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.     

Interaction of curcumin with phosphatidylcholine: A spectrofluorometric study

Curcumin [1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3, 5-dione], the main constituent of the rhizomes of the plant Curcume longa L. (turmeric), is a powerful antioxidant in both enzymatic and nonenzymatic systems. The interactions of curcumin with egg and soy phosphatidylcholine were followed by fluorescence spectroscopy. Curcumin had very weak fluorescence in aqueous system, which was enhanced in apolar environments. Curcumin emitted at 490 nm after being excited at 451 nm in phosphatidylcholine micelles. The equilibrium constants for the interaction of curcumin with egg and soy phosphatidylcholine were (3.26 +/- 0.2) x 10(5) and (2.64 +/- 0.2) x 10(5) M(-1), respectively. From the Scatchard plot of the fluorometric data, it was inferred that one molecule of curcumin could bind six molecules of phosphatidylcholine. The equilibrium constant for the phosphatidylcholine-curcumin interaction decreased with temperature, indicating the amphiphilic nature of curcumin. The DeltaG, DeltaH, and DeltaS values obtained for the interaction of egg phosphatidylcholine-curcumin were -7.8 +/- 0.3 kcal/mol, -9.6 +/- 0.4 kcal/mol, and -6.8 +/- 0.2 cal/mol/K, respectively. The fluorescence anisotropy measurements of curcumin with phosphatidylcholine suggested that the anisotropy of the curcumin molecule did not change in phosphatidylcholine. The interaction of divalent metal ions with phosphatidylcholine-curcumin in comparison with phosphatidylcholine-1-anilino-8-naphathalenesulfonic acid complex suggested the strong binding of curcumin to metal ions.     

Peroxidase-mediated cross-linking of a tyrosine-containing peptide with ferulic acid

The tyrosine-containing peptide Gly-Tyr-Gly (GYG) was oxidatively cross-linked by horseradish peroxidase in the presence of hydrogen peroxide. As products, covalently coupled di- to pentamers of the peptide were identified by LC-MS. Oxidative cross-linking of ferulic acid with horseradish peroxidase and hydrogen peroxide resulted in the formation of dehydrodimers. Kinetic studies of conversion rates of either the peptide or ferulic acid revealed conditions that allow formation of heteroadducts of GYG and ferulic acid. To a GYG-containing incubation mixture was added ferulic acid in small aliquots, therewith keeping the molar ratio of the substrates favorable for hetero-cross-linking. This resulted in a predominant product consisting of two ferulic acid molecules dehydrogenatively linked to a single peptide and, furthermore, two ferulic acids linked to peptide oligomers, ranging from dimers to pentamers. Also, mono- and dimers of the peptide were linked to one molecule of ferulic acid. A mechanism explaining the formation of all these products is proposed.     

Potential nitrite scavengers as inhibitors of the formation of N-nitrosamines in solution and tobacco matrix systems

The ability of 20 compounds, all but one tobacco constituents, to inhibit the formation of tobacco-specific N-nitrosamines (TSNA) was investigated in buffer and detergent solution and in tobacco midrib and lamina systems. In solution at pH 5.5, ascorbic acid and the phenolic acids caffeic and ferulic acid were the most potent inhibitors of the reaction between nornicotine and nitrite, with nearly complete inhibition at molar ratios test compound/nitrite > 1:1. Also, cysteine > dihydrocaffeic acid > protocatechuic acid approximately catechin acted as strong inhibitors with >90% inhibition at a ratio of 3:1. Lower inhibitions were observed with chlorogenic acid > p-coumaric acid > sclareol > serine. Rutin showed an inhibition of 34% at a ratio of 0.1:1. Sclareol, alanine, proline, and serine did not significantly affect the N-nitrosonornicotine (NNN) formation. alpha-Tocopherol and glutathione enhanced NNN formation at pH 5.5 but were inhibitors at pH 3. Cinnamic acid, vanillic acid, eugenol, and esculin enhanced NNN formation. Increased NNN formation was also observed for dihydrocaffeic acid, chlorogenic acid, protocatechuic acid, and catechin at a less-than-equimolar ratio of test compound to nitrite. The tobacco matrix experiments were performed with air-cured, ground tobacco midrib and lamina. Caffeic acid, ferulic acid, dihydrocaffeic acid and catechin were potent inhibitors of the formation of TSNA in the midrib as well as in the lamina. Also protocatechuic acid, glutathione, ascorbic acid, p-coumaric acid, chlorogenic acid and cysteine were inhibitors, while alpha-tocopherol and rutin inhibited the reaction in the midrib but not in the lamina. Cinnamic acid, vanillic acid, eugenol, alanine, proline and serine showed small effects only. The molar ratio of secondary alkaloid(s)/nitrite in the test systems were 0.1:1 (solution), approximately 0.25:1 (midrib), and approximately 1:1 (lamina) and is most likely the major contributor to the observed order of inhibition potency (solution > midrib > lamina) of the test compounds. The vicinal phenolic hydroxyl groups of polyphenols and the simultaneous presence of a phenol group and an olefinic bond in hydroxycinnamic acids were the most characteristic structural elements of the potent inhibitors.     

Investigation of the absorption mechanism of solubilized curcumin using Caco-2 cell monolayers

Curcumin is a bioactive compound with poor oral bioavailability. Low water solubility and rapid metabolism are two known limiting factors, but the absorption mechanism of solubilized curcumin remains unclear. This study investigated the permeation mechanism of solubilized curcumin using an in vitro Caco-2 cell monolayer model. It was shown that curcumin permeated across the monolayers fairly rapidly [P(app)(A-B) = (7.1 ± 0.7) × 10(-6) cm/s] and the permeation mechanism was found as passive diffusion [P(app)(B-A)/P(app)(A-B) = 1.4]. Furthermore, the permeation rates of curcumin complexed with bovine serum albumin and in the bile salts-fatty acids mixed micelles were also determined as P(app)(mixed micelle) > P(app)(DMSO) > P(app)(protein complex). These results suggested that solubilization agents play an important role in the permeation of solubilized curcumin, and stronger binding between the solubilization agents and curcumin may decrease the permeation rate. The results further suggest that lipid-based formulations, which solubilize curcumin in mixed micelles after lipid digestion, are promising vehicles for curcumin oral delivery.     

Inhibitory effects of naturally occurring compounds on aflatoxin B(1) biotransformation.

Effects of naturally occurring compounds from plants on biotransformation of a mycotoxin, aflatoxin B(1), were evaluated. Among 77 naturally occurring compounds tested, anthraquinones, coumarins, and flavone-type flavonoids were shown to be potent inhibitors of aflatoxin B(1)-8,9-epoxide formation. Addition of the flavonoids galangin, rhamnetin, and flavone strongly inhibited mouse liver microsomal conversion of aflatoxin B(1) to aflatoxin B(1)-8,9-epoxide, a metabolically activated mutagenic product. In contrast to these results, addition of isoflavonoids, catechins, terpenes, alkaloids, and quinones to mouse liver microsomes did not inhibit formation of aflatoxin B(1)-8,9-epoxide. Formation of the aflatoxin B(1) reductase product, aflatoxicol, by chicken liver cytosols was strongly inhibited by curcumin, the diferuloylmethane present in turmeric and other Curcuma species. Curcumin analogues also showed inhibitory effects, and a structure-activity study established that beta-diketone groups linked with two benzyl moieties were essential for inhibition of aflatoxicol formation. An additional 37 naturally occurring compounds tested did not inhibit formation of aflatoxicol. These results demonstrate that dietary constituents in certain fruits, vegetables, and spices may have significant inhibitory effects on metabolic transformation of aflatoxins to their hepatotoxic or carcinogenic derivatives or, alternatively, may promote their transformation into nontoxic products.     

Curcuma longa L. constituents inhibit sortase A and Staphylococcus aureus cell adhesion to fibronectin

The inhibitory activity of Curcuma longa L. (turmeric) rhizome constituents against sortase A, a bacterial surface protein anchoring transpeptidase, from Staphylococcus aureus ATCC 6538p was evaluated. The activity of the isolated compounds (1-4) was compared to that of the positive control,p-hydroxymecuribenzoic acid (pHMB). The biologically active components of C. longa rhizome were characterized by spectroscopic analysis as the curcuminoids curcumin (1), demethoxycurcumin (2), and bisdemethoxycurcumin (3). Curcumin was a potent inhibitor of sortase A, with an IC50 value of 13.8 +/- 0.7 microg/mL. Bisdemethoxycurcumin (IC50 = 31.9 +/- 1.2 microg/mL) and demethoxycurcumin (IC50 = 23.8 +/- 0.6 microg/mL) were more effective than pHMB (IC50 = 40.6 +/- 1.2 microg/mL). The three isolated compounds (1-3) showed no growth inhibitory activity against S. aureus strain Newman, with minimum inhibitory concentrations (MICs) greater than 200 microg/mL. Curcumin also exhibited potent inhibitory activity against S. aureus cell adhesion to fibronectin. The suppression of fibronectin-binding activity by curcumin highlights its potential for the treatment of S. aureus infections via inhibition of sortase activity. These results indicate that curcumin is a possible candidate in the development of a bacterial sortase A inhibitor.     

Improving the oral bioavailability of curcumin using novel organogel-based nanoemulsions

Curcumin is a natural bioactive compound with many health-promoting benefits. Its low oral bioavailability limits its application in functional foods. In the present study, novel organogel-based nanoemulsions have been developed for oral delivery of curcumin and improvement of its bioavailability. Recently developed curcumin organogel was used as the oil phase in the curcumin nanoemulsion formulation. Tween 20 was selected as the emulsifier on the basis of maximum in vitro bioaccessibility of curcumin in the nanoemulsion. In vitro lipolysis profile revealed that the digestion of nanoemulsion was significantly faster and more complete than the organogel. Permeation experiments on Caco-2 cell monolayers suggested that digestion-diffusion was the major absorption mechanism for curcumin in the nanoemulsion. Furthermore, in vivo pharmacokinetics analysis on mice confirmed that the oral bioavailability of curcumin in the nanoemulsion was increased by 9-fold compared with unformulated curcumin. This novel formulation approach may also be used for oral delivery of other poorly soluble nutraceuticals with high loading capacity, which has significant impact in functional foods, dietary supplements and pharmaceutical industries.     

Antioxidant properties of malt model systems

The aim of this study was to investigate the effect of kilning and roasting temperatures on antioxidant activity of malt model systems prepared from combinations of glucose, proline, and ferulic acid. Model systems (initial a(w) = 0.09, 6% moisture) were heated at 60 degrees C for up to 24 h, at 90 degrees C for up to 120 min, and at 220 degrees C for up to 15 min. The antioxidant activity of the glucose-proline-ferulic acid model system increased significantly on heating at 60 degrees C for 24 h or at 90 degrees C for 120 min. In contrast, the glucose-proline, ferulic acid-glucose, and ferulic acid-proline systems presented either nonsignificantly increased or unchanged antioxidant activity. The antioxidant activity of both the glucose-proline-ferulic acid and glucose-proline model systems increased significantly after heating at 220 degrees C for 10 min, followed by a significant decrease at 15 min. The data suggest that (1) at 60 degrees C, ferulic acid reacts with Maillard reaction products, resulting in a significant increase in antioxidant activity; (2) at 90 degrees C, the antioxidant activity of the glucose-proline-ferulic system comes from both ferulic acid and Maillard reaction products; and (3) at 220 degrees C, the major contributors to antioxidant activity in glucose-proline-ferulic acid and glucose-proline systems are glucose-proline reaction products.     

Characterization of two unknown compounds in methanol extracts of rosemary oil

In this study, two unknown compounds in rosemary oil, containing 3% carnosic acid and 0.3% carnosol, were identified and characterized. After methanol extraction, purification, and analysis by reversed-phase HPLC and LC-MS, a recovery of 92% (+/-8%) of carnosic acid was obtained, but no carnosol was found. However, two unknown compounds with a molecular weight of 330.2 and 302.2 were consistently detected. From additional LC-MS-MS, (1)H NMR, and elemental analyses, it became clear that the first compound (M(w) = 330.2) could not be carnosol. It was hypothesized that it originated from the breakdown of the intramolecular bond of carnosol, followed by the addition of a water molecule. Possibly, an unsaturated double bond was formed after dehydration. Assuming that this compound was an intermediate in the conversion to rosmanol, the second unknown compound (M(w) = 302.2) may have resulted from the breakdown of the intramolecular bond of rosmanol. Similarly, an unsaturated double bond may have been formed. After splitting off carbon oxide, a detectable molecule with a molecular weight of 302.2 was observed.     

Sugar beet waste and its component ferulic acid inhibits external mycelium of arbuscular mycorrhizal fungus

External arbuscular mycorrhiza (AM) mycelium plays an important role in soil while interacting with a range of biotic and abiotic factors. One example is the soil organic amendment sugar beet waste. The fermented Aspergillus niger-sugar beet waste (ASB) increases growth and P uptake by the AM mycelium in soil whereas non-fermented waste (SB) had a strong inhibitory effect. The underlying mechanisms are not understood.We used gas chromatography-mass spectrometry to identify differences in composition of water extracts of ASB and SB. The chromatograms showed that ferulic acid was present in SB and absent in ASB. We compared the effects of the water extracts of SB and ASB and ferulic acid upon the growth of Glomus intraradices in in vitro monoxenic cultures.Hyphal growth of the AM fungus G. intraradices was extremely reduced in ferulic acid and SB treatments. Moreover, AM hyphae appeared disorganized, undulated and tangled. In contrast, ASB increased hyphal length and numbers of branched absorbing structures and of spores. We conclude that ferulic acid is one compound in SB which is responsible for its inhibition of AM extraradical growth. The relevance of these findings is discussed.     

Curcumin bioavailability from enriched bread: the effect of microencapsulated ingredients

Human bioavailability of curcumin from breads enriched with 1 g/portion of free curcumin (FCB), encapsulated curcumin (ECB), or encapsulated curcumin plus other polyphenols (ECBB) was evaluated. Parental and metabolized curcuminoids and phenolic acids were quantified by HPLC/MS/MS in blood, urine, and feces collected over 24 h. The concentrations of serum curcuminoids were always below 4 nmol/L and those of glucuronides 10-fold less. Encapsulation delayed and increased curcuminoid absorption as compared to the free ingredient. Serum and urinary concentrations of ferulic and vanillic acid were between 2- and 1000-fold higher than those of curcuminoids, with ECBB eliciting the highest amounts. Fecal curcuminoids were 6-fold more abundant after ECB than FCB, while phenolic acids after ECBB quadruplicated those after ECB. Curcuminoid encapsulation increased their bioavailability from enriched bread, probably preventing their biotransformation, with combined compounds slightly reducing this effect. Phenolic acids are the major metabolites of curcuminoids and may contribute to their biological properties.     

Suppression of free fatty acid-induced insulin resistance by phytopolyphenols in C2C12 mouse skeletal muscle cells

It was reported that increased plasma levels of free fatty acids (FFAs) are associated with profound insulin resistance in skeletal muscle and may also play a critical role in the insulin resistance of obesity and type 2 diabetes mellitus. Skeletal muscle is the major site for insulin-stimulated glucose uptake and is involved in energy regulation and homeostasis. In this study, we used 12-O-tetradecanoylphorbol 13-acetate (TPA), a protein kinase C (PKC) activator, and palmitate to induce insulin resistance in C2C12 mouse skeletal muscle cells. Our data show that epigallocatechin gallate (EGCG) and curcumin treatment reduce insulin receptor substrate-1 (IRS-1) Ser307 phosphorylation, and curcumin is more potent to increase Akt phosphorylation in TPA induction. Moreover, we found that after 5 h of palmitate incubation, epicatechin gallate (ECG) can suppress IRS-1 Ser307 phosphorylation and significantly promote Akt, ERK1/2, p38 MAPK, and AMP-activated protein kinase activation. With a longer incubation with palmitate, IRS-1 exhibited a dramatic depletion, and treatment with EGCG, ECG, and curcumin could reverse IRS-1 expression, Akt phosphorylation, and MAPK signaling cascade activation and improve glucose uptake in C2C12 skeletal muscle cells, especially ECG and curcumin. In addition, treatment with these polyphenols can suppress acetyl-CoA carboxylase activation, but only EGCG could inhibit lipid accumulation in the intracellular site. These findings may suggest that curcumin shows the best capacity to improve FFA-induced insulin resistance than the other two, and ECG was more effective than EGCG in attenuating insulin resistance.     

Effect of curcumin on inflammation and oxidative stress in cisplatin-induced experimental nephrotoxicity

Nephrotoxicity is a major complication and a dose limiting factor for cisplatin therapy. Recent evidence suggests that inflammation and oxidative stress may contribute to the pathogenesis of cisplatin-induced acute renal failure. Curcumin is claimed to be a potent anti-inflammatory and antioxidant agent. The present study was performed to explore the effect of curcumin against cisplatin-induced experimental nephrotoxicity. Curcumin in the dosages of 15, 30, and 60 mg kg(-1) was administered 2 days before and 3 days after cisplatin administration. Renal injury was assessed by measuring serum creatinine, blood urea nitrogen, creatinine, urea clearance, and serum nitrite levels. Renal oxidative stress was assessed by determining renal malondialdehyde levels, reduced glutathione levels and enzymatic activities of superoxide dismutase and catalase. Systemic inflammation was assessed by tumor necrosis factor-alpha (TNF-alpha) levels. A single dose of cisplatin resulted in marked inflammation (486% rise in TNF-alpha level) and oxidative stress and significantly deranged renal functions as well as renal morphology. The serum TNF-alpha level was markedly reduced in curcumin-treated rats. Curcumin treatment significantly and dose-dependently restored renal function, reduced lipid peroxidation, and enhanced the levels of reduced glutathione and activities of superoxide dismutase and catalase. The present study demonstrates that curcumin has a protective effect on cisplatin-induced experimental nephrotoxicity, and this effect is attributed to its direct anti-inflammatory and strong antioxidant profile. Hence, curcumin has a strong potential to be used as a therapeutic adjuvant in cisplatin nephrotoxicity.     

Antioxidant activity of steryl ferulate extracts from rye and wheat bran

Antioxidant activity of steryl ferulates from other sources than rice have not yet been studied much, despite the fact that rice steryl ferulates (gamma-oryzanol) have been shown to possess good antioxidant activity. In this study, steryl ferulate extracts from wheat or rye bran were studied for their capability to inhibit hydroperoxide formation in bulk methyl linoleate and methyl linoleate emulsion. Further, their activity to scavenge DPPH radicals was analyzed. The activities were compared to synthetic steryl ferulates, rice steryl ferulates, ferulic acid, and alpha-tocopherol. Nonrice cereal extracts of steryl ferulates exhibited good antioxidant activity, especially in the bulk lipid system. The radical scavenging activity was similar to that of nonesterified ferulic acid, indicating that the ferulic acid moiety is responsible for the antioxidant properties. This study illustrates a new aspect to the health-promoting properties of rye and wheat.     

Regioselective dimerization of ferulic acid in a micellar solution.

Dehydrodimers of hydroxycinnamates play an important role in the cross-linking of plant cell walls. An aqueous solution of quaternary ammonium salts with a long aliphatic chain is known to spontaneously organize itself into micelles with the ionic part at the outer sphere. It is shown that regioisomeric ferulic acid dehydrodimers can be obtained in one step from trans-ferulic acid after attachment to these micelles and using the biomimetic peroxidase-H2O2 system. The surfactant hexadecyltrimethylammonium hydroxide yielded trans-4-(4-hydroxy-3-methoxybenzylidene)-2-(4-hydroxy-3-methoxyphenyl)-5-oxotetrahydrofuran-3-carboxylic acid (25%), (E,E)-4,4'-dihydroxy-5,5'-dimethoxy-3,3'-bicinnamic acid (21%), and trans-5-[(E)-2-carboxyvinyl]-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-2,3-dihydrobenzofuran-3-carboxylic acid (14%), whereas the surfactant tetradecyltrimethylammonium bromide gave 4-cis, 8-cis-bis(4-hydroxy-3-methoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane-2,6-dione (18%) as the main product. The use of micelles appears to be not only a new way to synthesize regioisomeric ferulic acid dehydrodimers but may also help to understand the regiospecificity of dimeric hydroxycinnamate formation in vivo.     

Intramolecular carbon isotope distribution of acetic acid in vinegar

Compound-specific carbon isotope analysis of acetic acid is useful for origin discrimination and quality control of vinegar. Intramolecular carbon isotope distributions, which are each carbon isotope ratios of the methyl and carboxyl carbons in the acetic acid molecule, may be required to obtain more detailed information to discriminate such origin. In this study, improved gas chromatography-pyrolysis-gas chromatography-combustion-isotope ratio mass spectrometry (GC-Py-GC-C-IRMS) combined with headspace solid-phase microextraction (HS-SPME) was used to measure the intramolecular carbon isotope distributions of acetic acid in 14 Japanese vinegars. The results demonstrated that the methyl carbons of acetic acid molecules in vinegars produced from plants were mostly isotopically depleted in (13)C relative to the carboxyl carbon. Moreover, isotopic differences (δ(13)C(carboxyl) - δ(13)C(methyl)) had a wide range from -0.3 to 18.2‰, and these values differed among botanical origins, C3, C4, and CAM plants.     

Biochar differentially affects the cycling and partitioning of low molecular weight carbon in contrasting soils

Biochar application to soils has received much attention due to the potential for dual benefits of improved fertility and carbon (C) sequestration. Whilst its effect on C and nitrogen (N) cycling in soils has been investigated previously, this has usually either focussed on the bulk soil organic matter, or a single compound such as glucose. Five low molecular weight dissolved organic C (LMWDOC) substrates (three sugars, one amino acid, one organic acid) were selected for a ¹⁴C-CLPP experiment from which turnover rate (t_1/2) and immediate carbon use efficiency (CUE) of the substrate were estimated. We demonstrated that whilst soil type had the greatest effect on soil microbial function, the addition of biochar also influenced microbial turnover and CUE of the substrates, most notably in the lowest fertility soil. We also identified that the relationship between turnover and CUE of the five substrates differed substantially, and the effect of biochar and soil type was more pronounced in the amino acid than the organic acid. This effect tended to be greatest in biochars produced at 450 °C, and less pronounced with the addition of biochars produced at 550 °C, though these trends were not consistent for all compounds in all soils tested. We conclude biochars and soils interact to manifest non-systematic differences in turnover rates of LMWDOCs, and thus a variety of mechanisms are likely responsible for this observation. As these compounds are most commonly found in the rhizosphere and can contribute a significant portion of photosynthetically-fixed C, and plant roots have been observed to grow preferentially around biochar particles, it is apparent that biochar may significantly affect the flow of LMWDOC through the microbial community in soils.