Examining the Performance of Two Extraction Solvent Systems on Phenolic Constituents from U.S. Southeastern Blackberries.

Two common extraction solvent systems, namely acidified aqueous methanol and acidified aqueous acetone, were used to extract blackberry phenolics, and the antioxidant properties of the recovered extracts were compared. The crude extracts were fractionated into low- and high-molecular-weight phenolics by Sephadex LH-20 column chromatography. The hydrophilic-oxygen radical absorbance capacity (H-ORACFL), ferric reducing antioxidant power (FRAP), and the cellular antioxidant activity (CAA) assays were employed as indices to assess antioxidant capacity of the extracts and their respective fractions. The methanolic solvent system displayed a greater efficiency at extracting anthocyanin and flavonol constituents from the blackberries, while the acetonic solvent system was better at extracting flavan-3-ols and tannins. Anthocyanins were the dominant phenolic class found in the blackberries with 138.7 ± 9.8 mg C3G eq./100 g f.w. when using methanol as the extractant and 114.6 ± 3.4 mg C3G eq./100 g f.w. when using acetone. In terms of overall antioxidant capacity of blackberry phenolics, the acetonic solvent system was superior. Though present only as a small percentage of the total phenolics in each crude extract, the flavan-3-ols (42.37 ± 2.44 and 51.44 ± 3.15 mg/100 g f.w. in MLF and ALF, respectively) and ellagitannins (5.15 ± 0.78 and 9.31 ± 0.63 mg/100 g f.w. in MHF and AHF, respectively) appear to account for the differences in the observed antioxidant activity between the two solvent systems.

The cellular antioxidant and anti-glycation capacities of phenolics from Georgia peaches.

Plant-based polyphenolics have been reported to bestow health benefits when consumed, which are partially ascribed to their antioxidant activity. Yet, many current in vitro chemical assays to characterize antioxidant potential do not truly reflect the physiological properties of food antioxidants in vivo. The present study employed biological approaches, including a cellular antioxidant activity (CAA) and protein glycation assays, to offer an improved picture of antioxidant potential of phenolic extracts from Georgia peach cultivars. The phenolic extracts from two peach varieties, showing contrasting antioxidant capacities according to hydrophilic-oxygen radical absorbance capacity (H-ORACFL) and ferric reducing antioxidant power (FRAP) assays, exhibited significant differences in two biological tests when the assays were performed on a fresh weight basis. The procyanidins fraction displayed notable antioxidant capacity, when compared to other phenolic classes in the peach extract, in these two biologically relevant assays.

Cellular evaluation of the antioxidant activity of U.S. Pecans [Carya illinoinensis (Wangenh.) K. Koch].

Clinical trials show an inverse relationship between the consumption of antioxidant-rich tree nuts and the development of chronic diseases. This study examined antioxidant efficacy of U.S. pecans using a modified cellular antioxidant activity (CAA) assay with comparisons to data from in vitro antioxidant assays (hydrophilic-oxygen radical absorbance capacity {H-ORACFL} and ferric reducing antioxidant power {FRAP}). Crude phenolic extracts from both raw and roasted pecans were analyzed. In the CAA assay, pecan phenolics were taken up by human colorectal adenocarcinoma (Caco-2) cells and bestowed CAA, determined by monitoring the fluorescence of 2',7'-dichlorofluorescein. Phenolics (25-100 µg/mL) demonstrated a reduction in fluorescence by 37-69% for raw and 26-68% for roasted pecans. The primary active phenolic constituents were determined by high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) to be epi(catechin) dimers and trimers. These oligomeric procyanidins, ranging in size from 560 to 840 g/mol appear to be small enough for cellular uptake, showing pecans are an effective antioxidant in biological systems, regardless of roasting.

Characterizing the phenolic constituents and antioxidant capacity of Georgia peaches.

Acetonic crude phenolic extracts of six Georgia peach cultivars were prepared and separated into low- and high-molecular-weight (LMW and HMW) fractions by Sephadex LH-20 column chromatography. Further characterization via RP-HPLC-ESI-MS identified the main phenolics as hydroxycinnamates, (+)-catechin, and proanthocyanidins with degrees of polymerization up to seven. The LMW phenolics of the commercial cultivar, 'July Prince', were further chromatographed and examined by RP-HPLC-ESI-MS. Derivatives of phenolic acids and flavan-3-ols, along with eriodictyol and quercetin diglycosides, were identified. Antioxidant capacities of the LMW and HMW fractions were determined using in vitro assays. H-ORACFL and FRAP assays gave values of 872 to 2428 µmol Trolox eq./100 g f.w. and 309 to 432 µmol Fe2+ eq./100 g f.w., respectively. The total phenolics content (TPC) was also measured; correlations between TPCs and antioxidant assays indicated that the HMW fractions of peach extracts were major contributors to the antioxidant capacity of the cultivars analyzed.

Modification of the cellular antioxidant activity (CAA) assay to study phenolic antioxidants in a Caco-2 cell line.

In vitro assays are widely used to analyze the antioxidant potential of compounds, but they cannot accurately predict antioxidant behavior in living systems. Cell-based assays, like the cellular antioxidant activity (CAA) assay, are gaining importance as they provide a biological perspective. When the CAA assay was employed to study phenolic antioxidants using hepatocarcinoma (HepG2) cells, quercetin showed antioxidant activity in HepG2 cells; 25 and 250µM quercetin reduced fluorescence by 17.1±0.9% and 58.6±2.4%, respectively. (+)-Catechin, a phenolic antioxidant present in many foods, bestowed virtually no CAA in HepG2 cells. When Caco-2 cells were employed, more robust antioxidant activity was observed; 50µM (+)-catechin and quercetin reduced fluorescence by 54.1±1.4% and 63.6±0.9%, respectively. Based on these results, likely due to differences in active membrane transport between the cell types, the Caco-2-based CAA assay appears to be a more appropriate method for the study of certain dietary phenolics.

Effect of pecan phenolics on the release of nitric oxide from murine RAW 264.7 macrophage cells.

Inflammation is linked to numerous chronic disease states. Phenolic compounds have attracted attention because a number of these compounds possess anti-inflammatory properties. A phenolic crude extract was prepared from pecans and separated by Sephadex LH-20 column chromatography into low- and high-molecular-weight (LMW/HMW) fractions. Anti-inflammatory properties of these fractions were assessed in LPS-stimulated RAW 264.7 murine macrophage cells. NO and reactive oxygen species (ROS) production was monitored after 3 different experimental protocols: (1) pre-treatment with Escherichia coli O111:B4 lipopolysaccharide (LPS); (2) pre-treatment with a pecan crude extract and its fractions; and (3) co-incubation of LPS with a pecan crude extract and its fractions. The LMW fraction displayed a dose-dependent decrease in NO production and a significant decrease from the LPS control in ROS production when cells were either co-incubated with or pre-treated with LPS. The phenolics were characterized by HPLC to help identify those responsible for the observed effect.

Separation and characterization of phenolic compounds from U.S. pecans by liquid chromatography-tandem mass spectrometry.

The phenolic acids and proanthocyanidins (PACs) of pecans possess bioactive properties, which might be useful in retarding the onset of and ameliorating the status of certain chronic disease states. There is a general lack of information in the literature regarding such compounds, especially the PACs. Crude phenolic extracts pooled from eight commercially significant cultivars were selected based on their relatively high antioxidant capacities. The pooled extracts were separated via Sephadex LH-20 column chromatography into five ethanolic low-molecular-weight (LMW) fractions and one acetonic high-molecular-weight (HMW) fraction. The preparations were then characterized using RP-HPLC-ESI-MS/MS and diol-phase HPLC-ESI-MS/MS in order to determine the key constituents present in the LMW and HMW fractions, respectively. As previously observed in pecan nutmeat, ellagic acid and (+)-catechin were found to be the major phenolics in the LMW fractions. The last eluting LMW fraction did not contain phenolic acids; rather it possessed PAC monomers and dimers. The HMW fraction comprised a majority of its PACs as dimers; yet, monomers, trimers, tetramers, pentamers, and hexamers were also separated and characterized.

Inhibition of nonenzymatic protein glycation by pomegranate and other fruit juices.

The nonenzymatic glycation of proteins and the formation of advanced glycation endproducts in diabetes leads to the crosslinking of proteins and disease complications. Our study sought to demonstrate the effect of commonly consumed juices (pomegranate, cranberry, black cherry, pineapple, apple, and Concord grape) on the fructose-mediated glycation of albumin. Albumin glycation decreased by 98% in the presence of 10 µL of pomegranate juice/mL; other juices inhibited glycation by only 20%. Pomegranate juice produced the greatest inhibition on protein glycation when incubated at both the same phenolic concentration and the same antioxidant potential. Both punicalagin and ellagic acid significantly inhibited the glycation of albumin by ~90% at 5 µg/mL. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that pomegranate, but not apple juice, protected albumin from modification. These results demonstrate that pomegranate juice and two of its major constituents are potent inhibitors of fructose-mediated protein glycation.

Inhibition of aromatase and α-amylase by flavonoids and proanthocyanidins from Sorghum bicolor bran extracts.

We compared the ability of simple flavonoids and proanthocyanidins in Sorghum bicolor bran extracts to inhibit enzymes in vitro. In particular, aromatase is a target for breast cancer therapy, and inhibition of α-amylase can reduce the glycemic effect of dietary starches. Proanthocyanidin-rich sumac sorghum bran extract inhibited α-amylase at a lower concentration (50% inhibitory concentration [IC50]=1.4 µg/mL) than did proanthocyanidin-free black sorghum bran extract (IC50=11.4 µg/mL). Sumac sorghum bran extract inhibited aromatase activity more strongly than black sorghum bran extract (IC50=12.1 µg/mL vs. 18.8 µg/mL, respectively). Bovine serum albumin (BSA), which binds proanthocyanidins, reduced inhibition by sumac but not black sorghum bran extract. When separated on Sephadex LH-20, sumac sorghum proanthocyanidins inhibited both enzymes but showed reduced inhibition with BSA. Flavonoids from either cultivar had higher IC50 values than proanthocyanidins, and BSA had little effect on their inhibition. Proanthocyanidins and simple flavonoids in LH-20 fractions both inhibited aromatase with mixed kinetics and affected K(m) and V(max). The results show that potential health benefits of sorghum bran may include actions of monomeric flavanoids as well as proanthocyanidins.

Anti-inflammatory activity of select sorghum (Sorghum bicolor) brans.

The bran fractions of certain varieties of sorghum (Sorghum bicolor) grain are rich sources of phytochemicals and antioxidants. In this article, the anti-inflammatory actions of extracts of select sorghum brans were evaluated in two experimental inflammatory systems: (1) the release of cytokines by lipopolysaccharide-activated peripheral blood mononuclear cells and (2) 12-O-tetradecanoylphorbol acetate (TPA)-induced ear edema in mice. A 1:200 dilution of a 10% (wt/vol) ethanol extract of black sorghum bran significantly inhibited the secretion of the pro-inflammatory cytokines interleukin-1beta and tumor necrosis factor-alpha. Ethanolic extracts of both black and sumac varieties of sorghum bran significantly reduced edema in inflamed ears as measured by ear thickness and ear punch weight 6 hours following TPA application. The degree of inhibition was similar to that observed with indomethacin. Black sorghum bran significantly diminished the increase in myeloperoxidase activity 24 hours following the application of TPA. No anti-inflammatory activity was observed with white and Mycogen sorghum bran varieties or with oat, wheat, or rice brans in the mouse ear model. The anti-inflammatory activity observed with these brans correlated with their phenolic content and antioxidant activity. These results demonstrate that select sorghum bran varieties possess significant anti-inflammatory activity.

Antioxidant and anti-inflammatory activities of polyphenolics from Southeastern U.S. range blackberry cultivars.

The antioxidant and topical anti-inflammatory activities of low and high molecular weight phenolic fractions (LMPF and HMPF, respectively) isolated from three blackberry cultivars (i.e., Navaho, Kiowa, and Ouachita), bred to tolerate the warm and humid climatic conditions of the southeastern United States, were investigated by the in vitro ferric reducing antioxidant power (FRAP) assay and an in vivo mouse ear edema model. Seventy percent (v/v) acidified acetone was employed to extract phenolics from the Georgia-grown blackberry cultivars, which were subsequently cleaned up on an Amberlite XAD-16 column and then further fractionated with Sephadex LH-20 to LMPF and HMPF. The anti-inflammatory response from topical application of solutions of the LMPF and HMPF as well as indomethacin, a potent nonsteroidal anti-inflammatory drug, was assessed in the TPA mouse ear model. All treatments significantly (P < 0.05) reduced TPA-induced irritation injury. Furthermore, mouse ear myeloperoxidase (MPO) activity, an indicator of polymorphonuclear leukocyte infiltration, was assessed and found to be significantly (P < 0.05) reduced after topical application of indomethacin and all blackberry preparations. Correlation coefficients of 0.925 and 0.923 (P < 0.01) were determined when the anti-inflammatory activities of the blackberry fractions were compared to their total phenolics contents and antioxidant activities (i.e., FRAP values), respectively.

Inhibition of protein glycation by extracts of culinary herbs and spices.

We tested whether polyphenolic substances in extracts of commercial culinary herbs and spices would inhibit fructose-mediated protein glycation. Extracts of 24 herbs and spices from a local supermarket were tested for the ability to inhibit glycation of albumin. Dry samples were ground and extracted with 10 volumes of 50% ethanol, and total phenolic content and ferric reducing antioxidant potential (FRAP) were measured. Aliquots were incubated in triplicate at pH 7.4 with 0.25 M fructose and 10 mg/mL fatty acid-free bovine albumin. Fluorescence at 370 nm/440 nm was used as an index of albumin glycation. In general, spice extracts inhibited glycation more than herb extracts, but inhibition was correlated with total phenolic content (R(2) = 0.89). The most potent inhibitors included extracts of cloves, ground Jamaican allspice, and cinnamon. Potent herbs tested included sage, marjoram, tarragon, and rosemary. Total phenolics were highly correlated with FRAP values (R(2) = 0.93). The concentration of phenolics that inhibited glycation by 50% was typically 4-12 microg/mL. Relative to total phenolic concentration, extracts of powdered ginger and bay leaf were less effective than expected, and black pepper was more effective. Prevention of protein glycation is an example of the antidiabetic potential for bioactive compounds in culinary herbs and spices.

Inhibition of hyaluronidase activity by select sorghum brans.

Hyaluronidase hydrolyzes glycosaminoglycans, including hyaluronan, in the extracellular matrix during tissue remodeling. Hyaluronidase activity increases in chronic inflammatory conditions, e.g., inflammatory joint disease. In this study, we tested the ability of ethanolic extracts (1:9 [wt/vol] of 50% ethanol) of bran from six cultivated varieties of Sorghum bicolor to inhibit hyaluronidase activity in vitro in comparison to extracts of wheat and rice bran. Each extract inhibited hyaluronidase activity with this order of potency: Sumac > Shanqui Red > Black > Mycogen > Fontanelle > White sorghum. Extracts of wheat and rice bran had weak inhibitory activities relative to the high phenolic sorghum brans. Hyaluronidase inhibition correlated positively with total phenolic content and ferric reducing antioxidant power values for each bran extract. Inhibition was not only due to condensed tannins (proanthocyanidins) because the Black sorghum cultivar lacks condensed tannins but has abundant anthocyanins and other polyphenols. Since hyaluronidase activity is important in conditions such as osteoarthritis and skin aging, these sorghum varieties deserve consideration for functional foods and beverages, and for nutraceutical and cosmeceutical ingredients.

A novel nutraceutical property of select sorghum (Sorghum bicolor) brans: inhibition of protein glycation.

Despite the high levels of polyphenolic phytochemicals in grain sorghum and its position as a major food staple, there has been a lack of research on its effects on both animal and human health and disease prevention. These phenolic compounds, mainly located in the bran fraction, result in the plant having substantial antioxidant properties. This study examined the effect of ethanol extracts of several varieties of sorghum (S. bicolor) bran on albumin glycation, a non-enzymatic process thought to be important in the pathogenesis of many diabetic complications. Sorghum brans with a high phenolic content and high antioxidant properties inhibited protein glycation, whereas sorghum brans that are low in these properties did not inhibit this process. Ethanol extracts of wheat, rice or oat bran did not inhibit protein glycation. Although one high phenolic sorghum bran variety (sumac) inhibited protein glycation by approximately 60%, it produced only a 20% decrease in methylglyoxal mediated albumin glycation. These results suggest that certain varieties of sorghum bran may affect critical biological processes that are important in diabetes and insulin resistance. These results distinguish select sorghum brans from the common food brans and suggest a nutraceutical rationale for its human consumption.

Topical anti-inflammatory activity of Polygonum cuspidatum extract in the TPA model of mouse ear inflammation.

BACKGROUND: This study tested the ability of a characterized extract of Polygonum cuspidatum (PCE) to inhibit mouse ear inflammation in response to topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA). METHODS: A 50% (wt:vol) ethanolic solution of commercial 200:1 PCE was applied to both ears of female Swiss mice (n = 8) at 0.075, 0.15, 0.3, 1.25 and 2.5 mg/ear 30 min after TPA administration (2 mug/ear). For comparison, 3 other groups were treated with TPA and either 1) the vehicle (50% ethanol) alone, 2) indomethacin (0.5 mg/ear), or 3) trans-resveratrol (0.62 mg/ear). Ear thickness was measured before TPA and at 4 and 24 h post-TPA administration to assess ear edema. Ear punch biopsies were collected at 24 h and weighed as a second index of edema. Myeloperoxidase activity was measured in each ear punch biopsy to assess neutrophil infiltration. RESULTS: PCE treatment at all doses significantly reduced ear edema compared to the TPA control. The PCE response was dose-dependent and 2.5 mg PCE significantly inhibited all markers of inflammation to a greater extent than indomethacin (0.5 mg). MPO activity was inhibited at PCE doses >/= 1.25 mg/ear. Trans-resveratrol inhibited inflammation at comparable doses. CONCLUSION: PCE inhibits development of edema and neutrophil infiltration in the TPA-treated mouse ear model of topical inflammation.

Topical anti-inflammatory activities of Vitis rotundifolia (muscadine grape) extracts in the tetradecanoylphorbol acetate model of ear inflammation.

The ability of muscadine grape skin, seed, or combined skin and seed extracts to inhibit mouse ear inflammation, edema, and polymorphonuclear leukocyte infiltration was tested following topical application of 12-O-tetradecanoylphorbol 13-acetate (TPA). Ethanolic extracts of skins, seeds, or a combination of these from purple (Ison) cultivars were applied to both ears of female Swiss mice 30 minutes after TPA (2 microg per ear) administration. Control mice were treated with indomethacin or 50% ethanol vehicle 30 minutes after TPA. Ear thickness was measured before TPA and at 4 and 24 hours post-TPA administration to assess ear edema. Ear punch biopsies were collected at 24 hours and weighed as a second marker of edema. Myeloperoxidase (MPO) (EC 1.11.1.7) activity was measured in each ear punch biopsy as an index of neutrophil infiltration. Extracts of muscadine skin, seed, and combination treatments significantly reduced ear edema, ear biopsy weight, and MPO activity compared to TPA vehicle control. There was no significant difference in anti-inflammatory activity of the skin and seed extracts. However, an additive effect was observed with the combination treatment that was statistically similar to the anti-inflammatory activity of indomethacin treatment. It can be concluded that muscadine skin, seed, and combination skin/seed extracts exhibit significant topical anti-inflammatory properties.

Inhibition of protein glycation by skins and seeds of the muscadine grape.

The formation of advanced glycation end products (AGEs) leading to protein glycation and cross-linking is associated with the pathogenesis of diabetic complications. The inhibition of protein glycation by phenolic and flavonoid antioxidants demonstrates that the process is mediated, in part, by oxidative processes. In this study, the effects of seed and skin extracts of the muscadine grape on AGEs formation were examined. Seeds and skins were extracted (10% w/v) with 50% ethanol and incubated at 37 degrees C with a solution containing 250 mM fructose and 10 mg/ml albumin. After 72 h, fluorescence was measured at the wavelength pair of 370 and 440 nm as an index of the formation of AGEs. Both seed and skin extracts were found to be efficacious inhibitors of AGE formation. A 1:300 dilution of the seed extract decreased fluorescence by approximately 65%, whereas muscadine grape skin extract produced a 40% lowering. This difference correlates with the greater antioxidant activity found in muscadine seeds in comparison to skins, however, on a mass basis, the inhibitory activities of the seeds and skins were found to be nearly equivalent. Gallic acid, catechin and epicatechin, the three major polyphenols in the seeds, all significantly decreased the AGE product related fluorescence at a concentration of 50 microM. Neither muscadine seed extract nor skin extract inhibited the methylglyoxal-mediated glycation of albumin. These results suggest that consumption of the muscadine grape may have some benefit in altering the progression of diabetic complications.

Antiinflammatory properties of the muscadine grape (Vitis rotundifolia).

The muscadine grape possesses one of the highest antioxidant levels among fruits; yet, the effect of this fruit on mammalian metabolic systems has not received significant attention. To examine the antiinflammatory properties of the muscadine, grape skins were dried, pulverized, and extracted (10% w/v) with 50% ethanol. The extract was then tested in two different assays: the release of superoxide in phorbol myristate acetate-activated neutrophils and the release of cytokines [tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-beta), and interleukin-6 (IL-6)] by lipopolysaccharide-activated peripheral blood mononuclear cells. The release of superoxide and cytokines was inhibited by increasing concentrations of the extract. A 1:100 dilution of the extract inhibited superoxide release by approximately 60% while the release of TNF-alpha and IL-1beta was reduced at a dilution of 1:200 by approximately 15 and 90%, respectively (all P < 0.05). The inhibition pattern on the release of IL-6 was similar to that seen with TNF-alpha. In a related in vivo study, rats were fed a diet containing 5% (wt/wt) dried muscadine grape skins for 14 days and then were injected with carrageenan in the foot pad. After 3 h, paw edema was measured and the rats on the grape skin diet had approximately 50% less paw edema than controls (P < 0.05). These results demonstrate that the muscadine grape skin powder possesses significant in vitro and in vivo antiinflammatory properties.

Nutritional significance and metabolism of very long chain fatty alcohols and acids from dietary waxes.

Very long chain fatty alcohols obtained from plant waxes and beeswax have been reported to lower plasma cholesterol in humans. This review discusses nutritional or regulatory effects produced by wax esters or aliphatic acids and alcohols found in unrefined cereal grains, beeswax, and many plant-derived foods. Reports suggest that 5-20 mg per day of mixed C24-C34 alcohols, including octacosanol and triacontanol, lower low-density lipoprotein (LDL) cholesterol by 21%-29% and raise high-density lipoprotein cholesterol by 8%-15%. Wax esters are hydrolyzed by a bile salt-dependent pancreatic carboxyl esterase, releasing long chain alcohols and fatty acids that are absorbed in the gastrointestinal tract. Studies of fatty alcohol metabolism in fibroblasts suggest that very long chain fatty alcohols, fatty aldehydes, and fatty acids are reversibly inter-converted in a fatty alcohol cycle. The metabolism of these compounds is impaired in several inherited human peroxisomal disorders, including adrenoleukodystrophy and Sjögren-Larsson syndrome. Reports on dietary management of these diseases confirm that very long chain fatty acids (VLCFA) are normal constituents of the human diet and are synthesized endogenously. Concentrations of VLCFA in blood plasma increase during fasting and when children are placed on ketogenic diets to suppress seizures. Existing data support the hypothesis that VLCFA exert regulatory roles in cholesterol metabolism in the peroxisome and also alter LDL uptake and metabolism.