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Nanogels are attractive biocompatible materials that enable local delivery of multiple drugs. In this study, we demonstrated that 3D printing technology could be used to precisely construct nanogel discs carrying paclitaxel and rapamycin. 3D-printed nanogel disc rounds (12 mm diameter × 1 mm thickness) carrying paclitaxel and rapamycin evaded premature gelation during storage and the initial burst release of the drugs in the dissolution medium. In vivo 3D-printed nanogel discs permitted successful intraperitoneal delivery of paclitaxel and rapamycin in ES-2-luc ovarian-cancer-bearing xenograft mice. They were also shown to be therapeutically effective and capable of preventing postsurgical peritoneal adhesions in the treated xenograft mice.

Melioidosis is an emerging infectious disease caused by Burkholderia pseudomallei and is associated with high morbidity and mortality rates in endemic areas. Antibiotic treatment is protracted and not always successful; even with appropriate therapy, up to 40% of individuals presenting with melioidosis in Thailand succumb to infection. In these circumstances, an effective vaccine has the potential to have a dramatic impact on both the scale and the severity of disease. Currently, no vaccines are licensed for human use. A leading vaccine candidate is the capsular polysaccharide consisting of a homopolymer of unbranched 1→3 linked 2-O-acetyl-6-deoxy-β-d-manno-heptopyranose. Here, we present the chemical synthesis of this challenging antigen using a novel modular disaccharide assembly approach. The resulting hexasaccharide was coupled to the nontoxic Hc domain of tetanus toxin as a carrier protein to promote recruitment of T-cell help and provide a scaffold for antigen display. Mice immunized with the glycoconjugate developed IgM and IgG responses capable of recognizing native capsule, and were protected against infection with over 120 × LD50 of B. pseudomallei strain K96243. This is the first report of the chemical synthesis of an immunologically relevant and protective hexasaccharide fragment of the capsular polysaccharide of B. pseudomallei and serves as the rational starting point for the development of an effective licensed vaccine for this emerging infectious disease.

Chokeberries are an excellent source of polyphenols, but their fate during juice processing and storage is unknown. The stability of anthocyanins, total proanthocyanidins, hydroxycinnamic acids, and flavonols at various stages of juice processing and over 6 months of storage at 25 °C was determined. Flavonols, total proanthocyanidins, and hydroxycinnamic acids were retained in the juice to a greater extent than anthocyanins, with losses mostly due to removal of seeds and skins following pressing. Anthocyanins were extensively degraded by thermal treatments during which time levels of protocatechuic acid and phloroglucinaldehyde increased, and additional losses occurred following pressing. Flavonols, total proanthocyanidins, and hydroxycinnamic acids were well retained in juices stored for 6 months at 25 °C, whereas anthocyanins declined linearly. Anthocyanin losses during storage were paralleled by increased polymeric color values, indicating that the small amounts of anthocyanins remaining were present in large part in polymeric forms.

Dietary polyphenolics can be converted into smaller phenolic acids (PA) by microorganisms in the colon and may contribute to health benefits associated with the parent polyphenolics. Urinary excretion of 18 PA and their conjugates was studied, using HPLC-MS/MS, in rats fed AIN93G-based diets containing 5% (dry weight basis) of either cranberry (CB), blueberry (BB), or black raspberry (BRB). Hippuric, 4-hydroxyphenylacetic, 3-methoxy-4-hydroxyphenylacetic, and 4-hydroxybenzoic acids were excreted in greatest quantity in the urine over a 24 h period in all diets. Primary PA excreted in the berry diets were 4-hydroxycinnamic acid for CB; chlorogenic, ferulic, and 3,4-dihydroxycinnamic acids for BB; and 3-hydroxyphenylpropionic, 3-hydroxybenzoic, and 3-hydroxycinnamic acids for BRB. PA were present in conjugated form with cinnamic acid derivatives being 50-70% and phenylacetic acid derivatives conjugated

Juice is the most common form in which cranberries are consumed; however there is limited information on the changes of polyphenolic content of the berries during juice processing. This study investigated the effects of three different pretreatments (grinding plus blanching; only grinding; only blanching) for cranberry juice processing on the concentrations of anthocyanins, flavonols, and procyanidins throughout processing. Flavonols and procyanidins were retained in the juice to a greater extent than anthocyanins, and pressing resulted in the most significant losses in polyphenolics due to removal of the seeds and skins. Flavonol aglycones were formed during processing as a result of heat treatment. Drying of cranberry pomace resulted in increased extraction of flavonols and procyanidin oligomers but lower extraction of polymeric procyanidins. The results indicate that cranberry polyphenolics are relatively stable during processing compared to other berries; however, more work is needed to determine their fate during storage of juices.

Changes in blackberry ellagitannin composition in response to juicing (clarified and nonclarified), pureeing, canning (in water or syrup), and freezing as well as changes in processed products during 6 months of storage were investigated. Canning, pureeing, and freezing had little effect on ellagitannins, but processing berries into nonclarified and clarified juices resulted in total ellagitannin losses of 70 and 82%, respectively, due to removal of ellagitannin-rich seeds in the presscake. Minimal changes in total ellagitannin content were observed during storage of thermally processed products, but compositional changes indicative of ellagitannin depolymerization were apparent. The ellagitannin content and composition of frozen berries remained stable over 6 months of storage. Ellagitannins are well retained in canned, pureed, and frozen blackberries, but methods are needed to prevent losses during juice processing and/or exploit the ellagitannin-rich coproducts.

Anthocyanins (ACNs) from various foods have been shown to minimize the development of obesity in some animal models. The objective of the current study was to compare the effects of feeding purified black raspberry (BRB) ACNs or the freeze-dried whole BRB on the development of obesity. Male C57BL/6J mice (25 days of age) were assigned at random to treatments (7/treatment; 3/cage). The treatments included (1) control low-fat diet (10% calories from fat) (LF); (2) LF plus BRB juice in place of drinking water; (3) LF diet plus purified BRB ACNs in drinking water (1.25 mg/mL); (4) control high-fat diet (60% calories from fat) (HF60); (5) HF60 diet + BRB juice in place of drinking water; (6) HF60 diet + ACNs in drinking water (1.25 mg/mL); and (7) HF60 + freeze-dried whole BRB powder (21.7 g/kg of diet). Body weight gains in mice fed HF60 diet plus purified BRB ACNs tended to be lower after 56, 63, and 70 days than in mice fed HF60 alone. Body weights were increased at time of sacrifice, but heart, liver, and kidney weights as a percentage of body weight were decreased in mice fed HF60 diet compared to LF fed mice. Weights (g or g/body weight) of epididymal and retroperitoneal fat were increased in the HF60 fed mice compared to LF fed mice. Fasting serum glucose, leptin, and insulin levels as well as homeostasis assessment of insulin resistance (HOMA-IR) were elevated in mice fed the HF60 diet relative to LF-fed controls. Serum cholesterol, triglycerides, and monocyte chemoattractant protein-1 (MCP-1) were not altered by diet. Serum levels of resistin were increased in mice fed the HF60 diet compared to mice fed the LF diet. None of the responses measured were altered by whole BRB powder included in the diet relative to the HF60 control diet. Cyanidin containing di- or triglycosides in BRB was ineffective in altering the development of obesity in contrast to cyanidin-monoglycosides, which have been shown to be effective. The sugar moiety on the anthocyanidins may be an important factor in determining the response in the development of obesity.

Male C57BL/6J mice (25 days of age) were fed either a low-fat diet (10% kcal from fat) (LF) or a high-fat diet (45% kcal from fat) (HF45) for a period of 72 days. Blueberry juice or purified blueberry anthocyanins (0.2 or 1.0 mg/mL) in the drinking water were included in LF or HF45 treatments. Sucrose was added to the drinking water of one treatment to test if the sugars in blueberry juice would affect development of obesity. Total body weights (g) and body fat (%) were higher and body lean tissue (%) was lower in the HF45 fed mice compared to the LF fed mice after 72 days, but in mice fed HF45 diet plus blueberry juice or blueberry anthocyanins (0.2 mg/mL), body fat (%) was not different from those mice fed the LF diet. Anthocyanins (ACNs) decreased retroperitoneal and epididymal adipose tissue weights. Fasting serum glucose concentrations were higher in mice fed the HF45 diet. However, it was reduced to LF levels in mice fed the HF45 diet plus 0.2 mg of ACNs/mL in the drinking water, but not with blueberry juice. beta cell function (HOMA-BCF) score was lowered with HF45 feeding but returned to normal levels in mice fed the HF45 diet plus purified ACNs (0.2 mg/mL). Serum leptin was elevated in mice fed HF45 diet, and feeding either blueberry juice or purified ACNs (0.2 mg/mL) decreased serum leptin levels relative to HF45 control. Sucrose in drinking water, when consumption was restricted to the volume of juice consumed, produced lower serum leptin and insulin levels, leptin/fat, and retroperitoneal and total fat (% BW). Blueberry juice was not as effective as the low dose of anthocyanins in the drinking water in preventing obesity. Additional studies are needed to determine factors responsible for the differing responses of blueberry juice and whole blueberry in preventing the development of obesity.

The proximate composition and identification and quantification of polyphenolic compounds in dried cranberry pomace were determined. Proximate analysis was conducted based on AOAC methods for moisture, protein, fat, dietary fiber, and ash. Other carbohydrates were determined by the difference method. Polyphenolic compounds were identified and quantified by HPLC-ESI-MS. The composition of dried cranberry pomace was 4.5% moisture, 2.2% protein, 12.0% fat, 65.5% insoluble fiber, 5.7% soluble fiber, 8.4% other carbohydrates, 1.1% ash, and 0.6% total polyphenolics. It contained six anthocyanins (111.5 mg/100 g of DW) including derivatives of cyanidin and peonidin. Thirteen flavonols were identified (358.4 mg/100 g of DW), and the aglycones myricetin (55.6 mg/100 g of DW) and quercetin (146.2 mg/100 g of DW) were the most prominent. Procyanidins with degrees of polymerization (DP) of 1-6 were identified (167.3 mg/100 g of DW), the most abundant being an A-type of DP2 (82.6 mg/100 g of DW).

Fruits and fruit byproducts are rich sources of polyphenols, including procyanidins, which are known to have numerous potential health benefits. This study investigated if varietal differences existed in the procyanidin composition of grape seed and if soaking in extraction solvent overnight prior to extraction improved the recovery of procyanidins from grape seed, grape pomace, and blueberry and cranberry powders. Riesling contained the highest amount of procyanidins, including the lower molecular weight monomers and dimers, followed by Chardonnay (60%), whereas Merlot contained much lower levels (14%) of individual and total procyanidins. A modified method of extraction whereby selected fruits and fruit byproducts were soaked in the extraction solvent overnight before the extraction process was begun increased procyanidins extracted by 24-100% from grape seeds and by 0-30% with berry procyanidin sources. The results indicate a wide variation in the procyanidin contents among different varieties of grape seeds that could have implications in the selection of procyanidin-rich germplasm. Soaking samples in the extraction solvent for 16 h resulted in increased procyanidins extracted and thus higher calculated concentrations in the food samples tested.

Blackberries ( Rubus sp.) were evaluated by high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) to identify the ellagitannins present in flesh, torus (receptacle tissue), and seeds. Most ellagitannins were present (or detectable) only in seed tissues. Ellagitannins identified by HPLC-ESI-MS in the seeds included pedunculagin, casuarictin/potentillin, castalagin/vescalagin, lambertianin A/sanguiin H-6, lambertianin C, and lambertianin D. For several of the ellagitannins, isomeric separation was also obtained. The MALDI-TOF-MS analysis was primarily utilized to evaluate and identify high molecular mass (>1000 Da) ellagitannins. The MALDI analysis verified the presence of the ellagitannins identified by HPLC-ESI-MS including lambertianin A/sanguiin H-6, lambertianin C, and lambertianin D, but the analysis also indicated the presence of several other compounds that were most likely ellagitannins based on the patterns observed in the masses (i.e., loss or addition of a gallic acid moiety to a known ellagitannin). This study determined the presence of several possible isomeric forms of ellagitannins previously unidentified in fruit and presents a possible analytical HPLC method for the analysis of the major ellagitannins present in the fruit.

Catechins and procyanidins are beneficial for human health; however, their bioavailability is low. The effect of food processing on catechin bioavailability from sources containing predominantly procyanidins has not been studied. The sumac sorghum mixture (50% whole grain+50% bran) used in this study contained catechins, procyanidins dimers, and polymers at 0.08, 0.6, and 26.4 mg/g, respectively. Extrusion decreased the polymeric procyanidins by 48% to 22 mg/g while increasing catechins (50%) and dimers (64%) to 0.12 and 1.0 mg/g, respectively. Six weanling pigs (8.9+/-1.1 kg) received a single dose by gavage of the sorghum mixture (7 g/kg0.75), the sorghum mixture extrudate, or white sorghum (50% whole grain+50% bran) in a randomized crossover design. Treatments were separated by a 7-day washout period. Blood was drawn at 0, 1, 2, and 4 h. Plasma catechin, 3'-O-methylcatechin, 4'-O-methylcatechin, epicatechin, 3'-O-methylepicatechin, and 4'-O-methylepicatechin peaked at 1 h and were 18, 43, 1, 0.7, 0.7, and 0.3 nmol/L for pigs receiving sorghum, respectively. Plasma levels in pigs receiving extruded sorghum were 66, 110, 2, 16, 8, and 11 nmol/L, respectively. Plasma levels of catechin, 3'-O-methylcatechin, and the total catechins were higher in pigs fed extruded sorghum at 1, 2, and 4 h postdose (Por=0.05). The majority of the absorbed catechins were excreted within 4 h after feeding. Urinary excretion of total catechins was significantly higher in pigs fed extruded sorghum than in those fed nonextruded sorghum. Procyanidin dimers were not detected in plasma or urine. The levels of catechins were close to zero in plasma and urine of pigs fed white sorghum. In conclusion, extrusion improved the bioavailability of catechins in sorghum.

Male C57BL/6J mice received diets with either 10% of calories from fat (LF) or a high-fat diet [45% (HF45) or 60% (HF60) calories from fat] for 92 days (expt 1) or 70 days (expt 2). These were given with or without freeze-dried powders from whole blueberries (BB) or strawberries (SB) (expt 1) or purified anthocyanin extracts from BB or SB (expt 2). Body composition was determined utilizing Echo MRI. Berries added to the LF diet did not alter weight gain, final body weights, body fat, or protein (percent body weight) or diet (grams) or energy (kilocalories) intake. However, in both HF45- and HF60-fed mice, weight gain, final weights, body fat (percent), and epididymal fat weights increased and body protein decreased ( p0.01) compared to LF mice. In mice fed HF45 diet plus BB, body weight gains, body fat (percent of BW), and epididymal fat weights were significantly greater than those in the HF45-fed controls, whereas weights of mice fed SB HF were similar to those of HF controls. SB or BB feeding did not alter glucose tolerance, although glucose tolerance decreased with age and in HF45 versus LF mice. Baseline plasma glucose was lower in SB- versus HF45-fed mice. After 8 weeks, mice fed the HF60 diet plus purified anthocyanins from BB in the drinking water had lower body weight gains and body fat than the HF60-fed controls. Anthocyanins fed as the whole blueberry did not prevent and may have actually increased obesity. However, feeding purified anthocyanins from blueberries or strawberries reduced obesity.

Sorghum bran is concentrated with procyanidins (predominately polymers), which may be beneficial for health in humans; however, the bioavailability of procyanidins is not well-understood. Female Sprague-Dawley rats were fed an AIN93G diet containing 0, 5, 10, 20, or 40% Hi-tannin sorghum bran (n = 5-7 for each group) for 50 days. Sorghum bran contained 23.3 mg/g of procyanidins. The urinary excretions of catechin, epicatechin, methylated catechins, and phenolic acids were analyzed using liquid chromatography-tandem mass spectrometry. Sorghum bran dose dependently increased the urinary excretion of catechin (0-2.2 nmol/day) and 3'-O-methylcatechin (0-9.5 nmol/day). Their serum concentrations also increased with dose (range of 0-14 nM for 3'-O-methylcatechin). Among the 14 phenolic acids analyzed, 3,4-dihydroxybenzoic acid, 3-methoxy-4-hydroxybenzoic acid, and 4-hydroxyphenylacetic acid dominated in the serum (1.8-8 micromol/L). In the urine, 3-methoxy-4-hydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, and 3-hydroxyphenylpropionic acid dominated and their excretion increased significantly with the level of sorghum bran in the diet. The summed phenolic acid excretion was 0.8 micromol/day in the control group and increased to 23 micromol/day for 40% sorghum bran group. The hippuric acid excretion ranged from 2.2 to 16.2 micromol/day and peaked in the 10% sorghum bran group. On the basis of chromic oxide, a nonabsorbable marker, total procyanidins and polymers disappeared progressively, and significant degradation occurred in the cecum and colon. Catechins and procyanidins in sorghum were bioavailable; however, bacteria-derived phenolic acids were the predominant metabolites of procyanidins. Procyanidins degraded in the gastrointestinal tract. Depolymerization was not observed.

Euterpe oleraceae is a large palm tree indigenous to the Amazon River and its tributaries and estuaries in South America. Its fruit, known as acai, is of great economic value to native people. In this study, a standardized freeze-dried acai fruit pulp/skin powder was used for all analyses and tests. Among many findings, anthocyanins (ACNs), proanthocyanidins (PACs), and other flavonoids were found to be the major phytochemicals. Two ACNs, cyandin 3-glucoside and cyanidin 3-rutinoside were found to be predominant ACNs; three others were also found as minor ACNs. The total content of ACNs was measured as 3.1919 mg/g dry weight (DW). Polymers were found to be the major PACs. The concentration of total PACs was calculated as 12.89 mg/g DW. Other flavonoids, namely, homoorientin, orientin, isovitexin, scoparin, and taxifolin deoxyhexose, along with several unknown flavonoids, were also detected. Resveratrol was found but at a very low concentration. In addition, components including fatty acids, amino acids, sterols, minerals, and other nutrients were analyzed and quantified. Total polyunsaturated fatty acid, total monounsaturated fatty acid, and total saturated fatty acids contributed to 11.1%, 60.2%, and 28.7% of total fatty acid. Oleic acid (53.9%) and palmitic acid (26.7%) were found to be the two dominant fatty acids. Nineteen amino acids were found; the total amino acid content was determined to be 7.59% of total weight. The total sterols accounted for 0.048% by weight of powder. The three sterols B-sitosterol, campesterol, and sigmasterol were identified. A complete nutrient analysis is also presented. Microbiological analysis was also performed.

Many fruits are rich in anthocyanins (ACNs). ACNs have high antioxidant capacity, but because of their apparent low bioavailability, their possible roles in health promotion in vivo are still in question. The objectives of these studies were to determine the fate of ACNs within the gastrointestinal (GI) tract and the effect on the bioavailability and subsequent metabolism of ACNs. Five weanling pigs were fed freeze-dried black raspberry (Rubus occidentalis L.) powder by oral administration, which provided 1146.1 ± 44.6 μmol TE of oxygen radical absorbance capacity with fluorescein as a fluorescent probe (ORACFL) per kg and 50.5 ± 3.7 mg per kg total ACNs. After 4 h, the pigs were sacrificed and the contents of five GI segments (duodenum, jejunum, ileum, cecum, and colon) were collected and analyzed for their total antioxidant capacity (TAC, measured as ORACFL) and ACNs. The recoveries of TAC and total ACNs were 46.5 ± 3.5 and 41.7 ± 4.9%, respectively. Both total ACNs and TAC were recovered primarily in t...

Proanthocyanidins and flavonoids were isolated and identified from seed coats of two aged and nonaged pinto bean lines: 1533-15 and CDC Pintium. The seed coat of 1533-15 darkens slowly and never darkens to the same extent as CDC Pintium. Analysis of the overall level of proanthocyanidins using a vanillin assay demonstrated that aged and nonaged seed coats of CDC Pintium had significantly higher levels of proanthocyanidins than aged and nonaged 1533-15 seed coats. Aged and nonaged seed coats of both lines were found to contain one main flavonol monomer, kaempferol, and three minor flavonols, kaempferol 3-O-glucoside, kaempferol 3-O-glucosylxylose, and kaempferol 3-O-acetylglucoside. These compounds were identified by NMR and ESI-MS analysis (except for kaempferol 3-O-acetylglucoside, which was tentatively identified only by ESI-MS analysis) and quantified using HPLC-DAD. The combined concentrations of all the kaempferol compounds in seed coats of CDC Pintium were significantly higher than in seed coats of 1533-15, and the combined contents did not change after aging. The content of kaempferol decreased nearly by half in the seed coats of CDC Pintium after aging, whereas no significant change was observed in the seed coats of 1533-15. Proanthocyanidin fractions from both lines, aged and nonaged, were subjected to LC-MS/MS analysis and found to be composed primarily of procyanidins. Procyanidins in the seed coats were predominantly polymers with the degree of polymers higher than 10. The proportion of these polymers decreased after aging, while that of the low-molecular-weight procyanidins increased. A catechin-kaempferol adduct was tentatively identified in both lines by LC-MS/MS, and the concentration increased in the seed coats after aging.

Anthocyanins in common foods in the United States, other than fruits and berries, were identified and characterized by high-performance liquid chromatography (HPLC)-electrospray ionization-tandem mass spectrometry coupled with diode array detection. Of all of the 40+ vegetables, nuts, and grains screened, seven vegetables, one nut, and one grain were found to contain anthocyanins; the number of anthocyanins detected varied from two in pistachio nuts to 34 in red radishes. The individual anthocyanins were identified by comparing their mass spectrometric data and retention times with those of standards, published data, and reference food samples. In all of the samples analyzed, except for sorghum, only six common anthocyanidins (delphinidin, cyanidin, pelargonidin, petunidin, peonidin, and malvidin) were found as their glycosides. Anthocyanins in certain vegetables such as red cabbage and red radish were highly conjugated with sugars and acylated groups, and thus, their structures were very complicated. Eight different either aliphatic or aromatic acylated groups (acetoyl, coumaroyl, malonoyl, p-hydroxybenzoyl, feruoyl, caffeoyl, sinapoyl, and oxaloyl) were identified in the anthocyanins. In addition to glucose, six other sugar moieties (galactose, xylose, rhamnose, rutinose, sambubiose, and laminaribiose) were observed. Three varieties of sorghum were found to contain 3-deoxyanthocyanidins and their derivatives as major anthocyanins. A number of new anthocyanins were identified in the foods studied. This paper presents complete HPLC profiles and MS spectrometric data, obtained under the same experimental conditions, for common vegetables, pistachio nuts, and sorghum that contain anthocyanins.

Anthocyanins were systematically identified and characterized by HPLC-ESI-MS/MS coupled with diode array detection in common fruits from U.S. food markets and other commercial sources. Of the 25 different fruits that were screened, 14 fruits were found to contain anthocyanins; the number of anthocyanins varied from 2 in peaches and nectarines to 31 in Concord grape. The individual anthocyanins were identified by comparing their mass spectral data and retention times with those of standards and published data. In all of the samples analyzed, only 6 common anthocyanidins, delphinidin, cyanidin, pelargonidin, petunidin, peonidin and malvidin, were found. In addition to the well-known major anthocyanins, a number of minor anthocyanins were identified for the first time. Some possible guidelines that help to identify anthocyanins in foods with complex anthocyanin composition were deduced and discussed. For the first time, this paper presents complete anthocyanin HPLC profiles and MS spectral data of common fruits using the same uniform experimental conditions.

This review summarizes the multifaceted aspects of antioxidants and the basic kinetic models of inhibited autoxidation and analyzes the chemical principles of antioxidant capacity assays. Depending upon the reactions involved, these assays can roughly be classified into two types: assays based on hydrogen atom transfer (HAT) reactions and assays based on electron transfer (ET). The majority of HAT-based assays apply a competitive reaction scheme, in which antioxidant and substrate compete for thermally generated peroxyl radicals through the decomposition of azo compounds. These assays include inhibition of induced low-density lipoprotein autoxidation, oxygen radical absorbance capacity (ORAC), total radical trapping antioxidant parameter (TRAP), and crocin bleaching assays. ET-based assays measure the capacity of an antioxidant in the reduction of an oxidant, which changes color when reduced. The degree of color change is correlated with the sample's antioxidant concentrations. ET-based assays include the total phenols assay by Folin-Ciocalteu reagent (FCR), Trolox equivalence antioxidant capacity (TEAC), ferric ion reducing antioxidant power (FRAP), "total antioxidant potential" assay using a Cu(II) complex as an oxidant, and DPPH. In addition, other assays intended to measure a sample's scavenging capacity of biologically relevant oxidants such as singlet oxygen, superoxide anion, peroxynitrite, and hydroxyl radical are also summarized. On the basis of this analysis, it is suggested that the total phenols assay by FCR be used to quantify an antioxidant's reducing capacity and the ORAC assay to quantify peroxyl radical scavenging capacity. To comprehensively study different aspects of antioxidants, validated and specific assays are needed in addition to these two commonly accepted assays.

Sorghum procyanidins were characterized and quantified from two brown sorghum varieties and their processed products by normal phase HPLC with fluorescence detection. The DP of the procyanidins was determined by thiolysis. Quantification was done by using purified oligomeric and polymeric cocoa procyanidins as external standards. Sorghum procyanidins were composed mostly of high MW (DP10) polymers. Significant differences were observed in levels as well as distribution of the different MW procyanidins between the sorghums. Processing of the sorghum brans into cookies and bread significantly reduced the levels of procyanidins; this effect was more pronounced in the higher MW polymers. Cookies had a higher retention of procyanidins (42-84%) than bread (13-69%). Extrusion of sorghum grain resulted in an increase in the levels of procyanidin oligomers with DP/= 4 and decrease in polymers with DP/= 6. This suggests a possible breakdown of the high MW polymers to the lower MW constituents during extrusion. Processing changes not only the content of procyanidins in sorghum products but also the relative ratio of the different molecular weights.

The polymeric procyanidins were fractionated from lowbush blueberry on a Sephadex LH-20 column. The degree of polymerization (DP) for the polymers was determined by thiolysis to be in a range of 19.9 to 114.1. Normal-phase HPLC analysis indicated that the polymeric procyanidins did not contain oligomeric procyanidins with DP10. The polymers eluted as a single peak at the end of the chromatogram. The normal-phase HPLC gradient was modified to improve the separation of procyanidin monomers through decamers and to elute all the polymers beyond those as a distinct peak. Monomers through decamers were quantified individually. All the polymers (DP10) were quantified using a mixture of purified polymers as an external standard. Polymers were found to be the dominant procyanidins in brown sorghum bran, cranberry, and blueberry. Thiolysis of the polymer peaks indicated that epicatechin was present as extension units in these foods, however, the composition of terminal units varied considerably between catechin and epicatechin, or an A-type dimer linkage in the case of cranberry.

The oxygen radical absorbance capacity (ORAC) assay has been widely accepted as a standard tool to measure the antioxidant activity in the nutraceutical, pharmaceutical, and food industries. However, the ORAC assay has been criticized for a lack of accessibility due to the unavailability of the COBAS FARA II analyzer, an instrument discontinued by the manufacturer. In addition, the manual sample preparation is time-consuming and labor-intensive. The objective of this study was to develop a high-throughput instrument platform that can fully automate the ORAC assay procedure. The new instrument platform consists of a robotic eight-channel liquid handling system and a microplate fluorescence reader. By using the high-throughput platform, the efficiency of the assay is improved with at least a 10-fold increase in sample throughput over the current procedure. The mean of intra- and interday CVs was ≤15%, and the limit of detection and limit of quantitation were 5 and 6.25 μM, respectively. Keywords: ORAC; anti...

Recent interest in the possible protective effects of dietary antioxidant compounds against human degenerative disease has prompted investigation of foods such as blueberries (Vaccinium sp.), which have a high antioxidant capacity. Fruit obtained from genotypes of highbush blueberries (Vaccinium corymbosum L.) and lowbush blueberries (Vaccinium angustifolium Aiton) were analyzed for their antioxidant capacity, their content of anthocyanins, and total phenolic compounds, to evaluate the intraspecific and interspecific variation in these parameters. The method of extraction influenced the composition of fruit extracts; the highest anthocyanin and total phenolic contents and antioxidant capacity were found in extracts obtained using a solvent of acidified aqueous methanol. Regardless of the method, lowbush blueberries were consistently higher in anthocyanins, total phenolics, and antioxidant capacity, compared with highbush blueberries. There was no relationship between fruit size and anthocyanin content in either species.

Antioxidant capacity, as measured by oxygen radical absorbance capacity (ORAC), and total phenolic and total anthocyanin contents were evaluated in fruit tissues of 87 highbush blueberry (Vacciniumcorymbosum L.) and species-introgressed highbush blueberry cultivars. ORAC and phenolic levels were evaluated in leaf tissues of the same materials. Average values for ORAC, phenolics, and anthocyanins in fruit were 15.9 ORAC units, 1.79 mg/g (gallic acid equivalents), and 0.95 mg/g (cyanidin-3-glucoside equivalents), respectively. Cv. Rubel had the highest ORAC per gram of fresh weight values, at 31.1 units, and cv. Elliott had the highest values on the basis of ORAC per square centimeter of surface area. In leaf tissue, values for both ORAC and phenolics were significantly higher than in fruit tissue, with mean values of 490 ORAC units and 44.80 mg/g (gallic acid equivalents), respectively. Leaf ORAC had a low, but significant, correlation with fruit phenolics and anthocyanins, but not with fruit ORAC. An analysis of ORAC values versus calculated midparent values in 11 plants from the 87-cultivar group in which all parents were tested suggested that, across cultivars, ORAC inheritance is additive. An investigation of ORAC values in a family of 44 cv. Rubel x Duke seedlings showed negative epistasis for ORAC values, suggesting Rubel may have gene combinations contributing to ORAC that are broken up during hybridization.

Milled oat groat pearlings, trichomes, flour, and bran were extracted with methanol and the fractions tested in vitro for antioxidant capacity against low-density lipoprotein (LDL) oxidation and R-phycoerythrin protein oxidation in the oxygen radical absorbance capacity (ORAC) assay. The oxidative reactions were generated by 2,2'-azobis(2-amidinopropane) HCl (AAPH) or Cu(2+) in the LDL assay and by AAPH or Cu(2+) + H(2)O(2) in the ORAC assay and calibrated against a Trolox standard to calculate Trolox equivalents (1 Trolox equivalent = 1 TE = activity of 1 micromol of Trolox). The antioxidant capacity of the oat fractions was generally consistent with a potency rank of pearlings (2.89-8.58 TE/g) > flour (1.00-3.54 TE/g) > trichome (1.74 TE/g) = bran (1.02-1.62 TE/g) in both LDL and ORAC assays regardless of the free radical generator employed. A portion of the oat antioxidant constituents may be heat labile as the greatest activity was found among non-steam-treated pearlings. The contribution of oat tocols from the fractions accounted for

Monomeric and oligomeric procyanidins present in cocoa liquors and chocolates were separated and quantified in four different laboratories using a normal-phase high-performance liquid chromatography (HPLC) method with fluorescence detection. Procyanidin standards through decamers were obtained by extraction from cocoa beans, enrichment by Sephadex LH-20 gel permeation chromatography, and final purification by preparative normal-phase HPLC. The purity of each oligomeric fraction was assessed using HPLC coupled to mass spectrometry. A composite standard was then prepared, and calibration curves were generated for each oligomeric class using a quadratic fit of area sum versus concentration. Results obtained by each of the laboratories were in close agreement, which suggests this method is reliable and reproducible for quantification of procyanidins. Furthermore, the procyanidin content of the samples was correlated to the antioxidant capacity measured using the ORAC assay as an indicator for potential biological activity.

Different cultivars of four Vaccinium species [Vaccinium corymbosum L (Highbush), Vaccinium ashei Reade (Rabbiteye), Vaccinium angustifolium (Lowbush), and Vaccinium myrtillus L (Bilberry)] were analyzed for total phenolics, total anthocyanins, and antioxidant capacity (oxygen radical absorbance capacity, ORAC). The total antioxidant capacity of different berries studied ranged from a low of 13.9 to 45.9 μmol Trolox equivalents (TE)/g of fresh berry (63.2−282.3 μmol TE/g of dry matter) in different species and cultivars of Vaccinium. Brightwell and Tifblue cultivars of rabbiteye blueberries were harvested at 2 times, 49 days apart. Increased maturity at harvest increased the ORAC, the anthocyanin, and the total phenolic content. The growing location (Oregon vs Michigan vs New Jersey) did not affect ORAC, anthocyanin or total phenolic content of the cv. Jersey of highbush blueberries. A linear relationship existed between ORAC and anthocyanin (rxy = 0.77) or total phenolic (rxy = 0.92) content. In general,...

An HPLC procedure utilizing reversed-phase chromatography coupled with a coulometric array detection system was developed for the characterization of overall antioxidant status in fruits and vegetables. The method was reliable and sensitive (20 pg to 1 ng detection limit) and can also be used to identify and simultaneously quantify multi-antioxidants including vitamin C, glutathione, phenolic acids, and flavonoids in fruits and vegetables. Each fruit and vegetable aqueous extract showed an unique distribution of chromatographic peaks that could serve as a “fingerprint” for the fruit or vegetable. A significant positive linear correlation was demonstrated in fruit and vegetable aqueous extracts between the total antioxidant activities determined by using oxygen radical absorbance capacity (ORACROO•) assay and the electrochemical data generated from the coulometric array detectors. From these data, the ORACROO• assay appears to be a valid and useful procedure for measuring total antioxidant activity in extr...

Anthocyanins are natural colorants belonging to the flavonoid family. They are widely distributed among flowers, fruits, and vegetables. Using the automated oxygen radical absorbance capacity (ORAC) assay developed in our laboratory, we determined the antioxidant capacity of 14 anthocyanins including the aglycons delphinidin, cyanindin, pelargonidin, malvidin, peonidin, and their derivatives with different sugar linkages. Among these anthocyanins, kuromanin (cyanidin-3-glucoside) had the highest ORAC activity, which was 3.5 times stronger than Trolox (vitamin E analogue), while pelargonin had the lowest antioxidant activity but was still as potent as Trolox. Different patterns of hydroxylation and glycosylation in anthocyanins appear to modulate their antioxidant properties. Therefore, in addition to their colorful characteristics, anthocyanins possess potent antioxidant properties.

Previously, some fruits were shown to contain high antioxidant activities. In this paper, we report the antioxidant activities of 22 common vegetables, one green tea, and one black tea measured using the automated oxygen radical absorbance capacity assay with three different reactive species: a peroxyl radical generator, a hydroxyl radical generator, and Cu2+, a transition metal. Based on the fresh weight of the vegetable, garlic had the highest antioxidant activity (μmol of Trolox equiv/g) against peroxyl radicals (19.4) followed by kale (17.7), spinach (12.6), Brussels sprouts, alfalfa sprouts, broccoli flowers, beets, red bell pepper, onion, corn, eggplant (9.8−3.9), cauliflower, potato, sweet potato, cabbage, leaf lettuce, string bean, carrot, yellow squash, iceberg lettuce, celery, and cucumber (3.8−0.5); kale had the highest antioxidant activity against hydroxyl radicals followed by Brussels sprouts, alfalfa sprouts, beets, spinach, broccoli flowers, and the others. The green and black teas had muc...

The total antioxidant activity of 12 fruits and 5 commercial fruit juices was measured in this study using automated oxygen radical absorbance capacity (ORAC) assay. On the basis of the wet weight of the fruits (edible portion), strawberry had the highest ORAC activity (micromoles of Trolox equivalents per gram) followed by plum, orange, red grape, kiwi fruit, pink grapefruit, white grape, banana, apple, tomato, pear, and honeydew melon. On the basis of the dry weight of the fruits, strawberry again had the highest ORAC activity followed by plum, orange, pink grapefruit, tomato, kiwi fruit, red grape, white grape, apple, honeydew melon, pear, and banana. Most of the antioxidant capacity of these fruits was from the juice fractions. The contribution of the fruit pulp fraction (extracted with acetone) to the total ORAC activity of a fruit was usually less than 10%. Among the commercial fruit juices, grape juice had the highest ORAC activity followed by grapefruit juice, tomato juice, orange juice, and apple juice.

A method for determining organic acid levels in biological materials by silica gel partition column chromatography (pcc) is described. It uses tetrabromophenolphthalein ethyl ester, which has E. high molar absorbency, for quantitating the acids in the column effluent. The concentrations of several organic acids were determined in two species of crested wheatgrass, Nordan (Agropyron ciesertorum) and Fairway (A. cristatum), grown. under different conditions of K fertilization. For comparison, aconitic, citric, and malic acids were also determined by polarography, spectrophotometry, and fluorometry, respectively. Results obtained by these methods agreed with the data obtained by pcc. The concentration of trans-aconitic acid averaged about 96 and 21 mequiv/kg of dry matter in Nordan and Fairway, respectively, when grown in the greenhouse without K. K fertilization (312 kg/ha) approximately doubled the trans-aconitic acid concentration in both species. Fairway contained 363 mequiv/kg of malic acid, while Nordan contained 280 mequiv/kg. K fertilization increased these concentrations to 611 and 446 mequiv/kg, respectively. Citric acid was increased by K fertilization, but there was no significant difference between species and in no case was the citric acid concentration greater than 80 mequiv/kg of dry matter.