Polyphenols bind to low density lipoprotein at biologically relevant concentrations that are protective for heart disease.

There is ample evidence in the epidemiological literature that polyphenols, the major non-vitamin antioxidants in plant foods and beverages, have a beneficial effect on heart disease. Until recently other mechanisms which polyphenols exhibit such as cell signaling and regulating nitric oxide bioavailability have been investigated. The oxidation theory of atherosclerosis implicates LDL oxidation as the beginning step in this process. Nine polyphenols from eight different classes and several of their O-methylether, O-glucuronide and O-sulfate metabolites have been shown in this study to bind to the lipoproteins and protect them from oxidation at lysosomal/inflammatory pH (5.2), and physiological pH (7.4). Polyphenols bind to the apoprotein at pH 7.4 with Kb > 106 M-1 and the number of molecules of polyphenols bound per LDL particle under saturation conditions varied from 0.4 for ferulic acid to 13.1 for quercetin. Competition studies between serum albumin and LDL show that substantial lipoprotein binding occurs even in the presence of a great molar excess of albumin, the major blood protein. These in vitro results are borne out by published human supplementation studies showing that polyphenol metabolites from red wine, olive oil and coffee are found in LDL even after an overnight fast. A single human supplementation with various fruit juices, coffee and tea also produced an ex vivo protection against lipoprotein oxidation under postprandial conditions. This in vivo binding is heart-protective based on published olive oil consumption studies. Relevant to heart disease, we hypothesize that the binding of polyphenols and metabolites to LDL functions as a transport mechanism to carry these antioxidants to the arterial intima, and into endothelial cells and macrophages. Extracellular and intracellular polyphenols and their metabolites are heart-protective by many mechanisms and can also function as potent "intraparticle" and intracellular antioxidants due to their localized concentrations that can reach as high as the micromolar level. Low plasma concentrations make polyphenols and their metabolites poor plasma antioxidants but their concentration in particles such as lipoproteins and cells is high enough for polyphenols to provide cardiovascular protection by direct antioxidant effects and by other mechanisms such as cell signaling.

Red Blood Cells and Lipoproteins: Important Reservoirs and Transporters of Polyphenols and Their Metabolites.

Dietary polyphenols are protective for chronic diseases. Their blood transport has not been well investigated. This work examines multiple classes of polyphenols and their interactions with albumin, lipoproteins, and red blood cell (RBC) compartments using four models and determines the % polyphenol in each compartment studied. The RBC alone model showed a dose-response polyphenol association with RBCs. A blood model with flavanones determined the % polyphenol that was inside RBCs and bound to the surface using a new albumin washing procedure. It was shown that RBCs can methylate flavanones. The whole blood model separated the polyphenol into four compartments with the aid of affinity chromatography. More polyphenols were found with albumin and lipoproteins (high-density lipoproteins and low-density lipoproteins) than with RBCs. In the plasma model, the polyphenols associated almost equally between lipoproteins and albumin. RBCs and lipoproteins are shown to be important reservoirs and transporters of polyphenols in blood.

Pure Polyphenols and Cranberry Juice High in Anthocyanins Increase Antioxidant Capacity in Animal Organs.

Anthocyanins and the broader class of polyphenols are strong antioxidants in vitro. Polyphenols are one of the major antioxidants in plant foods, and the beverages derived from them. There is extensive evidence in the literature that polyphenols are beneficial to health. In order to be bioactive in vivo, they need to be bioavailable and be transported from the circulation to target organs. To date, there have been few studies testing the extent to which polyphenols and especially anthocyanins affect the antioxidant capacity of animal organs. In our first pilot study, we investigated how three pure polyphenols (the flavonoids quercetin, catechin and hesperetin) given to rats by intraperitoneal injection (49 to 63 mg/kg) affected their organ antioxidant capacity. This was followed by a subsequent study that injected one ml of 100% cranberry juice (high in anthocyanins) to hamsters. Antioxidant capacity of animal organs was determined by using the ferric reducing antioxidant power (FRAP) colorimetric assay on methanolic extracts of select rat organs (i.e., liver, kidney, heart, prostate and brain) and in the hamster organs (i.e., liver, kidney, heart, bladder and brain). Overall the results showed that antioxidant capacity was significantly increased (p < 0.05) in experimental vs. control organs. Analysis of organs by high performance liquid chromatography (HPLC) from both animal studies provided evidence of polyphenol metabolites in the organ extracts. Taken together, this study provides data that the administration of anthocyanins and other polyphenols cause an increase in organ antioxidant capacity in two animal models. This result supports the growing evidence for the hypothesis that dietary polyphenols reduce the risk and extent of various chronic disease at the disease site.

Intracellular Polyphenols: How Little We Know.

In vitro cell studies are used by scientists to determine mechanisms that can be operating in vivo and pave the way for animal and human studies. Polyphenols, the major antioxidants in plants and the human diet, have been extensively studied in cells within thousands of publications. However, glucuronides, sulfates, and methyl metabolites and not the original polyphenols are generally present in the circulation and often have weaker beneficial effects in cell studies. The number of articles in the literature involving their cellular metabolites is quite small. We will critically examine the knowledge we have up to the present regarding polyphenols and cells in five areas: stability in in vitro cell culture media, metabolites, conjugation/deconjugation, transport to cells, and cells in vivo. Also, the future research needs in the field of polyphenols and their metabolites with cells will be outlined.

Determination of Total Chlorogenic Acids in Commercial Green Coffee Extracts.

Obesity and type II diabetes are serious health problems and are among the leading causes of death. There are a few prescription weight loss drugs, but they have a high cost and their adverse effects have limited their widespread use. For the consumer, the use of dietary supplements represents a natural and presumably safer means of losing weight. A high-pressure liquid chromatography (HPLC) method was developed to provide a simple, inexpensive method for analysis of 54 commercially available extracts of green coffee beans. Both chlorogenic acids (CGAs), which are the purported bioactives, and caffeine were measured using 5-chloroquinic acid as the standard and published extinction coefficients for the other monomeric and dimeric CGAs present. The average labeled dose of CGA was 233 mg, whereas the average calculated by HPLC analysis was only 157 mg. Thus, the consumer is likely to obtain product containing a little more than half of the reported label amount of CGA. Caffeine levels ranged from 0% to 17%. The marketing literature touts 50% CGA content as being the gold standard of green coffee bean extract products. Based on this value, only 28% of the commercial products we studied met this goal.

Binding of Plant Polyphenols to Serum Albumin and LDL: Healthy Implications for Heart Disease.

Cardiovascular disease (CVD) is the leading cause of death in industrialized nations. The initiating event in atherosclerosis, or hardening of the arteries, is oxidation of low density lipoprotein (LDL). Binding with serum albumin and LDL of 41 polyphenols (major antioxidants in plant foods) constituting four classes of flavonoids, three types of phenolic acids, and seven polyphenol conjugate metabolites was investigated indirectly by fluorescence quenching and directly by affinity separation/high-performance liquid chromatography (four of the polyphenols). Stern-Volmer plots yielded K values for the two proteins. Polyphenol binding was significantly stronger for albumin than with LDL. K values were highly correlated with the lipophilicity of the polyphenols. The number of polyphenol molecules determined by quenching was ∼1 for both proteins. Direct analysis under saturation conditions yielded from 2 to 13 molecules of polyphenols/LDL particle. Multiple substituent effects on binding were analyzed. Evidence was put forward that binding of polyphenols to these proteins is protective for CVD by multiple mechanisms.

Analysis of Popcorn (Zea mays L. var. everta) for Antioxidant Capacity and Total Phenolic Content.

Popcorn, one of the most popular snack foods in the world, is known for being a high fiber, healthy food. Our research has found that commercial popcorn also contains significant amounts of the class of antioxidants known as phenolic acids. The total antioxidant power of raw and popped popcorn extract has been quantified using the Folin-Ciocalteu and FRAP assays. The polyphenols were found exclusively in the pericarp of the kernel completely bound to the oligosaccharide fiber matrix. An in vitro digestion study was also performed to predict the phenolic acids' bioavailability. On average, nine commercial popcorn samples contain 5.93 ± 0.92 mg/g of total polyphenols after alkaline hydrolysis and 2.66 ± 0.15 mg/g after in vitro digestion as measured by the Folin-Ciocalteu assay. Furthermore, the popping process was found not to significantly decrease the antioxidant capacity. These results indicate that a considerable amount of the bound polyphenols are bioaccessible. Due to the high levels of bioaccessible polyphenols, popcorn may be a significant source of dietary polyphenol antioxidants.

Targeted intracellular delivery of resveratrol to glioblastoma cells using apolipoprotein E-containing reconstituted HDL as a nanovehicle.

The objective of this study is to transport and deliver resveratrol to intracellular sites using apolipoprotein E3 (apoE3). Reconstituted high-density lipoprotein (rHDL) bearing resveratrol (rHDL/res) was prepared using phospholipids and the low-density lipoprotein receptor (LDLr)-binding domain of apoE3. Biophysical characterization revealed that resveratrol was partitioned into the phospholipid bilayer of discoidal rHDL/res particles (~19 nm diameter). Co-immunoprecipitation studies indicated that the LDLr-binding ability of apoE3 was retained. Cellular uptake of resveratrol to intracellular sites was evaluated in glioblastoma A-172 cells by direct fluorescence using chemically synthesized NBD-labeled resveratrol (res/NBD) embedded in rHDL/res. Competition and inhibition studies indicate that the uptake is by receptor mediated endocytosis via the LDLr, with co-localization of apoE3 and res/NBD in late endosomes/lysosomes. We propose that rHDL provides an ideal hydrophobic milieu to sequester resveratrol and that rHDL containing apoE3 serves as an effective "nanovehicle" to transport and deliver resveratrol to targeted intracellular sites.

Effect of thermal processing on free and total phenolics in nine varieties of common beans.

Though much attention has been devoted to phenolics in beans, to our knowledge no studies have been done to determine the effect commercial canning has on these compounds. Free and total phenolic concentration in 18 cultivars of raw beans and 9 cultivars of commercial canned beans was determined. Alkaline hydrolysis was used on samples prior to analysis by Folin-Ciocalteu's reagent (FCR) and ferric-reducing antioxidant power (FRAP). A correction for non-phenolic antioxidants, interferences, was then made. There is a significant difference between the free phenolics of canned and raw beans using FCR (P = 0.006) but not FRAP. There is also a significant difference in the total phenolics of canned and raw beans using FRAP (P = 0.006) but not FCR. When considered on a single serving basis, the amount of phenolics in canned beans is comparable to that provided by raw fruits and vegetables.

Randomized, double-blind, placebo-controlled, linear dose, crossover study to evaluate the efficacy and safety of a green coffee bean extract in overweight subjects.

BACKGROUND: Adult weight gain and obesity have become worldwide problems. Issues of cost and potential side effects of prescription weight loss drugs have led overweight and obese adults to try nutraceuticals that may aid weight loss. One promising nutraceutical is green coffee extract, which contains high concentrations of chlorogenic acids that are known to have health benefits and to influence glucose and fat metabolism. A 22-week crossover study was conducted to examine the efficacy and safety of a commercial green coffee extract product GCA™ at reducing weight and body mass in 16 overweight adults. METHODS: Subjects received high-dose GCA (1050 mg), low-dose GCA (700 mg), or placebo in separate six-week treatment periods followed by two-week washout periods to reduce any influence of preceding treatment. Treatments were counterbalanced between subjects. Primary measurements were body weight, body mass index, and percent body fat. Heart rate and blood pressure were also measured. RESULTS: Significant reductions were observed in body weight (-8.04 ± 2.31 kg), body mass index (-2.92 ± 0.85 kg/m(2)), and percent body fat (-4.44% ± 2.00%), as well as a small decrease in heart rate (-2.56 ± 2.85 beats per minute), but with no significant changes to diet over the course of the study. Importantly, the decreases occurred when subjects were taking GCA. Body mass index for six subjects shifted from preobesity to the normal weight range (<25.00 kg/m(2)). CONCLUSION: The results are consistent with human and animal studies and a meta-analysis of the efficacy of green coffee extract in weight loss. The results suggest that GCA may be an effective nutraceutical in reducing weight in preobese adults, and may be an inexpensive means of preventing obesity in overweight adults.

High-antioxidant potatoes: acute in vivo antioxidant source and hypotensive agent in humans after supplementation to hypertensive subjects.

Potatoes have the highest daily per capita consumption of all vegetables in the U.S. diet. Pigmented potatoes contain high concentrations of antioxidants, including phenolic acids, anthocyanins, and carotenoids. In a single-dose study six to eight microwaved potatoes with skins or a comparable amount of refined starch as cooked biscuits was given to eight normal fasting subjects; repeated samples of blood were taken over an 8 h period. Plasma antioxidant capacity was measured by ferric reducing antioxidant power (FRAP). A 24 h urine was taken before and after each regimen. Urine antioxidant capacity due to polyphenol was measured by Folin reagent after correction for nonphenolic interferences with a solid phase (Polyclar) procedure. Potato caused an increase in plasma and urine antioxidant capacity, whereas refined potato starch caused a decrease in both; that is, it acted as a pro-oxidant. In a crossover study 18 hypertensive subjects with an average BMI of 29 were given either six to eight small microwaved purple potatoes twice daily or no potatoes for 4 weeks and then given the other regimen for another 4 weeks. There was no significant effect of potato on fasting plasma glucose, lipids, or HbA1c. There was no significant body weight increase. Diastolic blood pressure significantly decreased 4.3%, a 4 mm reduction. Systolic blood pressure decreased 3.5%, a 5 mm reduction. This blood pressure drop occurred despite the fact that 14 of 18 subjects were taking antihypertensive drugs. This is the first study to investigate the effect of potatoes on blood pressure. Thus, purple potatoes are an effective hypotensive agent and lower the risk of heart disease and stroke in hypertensive subjects without weight gain.

Antioxidant and anti-atherogenic activities of three Piper species on atherogenic diet fed hamsters.

Atherogenic diet is known to induce high plasma lipid concentration, oxidative stress and early atherosclerosis. Antioxidants have potentials to counter the effect of atherogenic diet. The present research aims at evaluating the antioxidant and anti-atherosclerotic activities of three Piper species (Piper guineense, Piper nigrum and Piper umbellatum) on atherogenic diet fed hamsters. Hamsters divided into 8 groups: normal control, atherosclerotic control and six test groups. The normal animals fed normal rodent chow, the atherosclerotic control animals fed the same rodent chow supplemented with 0.2% cholesterol and 10% coconut oil (high cholesterol diet). The 6 test groups' animals fed same diet as the atherosclerotic control group but with additional supplementation of 2 graded doses (1 and 0.25 mg/kg body weight, o.p.) of plant extracts for 12 weeks. The atherogenic diet induced a collapse of the erythrocyte antioxidant defense system (significant decrease in superoxide dismutase, catalase and glutathione peroxidase activities). Atherogenic diet also induced an increase in plasma total cholesterol, triglyceride, thiobarbituric acid reactive substances (TBARS), oxidation of low density lipoprotein cholesterol (LDL) and accumulation of foam cells in the aorta a hall mark for atherosclerosis. Administration of the Piper species prevented the collapse of the antioxidant system and the increase of plasma parameters maintaining them towards normality. The Piper species also prevented LDL oxidation by increasing the time (lag time) for its oxidation. The results suggest that these Piper species have significant antioxidant and anti-atherogenic effect against atherogenic diet intoxication.

Absorption and excretion of cranberry-derived phenolics in humans.

Absorption and excretion of twenty cranberry-derived phenolics were studied following the consumption of cranberry juice, sauces, and fruits by healthy human volunteers. Plasma and urine samples were collected and analysed by gas chromatography-mass spectrometry (GC-MS). A high performance liquid chromatography (HPLC) method was employed for analysing urinary creatinine, which was used as a normalisation agent. Significant increases in the sum of plasma phenolics were observed with different concentration peaks (between 0.5 and 2h) for individual subjects. Some of the phenolics, such as trans-cinnamic, vanillic, p-coumaric acids, and catechin showed second plasma concentration peaks. All of cranberry-derived phenolics increased significantly in urine samples after the intake of each cranberry product. The high molecular weight quercetin and myricetin, which were abundant in cranberry foodstuffs, were not found in either plasma or urine samples. This study provided the fundamental information for understanding the absorption and excretion of phenolics in the human gastrointestinal system after dietary intake of cranberry products.

Piper species protect cardiac, hepatic and renal antioxidant status of atherogenic diet fed hamsters.

Pre-clinical and clinical studies points to the use of antioxidants as an effective measure to reduce the progression of oxidative stress related disorders. The present study evaluate the effect of three Piper species (Piper guineense, Piper nigrum and Piper umbellatum) for the protection of cardiac, hepatic and renal antioxidant status of atherogenic diet fed hamsters. Hamsters were classified into eight groups: a normal control, atherogenic control and six other experimental groups (fed atherogenic diet supplemented with different doses of P. nigrum, P. guineense and P. umbellatum (1 and 0.25 g/kg) for 12 weeks. At the end of the feeding period the heart, liver and kidney from each group were analyzed for lipid profile and antioxidant enzymes activities. Atherogenic diet induced a significant (P<0.001) increase in the lipid profile across the board and equally significantly altered the antioxidant enzyme activities. Supplementation with Piper species significantly inhibited the alteration effect of atherogenic diet on the lipid profile and antioxidant enzymes activities. The Piper extracts may possess an antioxidant protective role against atherogenic diet induced oxidative stress in cardiac, hepatic and renal tissues.

Nuts, especially walnuts, have both antioxidant quantity and efficacy and exhibit significant potential health benefits.

Free and total (after basic hydrolysis) polyphenols in nine types of raw and roasted nuts and two types of peanut butter (54 commercial samples) were analyzed after methanol extraction by a single step Folin-Ciocalteu reagent using catechin as standard. Walnuts had the highest free and total polyphenols in both the combined raw and roasted samples. Total polyphenols in the nuts were significantly higher than free polyphenols. Roasting had little effect on either free or total polyphenols in nuts. Raw and roasted walnuts had the highest total polyphenols. The efficacy of raw and roasted nut antioxidants was assessed by measuring the ability of the free polyphenol nut extracts to inhibit the oxidation of lower density lipoproteins (LDL + VLDL). A nut polyphenol, catechin, was measured after binding of three nut extracts to lower density lipoproteins. Walnut polyphenols had the best efficacy among the nuts and also the highest lipoprotein-bound antioxidant activity. Based on USDA availability data, the per capita total polyphenols was 162 mg from nuts per day in 2008. This corresponds to 19% of the total polyphenols from fruits and vegetables, nuts, grains, oils and spices in the US diet. Nuts provided 158 mg of polyphenols per day to the European Union diet. Nuts are high in polyphenol antioxidants which by binding to lipoproteins would inhibit oxidative processes that lead to atherosclerosis in vivo. In human supplementation studies nuts have been shown to improve the lipid profile, increase endothelial function and reduce inflammation, all without causing weight gain. These qualities make nuts a nutritious healthy snack and food additive.

Vitamin enhanced waters and polyphenol rich beverages analyzed for antioxidant capacity and antioxidants/calorie.

The purpose of this study was to analyze polyphenol rich beverages (vitamin enhanced waters (VEWs), fruit juices and berry juices) to determine free polyphenol concentrations and free polyphenols per Calorie based on a serving size. The Folin-Ciocalteu reagent was used in a colorimetric assay based on a catechin standard. Fruit and berry juices contained, on average, more than eight-times the concentration of free polyphenols when compared to VEWs. When Calories per serving were taken into consideration, fruit and berry juices contained more than twice the free polyphenols per Calorie.

Salinity and sodicity of weathered minesoils in northwestern new Mexico and northeastern Arizona.

Evolving relationships between electrical conductivity (EC) and sodium adsorption ratio (SAR) in reconstructed soils at surface mines have been insufficiently documented in the literature. Some minesoils (i.e., rootzone material) are classified as saline, sodic, or saline-sodic and are considered unsuitable for revegetation. Weatherable minerals such as calcite and gypsum are common in alkaline minesoils and on dissolution tend to mitigate elevated SAR levels by maintaining or increasing electrolytes in the soil and providing sources of exchangeable calcium and magnesium. Topsoils (i.e., coversoils) contribute to mitigation of sodic conditions when soluble cations are translocated from coversoils into the underlying minesoils. This study evaluated the weathering characteristics of minesoils sites from three surface coal mines in northwestern New Mexico and northeastern Arizona. Minesoils were grouped into 11 classes based on EC and SAR. After 6 to 14 yr, differences between upper and lower halves of the coversoils suggest general increases occurred with EC, SAR, chloride (Cl(-)), and sulfate (SO(4)(2-)) with depth. Within the reclaimed minesoils, there were several significant (P < 0.05 or < 0.10) relationships among EC and SAR that related to Minesoil Class. Lower SAR levels with corresponding increases in EC compared to baseline minesoils were more apparent in upper minesoil depths (0-5 and 5-15 cm). Minesoil anion concentrations suggested coversoil leachates and gypsum dissolution influenced EC and SAR chemistry. Over time, chemical changes have increased the apparent stability of the saline and sodic reclaimed minesoils studied thereby reducing risks associated with potential aggregate slaking and clay particle dispersion.

Chocolate, lifestyle, and health.

Interest in the biological activities of cocoa polyphenols is increasing steadily. In fact, the high polyphenol content of cocoa, coupled with its widespread presence in many food items, render this food of particular interest from the nutritional and "pharmacological" viewpoints. This paper summarizes the new findings and developments regarding the effects of cocoa and chocolate consumption on human health as presented at the International Conference "Chocolate, Lifestyle, and Health" (Milan, Italy, March 2, 2007) regarding the effects of cocoa and chocolate consumption on human health.

Cranberries and cranberry products: powerful in vitro, ex vivo, and in vivo sources of antioxidants.

Cranberry products and especially cranberry juice (CJ) have been consumed for health reasons primarily due to their effect on urinary tract infections. We investigated the quantity of both free and total (after hydrolysis) phenolic antioxidants in cranberry products using the Folin assay. The order of amount of total polyphenols in cranberry foods on a fresh weight basis was as follows: dried > frozen > sauce > jellied sauce. On a serving size basis for all cranberry products, the order was as follows: frozen > 100% juice > dried > 27% juice > sauce > jellied sauce. High fructose corn syrup (HFCS) is a major source of sugar consumption in the U.S. and contains both glucose and fructose, potential mediators of oxidative stress. We investigated the effect of the consumption of HFCS and ascorbate with CJ antioxidants or without CJ (control) given to 10 normal individuals after an overnight fast. Plasma antioxidant capacity, glucose, triglycerides, and ascorbate were measured 6 times over 7 h after the consumption of a single 240 mL serving of the two different beverages. The control HFCS caused a slight decrease in plasma antioxidant capacity at all time points and thus an oxidative stress in spite of the presence of ascorbate. CJ produced an increase in plasma antioxidant capacity that was significantly greater than control HFCS at all time points. Postprandial triglycerides, due to fructose in the beverages, were mainly responsible for the oxidative stress and were significantly correlated with the oxidative stress as measured by the antioxidant capacity. Cranberries are an excellent source of high quality antioxidants and should be examined in human supplementation studies.