Absorption of N-phenylpropenoyl-L-amino acids in healthy humans by oral administration of cocoa (Theobroma cacao)

Besides flavan-3-ols, a family of N-phenylpropenoyl-L-amino acids (NPAs) has been recently identified as polyphenol/amino acid conjugates in the seeds of Theobroma cacao as well as in a variety of herbal drugs. Stimulated by reports on their biological activity, the purpose of this study was to investigate if these amides are absorbed by healthy volunteers after administration of a cocoa drink. For the first time, 12 NPAs were quantified in human urine by means of a stable isotope dilution analysis with LC-MS/MS (MRM) detection. A maximum amount was found in the urine taken 2 h after the cocoa consumption. The highest absolute amount of NPAs excreted with the urine was found for N-[4'-hydroxy-(E)-cinnamoyl]-L-aspartic acid (5), but the highest recovery rate (57.3 and 22.8%), that means the percentage amount of ingested amides excreted with the urine, were determined for N-[4'-hydroxy-(E)-cinnamoyl]-L-glutamic acid (6) and N-[4'-hydroxy-3'-methoxy-(E)-cinnamoyl]-L-tyrosine (13). In order to gain first insights into the NPA metabolism in vivo, urine samples were analyzed by LC-MS/MS before and after beta-glucuronidase/sulfatase treatment. As independent of the enzyme treatment the same NPA amounts were found in urine, there is strong evidence that these amides are metabolized neither via their O-glucuronides nor their O-sulfates. In order to screen for caffeic acid O-glucuronides as potential NPA metabolites, urine samples were screened by means of LC-MS/MS for caffeic acid 3-O-beta-D-glucuronide and 4-O-beta-D-glucuronide. But not even trace amounts of one of these glucuronides were detectable, thus excluding them as major NPA metabolites and underlining the importance of future investigations on a potential O-methylation or reduction of the N-phenylpropenoyl moiety in NPAs.     

Urinary 2-ethyl-3-oxohexanoic acid as major metabolite of orally administered 2-ethylhexanoic acid in human

Human metabolism of 2-ethylhexanoic acid (2-EHA), which is a known metabolite of important phthalates, was investigated using 2-EHA-contaminated food. The results of our studies reveal that the major catabolic pathway of 2-EHA in human is beta-oxidation. The dominant final urinary metabolite was identified and quantified as 3-oxo-2-ethylhexanoic acid (3-oxo-2-EHA), but only after immediate methylation of the extract from urine and prior to GC-MS analysis. Former studies without the precaution of immediate methylation had found 4-heptanone as the major metabolite, which is obviously an artifact arising from the decarboxylation of 3-oxo-2-EHA.     

Comparative in vivo antioxidant capacity of DL-2-hydroxy-4-methylthiobutanoic acid (HMTBA) and DL-methionine in male mice fed a high-fat diet

BACKGROUND: In animal diets, methionine (Met) is considered to be the first limiting amino acid, and the activity of synthetic Met is typically added either as DL ‐methionine (DLM) or as DL ‐2‐hydroxy‐4‐methylthiobutanoic acid (HMTBA). It has been demonstrated that HMTBA exhibits a higher antioxidant capability in vitro as compared to DLM. However, the difference in antioxidant capability between DLM and HMTBA in vivo is unknown. METHODS: In the present study, 60 male C57BL/6 mice were randomly divided into six groups and fed either a normal diet (NFD, 5.37% fat) or a high‐fat diet (HFD, 19.7% fat) in conjunction with 0.2% DLM, 0.2% HMTBA or 0.1% DLM and 0.1% HMTBA for 4 weeks. RESULTS: HFD supplemented with 2% DLM and NFD with 2% HMTBA both induced adverse affects in relation to serum lipid parameters and depressed antioxidant defense systems in the digestive system. However, these changes were restored in the 0.2% HMTBA‐treated HFD group. Furthermore, no significant differences were found in the lipid parameters and antioxidant status in the NFD and HFD group supplemented with 0.1% DLM and 0.1% HMTBA. CONCLUSION: HMTBA restored oxidative redox status under OS conditions and its antioxidant properties were positively correlated with the dosage included in diet. Copyright © 2011 Society of Chemical Industry     

Improved determination of D-glucosamine hydrochloride in health foods by HPLC using 7-fluoro-4-nitrobenzo-2-oxa-l,3-diazole as a derivative

Abstract: This article presents an improved method to detect d ‐glucosamine hydrochloride in health foods. A simple precolumn derivatization procedure with 7‐flouro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD‐F) reagent was employed. The separation of the derivatized d ‐glucosamine hydrochloride (NBD‐d ‐glucosamine hydrochloride) was performed using a mobile phase consisting of acetonitrile, potassium dihydrogen phosphate (0.01 mol/L), and trifluoroacetic acid (350:649.74:0.26, volume ratio) at a flow rate of 1.0 mL/min with the column temperature 35 °C. Under the optimum chromatographic conditions, the peak area of NBD‐d ‐glucosamine hydrochloride compared with its absolute value of the peak area of NBD‐d ‐glucosamine hydrochloride in a standard solution concentration range from 1.0 to 500.0 mg/L showed a good linear calibration (R = 0.9999). Recoveries, at spiked concentrations of 10.0, 40.0, and 500.0 mg/L, varied between 97.2% and 102.6% with relative standard deviations ranging from 0.4% to 1.5%. The present method provides sufficient sensitivity as reflected by the values of limit of detection (LOD) and limit of quantification (LOQ). LOD was determined from the signal‐to‐noise ratios (S/N) of NBD‐d ‐glucosamine hydrochloride peak of at least 3 in the recovery test at 0.02 mg/L, and the estimated LOQ was 0.06 mg/L (S/N = 10). The proposed method was successfully applicable to detect d ‐glucosamine hydrochloride in health foods and drugs containing a variety of complex materials.     

Anthocyanin content and antioxidant capacity in bran extracts of some Thai black rice varieties

This study investigated both the anthocyanin content and the antioxidant capacity of a set of genetically related glutinous and nonglutinous Thai black rice varieties. The ethanol/water extracts of the brans of these black rice varieties showed relatively potent antioxidant activities compared with those of tocopherol. These antioxidant activities were determined by thiocyanate, H₂O₂‐scavenging chemiluminescence (XYZ), Cu⁺⁺/bathocuproine colorimetry (PAO) and 1,1‐diphenyl‐2‐picrylhydrazyl radical‐scavenging assay. The structural identification and quantification of the black rice anthocyanins performed by high‐performance liquid chromatography coupled with electrospray ionisation and tandem mass spectrometry found cyanidin‐3‐O‐glucoside and peonidin‐3‐O‐glucoside as the major anthocyanins in the ranges of 16.01–34.40 and 2.43–7.36 μg mL^?1, respectively. The comparative study in terms of quantity of these phytochemicals and antioxidant capacity of the black rice bran extracts suggested the contribution of overall phenolic components rather than that of the particular anthocyanin pigments.     

Age‐related changes in pro‐opiomelanocortin (POMC) and related receptors in human epidermis

Much effort has been placed in cosmetic research for better understanding of the effects of ageing on skin’s appearance, structure, mechanical properties and function. It is now of common knowledge that UV radiations induce pre‐mature skin ageing notably in the epidermis where UV radiations induce keratinocyte differentiation. As UV radiations have also been shown to regulate the pro‐opiomelanocortin (POMC) peptide family in the skin and because no study has been conducted so far to investigate the age‐related changes in POMC and related receptors, we analysed POMC, MC‐1R, MC‐2R and MOR‐1 at mRNA level and MC‐1R, MC‐2R and MOR‐1 at protein level too in primary cultures of normal human keratinocytes obtained from female donors aged from 17 to 75 years old. Regarding the gene expressions, we observed that MC‐1R, MC‐2R and MOR‐1 suffered a dramatic decrease after 50 years of age, whereas POMC increased five‐fold. Western blot analysis confirmed these results except for MOR‐1 whose expression appeared to decrease at older age, around 70 years old. Immunostainings specific to MC‐1R, MC‐2R and MOR‐1 performed on full‐thickness skin biopsies also revealed an intense staining in the basal and spinous layers of a 30‐year‐old donor, whereas no reactivity could be observed in a 60‐year‐old one. We conclude that POMC and POMC‐related receptors suffer a dramatically disturbed balance with ageing and that this may be implicated in the general process of skin ageing.     

HPLC-DAD-ESI-MS analysis of flavonoid compounds in 5 seedless table grapes grown in Apulian Region

Flavonoids present in skin extracts of red seedless table grape varieties Summer Royal, Autumn Royal, and Crimson, and white seedless varieties Carati and Thompson were analyzed by HPLC-DAD-MS, in 3 y of study (2006 to 2008). The anthocyanins, delphinidin-3-O-glucoside, cyanidin-3-O-glucoside, petunidin-3-O-glucoside (with their corresponding p-coumaroyl derivatives), peonidin-3-O-glucoside, and malvidin-3-O-glucoside (with their corresponding acetyl, caffeoyl, and p-coumaroyl derivatives) were found. In addition the flavonols quercetin-3-O-glucuronide, quercetin-3-O-rutinoside, quercetin-3-O-glucoside, kaempferol-3-O-glucoside, kaempferol-3-O-galactoside, and the flavan-3-ols procyanidin B1, procyanidin B2, and catechin were also detected. Anthocyanins were the main flavonoids in red grapes ranging from 24 (Crimson) to 500 (Summer Royal) mg/kg fresh weight of grapes; consistent levels of flavonols and flavan-3-ols were also quantified in all varieties. To determine the effective climatic influence on flavonoids content in field conditions, viticultural practices have been developed, that could exclude the effects of direct solar radiation from confounding the assessment of those related to thermal conditions alone. A strong positive correlation was determined between flavonoids and temperature data that seem to be responsible for the difference of these metabolites along the years; furthermore, it has been possible to define a linear relationship (R(2) = 0.6871, P = 0.0057) between thermal amplitude and total flavonoids values in the red grapes. PRACTICAL APPLICATION: Grapes are economically the most important fruit species in the world and approximately 30% of its production is used as fresh fruit. Because of the very important role of flavonoids in food quality as well as their health-promoting properties, and considering that our experiments were performed along 3 consecutive years, gathered results in this research are quite promising to give a useful information on the flavonoid contents and their evolution in 5 seedless table grapes that are widespread in Mediterranean regions but also in California and South America, and are grown in a viticultural climate (Apulia, South Italy) very close to some regions of Spain, Turkey, Tunisia, and Israel.     

In vitro antioxidant activity of different cultivars of banana flower (Musa paradicicus L.) extracts available in India

Abstract: Six different cultivars of banana flowers (Musa paradicicus) (Kathali, Bichi, Shingapuri, Kacha, Champa, and Kalabou) were analyzed for the content of polyphenol expressed as gallic acid equivalent and flavonoid expressed as quercetein equivalent, and the in vitro total antioxidative activities of the flower extracts were compared with standard and expressed as trolox equivalent. The reducing power, 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) and 2,2‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid) radical cation (ABTS?⁺) scavenging activities, inhibition of lipid peroxidation in a linoleic acid emulsion system, and liposome peroxidation system were measured and compared with respective standard antioxidants. Iron‐mediated Fenton reaction was carried out to evaluate the protective effect of the extract of banana flower (Kacha cultivar) against H₂O₂‐induced DNA damage. The Kacha variety contains the maximum amount of polyphenol (11.94 ± 0.03 mg of gallic acid equivalent/g of dry weight) and flavonoid (0.174 ± 0.001 g of quercetin equivalent/g of polyphenol). It also has the highest total antioxidant capacity, DPPH radical scavenging activity, and ABTS?⁺ radical scavenging activity with a least EC₅₀ value of 0.051 mg/mL. Hepatic cell damage in iron‐mediated Fenton reaction caused by free radicals is reduced by the banana flower extract. On the basis of the results obtained, the banana flowers are found to be a potential source of natural antioxidants. This is the first report on the antioxidant properties of the extracts from banana flowers. The study suggests that the flowers of M. paradicicus that are found in India and consumed as vegetable can provide valuable functional ingredients that help in the prevention of oxidative stress.     

Inhibitory effects of black tea theaflavin derivatives on 12-O-tetradecanoylphorbol-13-acetate-induced inflammation and arachidonic acid metabolism in mouse ears

Tea has been shown to possess several health beneficial properties primarily due to its polyphenolic content. The major polyphenolic compounds in black tea leaves are theaflavins (TFs) formed by oxidative coupling of catechins in tea leaves during its processing. In this paper, we report the characterization of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse ear inflammatory model and the inhibitory effects of major black tea TFs derivatives on this inflammation. In addition, the effect on inflammatory biomarkers, such as proinflammatory cytokines and arachidonic acid metabolites, are reported as well. A single topical application of TPA to ears of CD-1 mice induced a time- and dose-dependent increase in edema as well as formation of proinflammatory cytokine proteins interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) in mouse ears. A single topical application of equimolar of black tea constituents (TF, theaflavin-3-gallate, theaflavin-3'-gallate, and theaflavin-3,3'-digallate) strongly inhibited TPA-induced edema of mouse ears. Application of TFs mixture to mouse ears 20 min prior to each TPA application once a day for 4 days inhibited TPA-induced persistent inflammation, as well as TPA-induced increase in IL-1beta and IL-6 protein levels. TFs also inhibited arachidonic acid (AA) metabolism via both cyclooxygenase (COX) and lipoxygenase pathways. This observation was substantiated by decreased amounts of AA metabolites prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) levels. Combined application of TF and sulindac, a nonsteroidal anti-inflammatory drug resulted a significant synergetic anti-inflammatory effect. Oral administration of TFs or the hot water extract of black tea leaves also significantly inhibited TPA-induced edema in mouse ears. In conclusion, proinflammatory cytokines, IL-1beta and IL-6, as well as the intermediated metabolites of AA, PGE2, and LTB4 are good biomarkers for inflammation. Black tea constituents, TF and its derivatives, had strongly anti-inflammatory activity in vivo which may be due to their ability to inhibit AA metabolism via lipoxygenase and COX pathways.     

Fate of 14C-acrylamide in roasted and ground coffee during storage

Acrylamide (AA) is formed during heating of carbohydrate rich foods in the course of the Maillard reaction. AA has been classified as probably carcinogenic to humans. Storage experiments with roasted coffee have shown that AA levels decrease depending on storage time and temperature. In the present study the fate of AA lost during storage of roasted and ground (R&G) coffee was studied, using 14C-labeled AA as radiotracer. Radiolabel was measured in coffee brew, filter residue, and volatiles. In the brew, total (14)C-label decreased during storage of R&G coffee, while activity in the filter residue built up concomitantly. [2,3-14C]-AA (14C-AA) was the only 14C-related water extractable low molecular compound in the brew detected by radio-HPLC. No formation of volatile 14C-AA-related compounds was detected during storage and coffee brewing. Close to 90% of the radiolabel in the filter residue (spent R&G coffee, spent grounds) remained firmly bound to the matrix, largely resisting extraction by aqueous ammonia, ethyl acetate, chloroform, hexane, and sequential polyenzymatic digest. Furanthiols, which are abundant as aroma components in roasted coffee, have not been found to be involved in the formation of covalent AA adducts and thus do not contribute substantially to the decrease of AA during storage.     

Heme oxygenase induction by cyanidin-3-O-beta-glucoside in cultured human endothelial cells

The aim of the present research was to investigate the effect of cyanidin-3-O-beta-glucoside (C3G) on heme oxygenase-1 (HO-1), endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS) and dimethylarginine dimethylamino hydrolase-2 (DDAH-2) expression in cultured endothelial cells. Different concentrations (0.00625-250 microM) of C3G were tested in order to investigate possible beneficial and harmful effects of C3G. Our data demonstrated that C3G increased the induction of eNOS and HO-1 in a dose-dependent manner. Higher concentration (62.5-250 microM) also resulted in increase of isoprostane, cGMP and PGE2 levels and in induction of iNOS with consequent oxidative stress. In conclusion, our data evidence that C3G may exert various protective effects against endothelial dysfunction, whereas potentially harmful effects of C3G appear to be limited to concentrations very difficult to be reached in physiological conditions unless there is abundant oral supplementation.     

Increased ethylene biosynthesis elevates incidence of chilling injury in cold‐stored ‘Amber Jewel’ Japanese plum (Prunus salicina Lindl.) during fruit ripening

Mature ‘Amber Jewel’ Japanese plum fruit were stored at 0 or 5 °C for 3 and 6 weeks to investigate their effects on ethylene (C₂H₄) biosynthesis during ripening at ambient temperature in the development of chilling injury (CI) and fruit quality. CI (internal breakdown and browning) and fruit softening were higher during ripening, in the fruit stored at 5 °C than 0 °C, irrespective of storage period (SP). C₂H₄ production and activities of 1‐aminocyclopropane‐1‐carboxylic acid synthase (ACS) and 1‐aminocyclopropane‐1‐carboxylic acid oxidase (ACO) enzymes, and 1‐aminocyclopropane‐1‐carboxylic acid (ACC) content were higher during ripening in fruit stored at 5 °C than 0 °C. The fruit stored at 5 °C also exhibited higher respiration rate and higher soluble solids concentration/titratable acidity ratio. In conclusion, increase in storage temperature and SP elevates the activities of ACS and ACO enzymes and consequently C₂H₄ production which leads to the development of CI in plum fruit with advancement of ripening.     

Characterization of volatile compounds in Fen‐Daqu – a traditional Chinese liquor fermentation starter

Fen‐Daqu is a saccharifying agent and fermentation starter for the production of Chinese liquor Fen (alcoholic spirit) and Fen traditional vinegar. The volatile compounds produced at seven incubation steps were analysed by HS‐SPME‐GC‐MS. A total of 83 major volatile compounds were identified, including 23 esters, 8 acids, 24 alcohols, 18 ketones and aldehydes, 6 pyrazines and 4 acetals. Data obtained by HS‐SPME‐GC‐MS were subjected to principal component analysis. The trajectory plots of volatile compounds in Fen‐Daqu samples obtained during successive steps of incubation were revealed. The major compounds that contributed to discrimination were hexanal, (E)‐2‐octenal, (Z)‐2‐octen‐1‐ol, nonanoic acid, 1‐octanol, 2‐decen‐1‐ol, hexyl acetate, (E)‐2‐octen‐1‐ol, acetic acid, ethyl acetate, phenylethyl alcohol, ethyl alcohol, octanoic acid, 1‐octanol, 3‐methyl‐2‐buten‐1‐ol and pyrazines. Copyright © 2012 The Institute of Brewing & Distilling     

Comparative proteome analysis of glutenin synthesis and accumulation in developing grains between superior and poor quality bread wheat cultivars

BACKGROUND: Wheat glutenins are the major determinants of wheat quality. In this study, grains at the development stage from three wheat cultivars (Jimai 20, Jin 411 and Zhoumai 16) with different bread‐making quality were harvested based on thermal times from 150 °C_d to 750 °C_d, and were used to investigate glutenin accumulation patterns and their relationships with wheat quality. RESULTS: High and low molecular weight glutenin subunits (HMW‐GSs and LMW‐GSs) were synthesised concurrently. No obvious correlations between HMW/LMW glutenin ratios and dough property were observed. Accumulation levels of HMW‐GSs and LMW‐GSs as well as 1Bx13 + 1By16 and 1Dx4 + 1Dy12 subunits were higher in superior gluten quality cultivar Jimain 20 than in poor quality cultivar Jing 411 and Zhoumai 16. According to the results of two‐dimensional gel electrophoresis, six types of accumulation patterns in LMW‐GSs were identified and classified. The possible relationships between individual LMW‐GSs and gluten quality were established. CONCLUSION: The high accumulation level of HMW‐GSs and LMW‐GSs as well as 1Bx13 + 1By16 and 1Dx4 + 1Dy12 subunits contributed to the superior gluten quality of Jimai 20. Two highly expressed and 16 specifically expressed LMW glutenin subunits in Jimain 20 had positive effects on dough quality, while 17 specifically expressed subunits in Zhoumai 16 and Jing 411 appeared to have negative effects on gluten quality. Copyright © 2011 Society of Chemical Industry