro under normoxic or hypoxic conditions. Rhein inhibited vascular endothelial growth factor (VEGF(165))-stimulated human umbilical vein endothelial cell (HUVEC) tube formation, proliferation and migration under normoxic and hypoxic conditions. In addition, rhein inhibited in vitro angiogenesis by suppressing the activation of phosphatidylinositol 3-kinase (PI3K), phosphorylated-AKT (p-AKT) and phosphorylated extracellular signal-regulated kinase (p-ERK) but showed no inhibitory effects on total AKT or ERK. Rhein dose-dependently inhibited the viability of MCF-7 and MDA-MB-435s breast cancer cells under normoxic or hypoxic conditions, and inhibited cell cycle in both cell lines. Furthermore, Western blotting demonstrated that rhein inhibited heat shock protein 90alpha (Hsp90α) activity to induce degradation of Hsp90 client proteins including nuclear factor-kappa B (NF-κB), COX-2, and HER-2. Rhein also inhibited the expression of hypoxia-inducible factor-1 alpha (HIF-1α), vascular endothelial growth factor (VEGF(165)), epidermal growth factor (EGF), and the phosphorylation of inhibitor of NF-κB (I-κB) under normoxic or hypoxic conditions. Taken together, these data indicate that rhein is a promising anti-angiogenic compound for breast cancer cell viability and growth. Therefore, further studies including in vivo and pre-clinical need to be performed.

trans-Resveratrol inhibits hyperglycemia-induced inflammation and connexin downregulation in retinal pigment epithelial cells.

The purpose of this study was to determine the inhibitory activity of trans-resveratrol against hyperglycemia-induced inflammation and degradation of gap junction intercellular communication in retinal pigment epithelial cells. Retinal (ARPE-19) cells were incubated with 5.5 mM glucose, 5.5 mM glucose and 10 microM resveratrol, 33 mM glucose, or 33 mM glucose and 0-10 microM trans-resveratrol at 37 degrees C and 5% CO(2) for 9 days. Cell viability was determined by the crystal violet assay. The levels of low-grade inflammation biomarkers interleukin-6 and interleukin-8 (IL-6 and IL-8), angiogenic factors, and vascular endothelial growth factor (VEGF) were determined by the enzyme-linked immunosorbent assay (ELISA). Gap junction intercellular communication (GJIC) was determined by the scrape-load/dye transfer method. The expression levels of protein kinase Cbeta (PKCbeta), connexin 43 (Cx43), transforming growth factor-beta1 (TGF-beta1), and cyclooxygenase-2 (COX-2) were determined by Western blot. Incubation of retinal cells with 10 microM trans-resveratrol in the presence of 5.5 mM glucose did not affect any of the biomarkers investigated. Incubation of ARPE-19 cells with 33 mM glucose for 9 days significantly induced the accumulation of VEGF, IL-6, IL-8, TGF-beta, and COX-2, activation of PKCbeta, and reduction of Cx43 and GJIC. Incubation of ARPE-19 cells with 33 mM glucose in the presence of 0-10 microM trans-resveratrol dose-dependently inhibited VEGF, TGF-beta1, COX-2, IL-6, and IL-8 accumulation, PKCbeta activation, and Cx43 degradation and enhanced GJIC. These data suggest that trans-resveratrol can protect the retinal pigment epithelial cells against hyperglycemia-induced low-grade inflammation and GJIC degradation.

Tunable Cytotoxicity of Rhodamine 6G via Anion Variations

Chemotherapeutic agents with low toxicity to normal tissues are a major goal in cancer research. In this regard, the therapeutic activities of cationic dyes, such as rhodamine 6G, toward cancer cells have been studied for decades with observed toxicities toward normal and cancer cells. Herein, we report rhodamine 6G-based organic salts with varying counteranions that are stable under physiological conditions, display excellent fluorescence photostability, and more importantly have tunable chemotherapeutic properties. Our in vitro studies indicate that the hydrophobic compounds of this series allow production of nanoparticles which are nontoxic to normal cells and toxic to cancer cells. Furthermore, the anions, in combination with cations such as sodium, were observed to be nontoxic to both normal and cancer cells. To the best of our knowledge, this is the first demonstration that both the cation and anion play an extremely important and cooperative role in the antitumor properties of these compounds.

Fenugreek bread: a treatment for diabetes mellitus.

Use of fenugreek, a food with demonstrated efficacy in lowering blood sugar, is limited by its bitter taste and strong flavor. A bread incorporating fenugreek using a proprietary process was tested for its taste acceptability and its effect on carbohydrate metabolism. We developed a fenugreek bread formula that was produced in a commercial bakery by incorporating fenugreek flour into a standard wheat bread formula. Whole wheat bread was prepared by the same formula in the same bakery using wheat flour. Eight diet-controlled diabetic subjects were served two slices (56 g) and 5% fenugreek. Blood glucose and insulin were tested periodically over a 4-hour period after consumption. The tests were run on two occasions 1 week apart, once with the fenugreek bread and once with regular bread. The study was double-blind, and the order was randomized and balanced. Fenugreek and whole wheat bread samples were evaluated for sensory attributes and nutrient composition. There was no statistically significant difference in proximate composition, color, firmness, texture, and flavor intensity between the fenugreek and wheat bread (P > .05). The area under the curve for glucose and insulin was lower in the fenugreek condition, but only reached significance with insulin (P < .05). The fenugreek-containing bread was indistinguishable from the whole wheat bread control. Normally, fenugreek flour impacts bread quality negatively. The bread maintained fenugreeks functional property of reducing insulin resistance. Acceptable baked products can be prepared with added fenugreek, which will reduce insulin resistance and treat type 2 diabetes.

Differential effect of 14 free fatty acids in the expression of inflammation markers on human arterial coronary cells.

Cardiovascular disease is the leading cause of death in the United States, and circulating free fatty acids (FFAs) are known risk factors associated with cardiovascular inflammation. The influence of 14 dietary FFAs (including saturated, mono- and polyunsaturated, and trans) on the expression of inflammatory markers in human coronary arterial smooth muscle (HCASM) and endothelial (HCEC) cells using a cell culture model was investigated. HCASM and HCEC cell cultures were incubated with 200 μM of each FFA for 8 or 24 h, respectively, at 37 °C in a 5% CO2 humidified incubator. Inflammatory markers were assessed by ELISA or Western blot in the supernatant or cell lysates respectively. Results showed significant differences in the expression of inflammatory markers among the fatty acid treatments and the control, with myristic and palmitic acids being identified as the most and linoleic acid as the least pro-inflammatory. This suggests that FFAs may induce low-grade inflammation in human coronary arterial cells and provides more information on mode of action.

In vitro and in vivo anti-angiogenic activities and inhibition of hormone-dependent and -independent breast cancer cells by ceramide methylaminoethylphosphonate.

Ceramide methylaminoethylphosphonate (CMAEPn) was isolated from eastern oyster ( Crassostrea virginica ) and screened against in vitro and in vivo angiogenesis and against MCF-7 and MDA-MB-435s breast cancer cell lines. In vitro angiogenesis was evaluated by the vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cell (HUVEC) tube formation assay. MCF-7 and MDA-MB-435s cell viability was evaluated by the CellTiter 96 AQ(ueous) One Solution Cell Proliferation assay. Apoptosis was evaluated by the caspase-9 assay, autophagy by acridine orange staining and beclin-1 level. Our study indicates that CMAEPn at 50 microM inhibited VEGF-induced tube formation by HUVEC. The viability of MCF-7 and MDA-MB-435s breast cancer cells exposed to 125 microM CMAEPn for 48 h was reduced to 76 and 85%, respectively. The viability of MCF-7 and MDA-MB-435s cells exposed to 250 microM CMAEPn for 48 h under the same conditions was reduced to 38 and 45%, respectively. CMAEPn at 125 microM inhibited VEGF-induced MDA-MB-435s cell migration and invasion. CMAEPn at 125 microM also decreased VEGF, EGF levels in the conditioned media, PI3K, IkappaB phosphorylation and degradation in the cytoplasmic extracts, and NFkappaB nuclear translocation. Both acridine orange staining and beclin-1 indicated autophagic cell death in MCF-7 and MDA-MB-435s cells, respectively. In vivo, CMAEPn at 30 mg/kg body weight inhibited bFGF-induced angiogenesis and caused a 57% reduction in hemoglobin levels in the matrigel plug assay within 7 days. This is the first report on CMAEPn-inhibited angiogenesis both in vitro and in vivo.

Positive cooperative mechanistic binding of proteins at low concentrations: a comparison of poly (sodium N-undecanoyl sulfate) and sodium dodecyl sulfate.

The interactions of the negatively charged achiral molecular micelle, poly (sodium N-undecanoyl sulfate) (poly-SUS), with four different proteins using intrinsic and extrinsic fluorescence spectroscopic probes, are studied. A comparison of poly-SUS with the conventional surfactant, sodium dodecyl sulfate (SDS), and the monomeric species, SUS, is also reported. In this work, we observed that poly-SUS preferentially binds to acidic proteins, exhibiting positive cooperativity at concentrations less than 1 mM for all proteins studied. Moreover, it appears that the hydrophobic microdomain formed through polymerization of the terminal vinyl group of the monomer, SUS, is largely responsible for the superior binding capacity of poly-SUS. From these results, we conclude that the interactions of poly-SUS with the acidic proteins are predominantly hydrophobic and postulate that poly-SUS would produce superior interactions relative to SDS at low concentrations in polyacrylamide gel electrophoresis (PAGE). As predicted, use of poly-SUS allowed separation of the His-tagged tumor suppressor protein, p53, at sample buffer concentrations as low as 0.08% w/v (2.9 mM), which is 24 times lower than required for SDS in the standard reducing PAGE protocol. This work highlights the use of poly-SUS as an effective surfactant in 1D biochemical analysis.

Ionic liquids as buffer additives in ionic liquid-polyacrylamide gel electrophoresis separation of mixtures of low and high molecular weight proteins

This study aims at investigating methodologies for better separation of proteins using novel hydrophobic ionic liquids (ILs). In this regard, hydrophobic ILs [CnPBr] (n = 4, 6, 8) were synthesized and examined in ionic liquid-polyacrylamide gel electrophoresis (IL-PAGE) as buffer additives for separation of catalase (Cat), transferrin (Tf), bovine serum albumin (BSA), ovalbumin (Ova) and α-lactalbumin (α-Lact). The influence of alkyl chain length of the cation of these ILs and their concentration in running and sample buffers on protein separation was investigated. Separation using ILs as additives was achieved at lower concentrations as compared to standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The IL concentrations were 100-fold less in sample buffer and 5-fold less in running buffer as compared to conventional SDS-PAGE. The results demonstrated that ILs additives played a role in improving some protein separation, IL-PAGE provided higher resolution and separation efficiency than SDS-PAGE for Tf and Ova. Fluorescence studies were performed in order to understand protein–IL interactions and were used to determine the appropriate IL for use as a buffer additive in PAGE. When compared with standard SDS-PAGE, no heating of sample buffer was required in IL-PAGE, which revealed that proteins could be efficiently denatured by use of IL, which was later confirmed by use of circular dichroism (CD) studies.