Chemical studies on antioxidant mechanism of curcuminoid: analysis of radical reaction products from curcumin

In the course of studies on the antioxidant mechanism of curcumin, its radical reaction was investigated. Curcumin was reacted with radical species, which were generated from the pyrolysis of 2, 2'-azobis(isobutyronitrile) under an oxygen atmosphere, and the reaction products from curcumin were followed by HPLC. The reaction at 70 degrees C gave several products, three of which were structurally identified to be vanillin, ferulic acid, and a dimer of curcumin after their isolation. The dimer was a newly identified compound bearing a dihydrofuran moiety, and its chemical structure was elucidated using spectroscopic analyses, especially 2D NMR techniques. A mechanism for the dimer production is proposed and its relation to curcumin's antioxidant activity discussed. The time course and gel permeation chromatography studies of the reaction were also investigated, and the results indicate that the dimer is a radical-terminated product in the initial stage.     

Antioxidant capacity of oat (Avena sativa L.) extracts. 2. In vitro antioxidant activity and contents of phenolic and tocol antioxidants

Oat milling fractions were examined for concentrations of total phenolics, tocols, and phenolic acids and in vitro antioxidant activity to determine their potential as dietary antioxidants. Methanolic extracts of pearling fractions, flour and aspirations from flaking, and trichomes had high, intermediate, and low antioxidant activities, respectively, evaluated by the beta-carotene bleaching method. Pearling fractions were also highest in total phenolics and tocols. p-Hydroxybenzoic acid, vanillic acid, caffeic acid, vanillin, p-coumaric acid, and ferulic acid were identified and quantified by HPLC. Three avenanthramides and an unidentified ferulate derivative were also detected. Total phenolic content was significantly correlated with antioxidant activity, and regression equations that predicted antioxidant activity from phenolic and tocol concentrations were calculated. Antioxidant activity, evaluated by beta-carotene bleaching, was correlated with measures of oxygen radical absorbance capacity and low-density lipoprotein oxidation. These data indicate a potential for oat products, especially those enriched in outer layers of the groat, to contribute to dietary intakes of antioxidant phytonutrients.     

利用黑曲霉β-葡萄糖苷酶催化香兰素葡萄糖苷水解

本文通过在香兰素葡萄糖苷溶液中加入黑曲霉菌种发酵制取的β-葡萄糖苷酶,进行酶促反应实验,定时取样进行高效液相分析检测,结果表明在反应过程中,溶液中香兰素葡萄糖苷的含量呈逐步下降趋势,香兰素的量呈逐步增加趋势;而在没有加β-葡萄糖苷酶的对照实验中,整个反应过程中香兰素葡萄糖苷的含量基本没有出现什么变化,在反应液中也没有检测到香兰素,这说明黑曲霉β-葡萄糖苷酶能够催化香兰素葡萄糖苷分解为香兰素的反应。     

Relationship between phenolic compounds, anthocyanins content and antioxidant activity in colored barley germplasm

Barley and its products are good sources of antioxidants. This experiment was conducted to examine the classification and concentration of phenolic compounds, proanthocyanidins, and anthocyanins in 127 lines of colored barley. Their relationship with 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity was also examined. Barley was placed into seven groups using the colorimeter: hulled (black 1, black 2, black 3, and purple) and unhulled (black, blue, and purple). The contents of phenolic compounds and anthocyanins were analyzed by using HPLC. The average content of phenolic compounds in unhulled barley groups (268.6 microg/g) was higher than that in hulled (207.0 microg/g) (P > 0.05). The proanthocyanidins content was determined by modified vanillin assay. The average content of proanthocyanidins was significantly higher in purple and blue barley groups compared with black (P < 0.05). The content of anthocyanins varied from 13.0 to 1037.8 microg/g. Purple and blue barley groups contained higher average contents of anthocyanins than black (P < 0.05). The most common anthocyanin in the purple barley groups was cyanidin 3-glucoside, whereas delphinidin 3-glucoside was the most abundant anthocyanin in the blue and black groups. In colored barley, DPPH radical scavenging activity had high positive correlation to the content of phenolic compounds and proanthocyanidins.     

The role of oxylipins and antioxidants on off-flavor precursor formation during potato flake processing

The impact of processing on nonenzymatic antioxidant degradation and lipid oxidation leading to off-flavor development in potato flakes during storage was investigated. Lipoxygenase activity measurements in parallel with the analysis of lipid oxidation products (oxylipins) profiles using HPLC showed that the processing conditions used inhibited efficiently enzymatic lipid oxidation. However, nonenzymatic lipid oxidation products were found throughout processing and in fresh potato flakes. Furthermore, these autoxidative processes cannot be inactivated by the main endogenous nonenzymatic antioxidants in potato tubers (ascorbic acid, phenolic compounds and carotenoids), as these antioxidants are degraded during processing. Indeed, leaching and thermal treatments taking place during processing lead to a decrease of about 95%, 82% and 27% in the content of ascorbic acid, phenolic compounds and carotenoids, respectively. Therefore, storage is a critical step to prevent off-flavor development in potato flakes. Specific attention has thus to be paid on the use of efficient exogenous antioxidants as well as on storage conditions.     

Vanillin content in boiled peanuts

A high-performance liquid chromatographic (HPLC) method for determination of vanillin in boiled peanuts has been developed. Vanillin was extracted with acetonitrile by blending at high speed followed by purification of an aliquot of the extract on a minicolumn packed with Al(2)O(3). Vanillin was quantitated by HPLC on silica gel with n-hexane/2-propanol/water/acetic acid (2100/540/37/2, v/v) as a mobile phase. The recovery of vanillin added to fresh peanut hulls at 0.50 and 2.50 microg/g was 78.7 +/- 2.7 and 79.9 +/- 3.1%, respectively. The detection limit of vanillin in boiled peanuts was estimated at 0.05 microg/g. UV-detector response to vanillin was linear to at least 2.5 microg/injection. Free vanillin has been found in two commercial brands of boiled peanuts at low ppm levels. Both the kernels and the hulls contained vanillin, which was formed during hydrolysis of lignin, one of the major constituents of the peanut hulls. Since vanillin has a low flavor threshold, it could be considered as one of the major ingredients that determines the flavor of boiled peanuts.     

Screening and identification of antioxidants in biological samples using high-performance liquid chromatography-mass spectrometry and its application on Salacca edulis Reinw

In this study, a new approach was developed for screening and identifying antioxidants in biological samples. The approach was based on significant decreases of the intensities of ion peaks obtained from high-performance liquid chromatography (HPLC) coupled with mass spectrometry (MS) upon reaction with 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radicals. HPLC-MS/MS was further applied to elucidate structures of antioxidant peaks characterized in a spiking test. The new approach could also be used to monitor the reactivity of antioxidants in biological sample with free radicals. The approach was successfully applied to the identification of antioxidants in salak (Salacca edulis Reinw), a tropical fruit that is reported to be a very good source of natural antioxidants, but it was still not clear which compounds were responsible for its antioxidant property. The antioxidants in salak were identified to be chlorogenic acid, (-)-epicatechin, and singly linked proanthocyanidins that mainly existed as dimers through hexamers of catechin or epicatechin. In salak, chlorogenic acid was identified to be an antioxidant of the slow reaction type as it reacted with free radicals much more slowly than either (-)-epicatechin or proanthocyanidins. The new approach was proved to be useful for the characterization and identification of antioxidants in biological samples as a mass detector combined with an HPLC separation system not only serves as an ideal tool to monitor free radical active components but also provides their possible chemical structures in a biological sample.     

Enzymatic extraction and transformation of glucovanillin to vanillin from vanilla green pods.

Glucovanillin was extracted from green pods and simultaneously transformed to vanillin by a combination of enzyme activities involving cell wall degradation and glucovanillin hydrolysis. The reaction is best carried out with 47.5% v/v aqueous ethanol solution during 8 h at 70 degrees C, in a two-step enzymatic reaction using Viscozyme followed by Celluclast, two commercial enzymatic products containing mainly pectinase and cellulase activities, respectively. The extractive reaction proceeded with high efficiency with an amount of extracted vanillin 3.13 times higher than the one obtained with the Soxhlet method. The classical curing/extraction process results in 1.1-1.8 g of vanillin/100 g of dry pods. It is concluded that the enzymatic reaction may substitute the microbial process involved in tissue fermentation previous to vanillin extraction with the simultaneous hydrolysis of glucovanillin.     

L-tryptophan reacts with naturally occurring and food-occurring phenolic aldehydes to give phenolic tetrahydro-beta-carboline alkaloids: activity as antioxidants and free radical scavengers

The reaction between the essential amino acid l-tryptophan and flavoring or naturally occurring phenyl and phenolic aldehydes was studied, and the alkaloidal reaction products were characterized by NMR and HPLC-MS. Benzaldehyde, vanillin, syringaldehyde, salicylaldehyde, and anisaldehyde condensed with l-tryptophan in aqueous-acidic media affording the corresponding phenolic tetrahydro-beta-carboline-3-carboxylic acid as two diastereoisomers, 1S,3S-cis and 1R,3S-trans. With the exception of benzaldehyde, the rest of the aldehydes needed heating conditions (70 degrees C) to significantly form tetrahydro-beta-carbolines over time with the cyclization highly favored at low pH. This suggests a likely formation of these compounds under conditions that may occur in foods, food processing, or cooking. The new phenolic tetrahydro-beta-carboline alkaloids were assayed, for the first time, for their activity as free radical scavengers and antioxidants and showed good antioxidant properties with Trolox equivalent antioxidant capacity (TEAC) values much higher than those of ascorbic acid and the water soluble vitamin E analogue, Trolox, in the 2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) assay.     

Modification of ascorbic acid using transglycosylation activity of Bacillus stearothermophilus maltogenic amylase to enhance its oxidative stability

Ascorbic acid (1), a natural antioxidant, was modified by employing transglycosylation activity of Bacillus stearothermophilus maltogenic amylase with maltotriose and acarbose as donor molecules to enhance its oxidative stability. The transglycosylation reaction with maltotriose as donor created mono- and di-glycosyl transfer products with an alpha-(1,6)-glycosidic linkage. In addition, two acarviosine-glucosyl transfer products were generated when transglycosylation was performed with acarbose as a donor. All transfer products were observed by TLC and HPLC, and purified by Q-sepharose anion exchange and Biogel P-2 gel permeation chromatographies. LC/MS and (13)C NMR analyses revealed that the structures of the transfer products were 6-O-alpha-D-glucosyl- (2) and 6-O-alpha-D-maltosyl-ascorbic acids (3) in the reaction of maltotriose, and 6-O-alpha-acarviosine-D-glucosyl- (4) and 2-O-alpha-acarviosine-D-glucosyl ascorbic acids (5) in the reaction of acarbose. The stability of the transglycosylated ascorbic acid derivatives was greatly enhanced against oxidation by Cu(2+) ion and ascorbate oxidase. Among them, compound 3 proved to be the most stable against in vitro oxidation. The antioxidant effects of glycosyl-derivatives of ascorbic acid on the lipid oxidation in cooked chicken breast meat patties indicated that they had antioxidant activities similar to that of ascorbic acid. It is suggested that the transglycosylated ascorbic acids can possibly be applied as effective antioxidants with improved stability in food, cosmetic, and other applications.     

Characterization of antioxidants and change of antioxidant levels during storage of Manilkara zapota L

Antioxidants found in fruits and vegetables play an important role via their protective effects against the onset of aging-related chronic diseases. Our previous research has indicated that unripe ciku fruits (Manilkara zapota L.) are an excellent source of antioxidants, with over 3000 mg of L-ascorbic acid equivalent antioxidant capacity (AEAC) per 100 g of fresh sample. In this study, 24 antioxidants in an extract of ciku king were characterized through a free radical spiking test. Their chemical structures were proposed using high-performance liquid chromatography-mass spectrometry (HPLC-MS) and tandem MS (HPLC/MSn). The antioxidant capacity of ciku king fruits was mainly attributed to polyphenolics with basic blocks of gallocatechin or catechin or both. The changes of total antioxidant capacity (TAC) and total phenolics content (TPC) of ciku king fruits with storage time were also investigated. It was found that the TAC and TPC decreased significantly as the fruits gradually changed from the unripe to the overripe stage. The best time for one to consume ciku king fruits at a flavorful stage with high amounts of antioxidants with AEAC values ranging from 600 to 1200 mg per 100 g fresh sample is suggested. The change of the content of major antioxidant peaks was also consistent with changes of antioxidant levels during storage.     

Glabridin protects paraoxonase 1 from linoleic acid hydroperoxide inhibition via specific interaction: a fluorescence-quenching study

The enzyme paraoxonase 1 (PON1) binds to high-density lipoprotein (HDL) and is responsible for many of HDL's antiatherogenic properties. We previously showed that recombinant PON1 is inhibited by linoleic acid hydroperoxide (LA-OOH) present in the lipid fraction of the human carotid plaque (LLE) via oxidation of the enzyme's Cys284 thiol. Here we explore the effect of glabridin, an isoflavan isolated from licorice root, on preventing LA-OOH's inhibitory effect on rePON1 using the tryptophan-fluorescence-quenching technique and modeling calculations. Glabridin significantly prevented rePON1 inhibition by LLE or oxidized linoleic acid (by 22% and 15%, respectively), whereas ascorbic acid and Trolox, strong antioxidants, had no effect. Glabridin quenched the intrinsic fluorescence of rePON1 in a concentration-dependent manner. Binding parameters and modeling calculations demonstrated a major role for hydrophobic forces in the rePON1-glabridin interaction, indicating that it is not the antioxidant capacity of glabridin that protects rePON1 from LA-OOH inhibition, but rather its specific interaction with the enzyme.     

On-line HPLC detection of tocopherols and other antioxidants through the formation of a phosphomolybdenum complex

An on-line method to detect and quantify antioxidant species in complex extracts has been developed as a combination of conventional HPLC separation and a postcolumn reaction with phosphomolybdenum reagent at acidic pH. Sample analytes were chromatographed by HPLC, and the postcolumn formation of a phosphate/Mo(V) complex was detected at 598 nm with an on-line absorbance detector. An optimized instrumental system was set up using pure alpha-tocopherol, and it was successfully tested with complex food extracts including lettuce, tomato, red pepper, and soybean seed, where several tocopherols and carotenoids were identified. A potential application of this detection method to quantitatively determine different antioxidants was considered, and a specific application to the determination of tocopherols was developed. The new method was characterized with respect to linearity interval, repeatability, and reproducibility, the quantitative results obtained were validated by comparison with a conventional HPLC method with fluorometric detection, and it was applied to the determination of tocopherols in different foods. The results suggest that the proposed on-line HPLC method can be a powerful instrument for the detection, purification, and characterization of natural antioxidants.     

Inhibition of oxidation of human low-density lipoproteins by phenolic substances in different essential oils varieties

Phenolics antioxidant phytochemicals have been recently implicated for the lower rates of cardiac disease mortality among people consuming a Mediterranean diet. Essential oils are natural products extracted from vegetable materials, which can be used as antibacterial, antifungal, antioxidants, and anti-carcinogenic agents or to preserve and give specific flavors to foods. The activities of 23 selected essential oils in inhibiting the copper-catalyzed oxidation of human-low-density lipoproteins (LDL) were determined in vitro. LDL oxidation was inhibited between 6, 2, and 83% by 2 microM (GAE) total phenolics. The relative inhibition of LDL oxidation was used to categorize the essential oils into four groups below 2% when they contained methylchavicol, anethol, p-cymen, apiole, cinnamic ether; 6-10% if they possessed a majority of carvacrol, thymol, p-cymene, or vanillin; 10-50% for moderate amounts of thymol, carvacrol, cuminol, or eugenol; and 50-100% when eugenol is the major component. Total phenol content of essential oils gave a correlation with LDL antioxidant activity of r = 0.75. The Activity of each phenolics compound could play a role in protecting LDL against oxidation if the substance is absorbed by the body.     

Reactivity of epicatechin in aqueous glycine and glucose maillard reaction models: quenching of C2, C3, and C4 sugar fragments

Mechanisms of how epicatechin alters the pathways of the Maillard reaction were investigated. Carbon-13 and nitrogen-15 labeling studies were utilized to define the reactivity of epicatechin with glucose, glycine, and/or reaction products in an aqueous model (pH 7, 125 degrees C for 30 min) via GC, GC/MS and HPLC/MS analysis. Quantification of the volatile reaction product isotopomers by GC/MS from a 1:1 labeled to unlabeled glucose (carbohydrate module labeling technique) plus glycine model system indicated the formation of 2,3-butanedione and acetol were primarily formed via intact C4 and C3 sugar fragments, whereas pyrazine, methylpyrazine, 2,5-dimethylpyrazine, 2,3,5-trimethylpyrazine, and cyclotene were primarily formed via intact C2/C2, C2/C3, C3/C3, C3/C3, and C3/C3 sugar fragment pairs, respectively. The formation of these seven compounds was also reported by GC analysis to be dramatically inhibited when epicatechin was added to the glucose/glycine model system (observed 9-113-fold reduction). HPLC/MS analysis of both the glucose-labeled and glycine-labeled model systems with and without epicatechin indicated that epicatechin reacted directly with C2, C3, and C4 sugar fragments, while epicatechin did not report any direct reactivity with glycine. In conclusion, the quenching of sugar fragmentation products via epicatechin was correlated with the observed inhibition on volatile compound formation when epicatechin was added to a glucose/glycine aqueous reaction model system.     

Extraction and colorimetric determination of azadirachtin-related limonoids in neem seed kernel.

A colorimetric method was developed for the determination of total azadirachtin-related limonoids (AZRL) in neem seed kernel extracts. The method employed acidified vanillin solution in methanol for the colorization of the standard azadirachtin or neem seed kernel extracts in dichloromethane. Through the investigation of various factors influencing the sensitivity of detection, such as the concentration of vanillin, acid, and the time required for the formation of color, optimum conditions were selected to perform the assay. Under the optimum conditions, a good linearity was found between the absorbance at 577 nm and the concentration of standard azadirachtin solution in the range of 0.01-0.10 mg/mL. In addition, different extraction procedures were evaluated using the vanillin assay. The HPLC analysis of the extracts indicated that if the extractions were performed in methanol followed by partitioning in dichloromethane, approximately 50% of the value determined by the vanillin assay represents azadirachtin content.     

On-line HPLC-DPPH screening method for evaluation of radical scavenging phenols extracted from apples (Malus domestica L.)

An on-line HPLC-DPPH screening method for phenolic antioxidants in apple methanol/water (80:20, v/v) extracts was applied. The determination of antioxidants was based on a decrease in absorbance at 515 nm after postcolumn reaction of HPLC-separated antioxidants with the 2,2'-diphenyl-1-picrylhydrazyl radicals (DPPH*). Each of the antioxidants separated by the HPLC column was observed as a negative peak corresponding to its antioxidative activity. The on-line method was applied for quantitative analysis of the antioxidants. A linear dependence of negative peak area on concentration of the reference antioxidants was observed. For validation of the on-line method the limit of detection, LOD (microg/mL), and the limit of quantification, LOQ (microg/mL), of the phenolic compounds were determined. Comparison of the UV and DPPH radical quenching chromatograms with authentic compounds identified catechin, chlorogenic acid, caffeic acid, epicatechin, and phloridzin in the apple cultivars (Lobo, Golden Delicious, and Boskoop), and the distribution of total antioxidant activity was calculated.     

Degradation Study of Carnosic Acid, Carnosol, Rosmarinic Acid, and Rosemary Extract (Rosmarinus officinalis L.) Assessed Using HPLC

Rosemary, whose major caffeoyl-derived and diterpenoid ingredients are rosmarinic acid, carnosol, and carnosic acid, is an important source of natural antioxidants and is being recognized increasingly as a useful preservative, protectant, and even as a potential medicinal agent. Understanding the stability of these components and their mode of interaction in mixtures is important if they are to be utilized to greatest effect. A study of the degradation of rosmarinic acid, carnosol, carnosic acid, and a mixture of the three was conducted in ethanolic solutions at different temperatures and light exposure. As expected, degradation increased with temperature. Some unique degradation products were formed with exposure to light. Several degradation products were reported for the first time. The degradation products were identified by HPLC/MS/MS, UV, and NMR. The degradation of rosemary extract in fish oil also was investigated, and much slower rates of degradation were observed for carnosic acid. In the mixture of the three antioxidants, carnosic acid serves to maintain levels of carnosol, though it does so at least in part at the cost of its own degradation.     

Phenolic composition and antioxidant properties of Polish blue-berried honeysuckle genotypes by HPLC-DAD-MS, HPLC postcolumn derivatization with ABTS or FC, and TLC with DPPH visualization

In this study, different Polish cultivars of blue-berried honeysuckles (Lonicera caerulea L.), wild and bog bilberry, were analyzed for bioactive compounds. The chemical properties verified included composition of anthocyanins and other polyphenols, antioxidant activity, and profiles of antioxidants by HPLC postcolumn derivatization or TLC. The antioxidant activities of different blue-berried honeysuckle cultivars were similar to that of wild-growing bilberries (ranging from 170 to 417 μmol TE/g dm in ABTS and from 93 to 166 μmol TE/g dm in DPPH and Folin-Ciocalteu tests). The major anthocyanin in the blue-berried honeysuckle was cyanidin-3-glucoside, which constituted 84-92% of the total anthocyanins. The TLC and HPLC postcolumn antioxidant profiles indicated that anthocyanins are the major antioxidants in all berries studied. Wild berries and the cultivars of the blue-berried honeysuckles are also a similar source of such minerals as K, Mg, and Ca.     

Comparative study of the products of the peroxidase-catalyzed and the polyphenoloxidase-catalyzed (+)-catechin oxidation. Their possible implications in strawberry (Fragaria x ananassa) browning reactions

The peroxidase- and polyphenoloxidase-catalyzed oxidations of (+)-catechin yield several products showing different degrees of polymerization, which are apparently responsible for the pigment decay and the associated browning reaction that occurs in processed strawberry fruits and their derived foods. In this work, we have purified both peroxidase and polyphenoloxidase from Oso Grande cv. strawberry fruits, and comparatively analyzed the products of their enzyme-mediated (+)-catechin oxidation. The joint analysis by reversed-phase and size-exclusion HPLC of the (+)-catechin oxidation products obtained with both enzymes indicate that they were qualitatively the same: dehydrodicatechin B4, a (+)-catechin quinone methide, dehydrodicatechin A, a (+)-catechin trimer, and a (+)-catechin oligomer with polymerization degree equal to or greater than 5. The main quantitative differences between the oxidative reactions were the great amount of oligomer formed in the case of the polyphenoloxidase-mediated reaction and the low amount of (+)-catechin reacted in the case of the peroxidase-mediated reaction. One of the possible reasons for such low levels of (+)-catechin consumption in the case of the peroxidase-mediated reaction was the possible inhibition by products of the enzyme-catalyzed oxidation. In fact, the peroxidase-mediated (+)-catechin oxidation was differentially inhibited by dehydrodicatechin A, showing a competitive type inhibition and a k(I) of 6.4 microM. In light of these observations, these results suggest that brown polymer formation, estimated as oligomeric compounds resulting from (+)-catechin oxidation, in strawberries is mainly due to polyphenoloxidase, and although peroxidase also plays an important role, it is apparently auto-regulated by product (dehydrodicatechin A) inhibition.