Enzymatic production of caffeic acid by koji from plant resources containing caffeoylquinic acid derivatives

The effect of a koji (Aspergillus awamori mut.) extract on the caffeoylquinic acid derivatives purified from sweetpotato (Ipomoea batatas L.) leaves was examined to develop the mass production of caffeic acid. A koji extract hydrolyzed the caffeoylquinic acid derivatives, chlorogenic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid and 3,4,5-tri-O-caffeoylquinic acid, to caffeic acid. Furthermore, the koji extract also converted the major polyphenolic components from sweetpotato, burdock (Arctium lappa L.), and mugwort (Artemisia indica var. maximowiczii) leaves to caffeic acid. These results suggest that the production of caffeic acid from plant resources containing caffeoylquinic acid derivatives is possible.     

Labeling of Polyethylenimine with Fluorescent Dye to Image Nucleus,Nucleolus, and Chromosomes in Digitonin-Permeabilized HeLa Cells

The effect of a koji (Aspergillus awamori mut.) extract on the caffeoylquinic acid derivatives purified from sweetpotato (Ipomoea batatas L.) leaves was examined to develop the mass production of caffeic acid. A koji extract hydrolyzed the caffeoylquinic acid derivatives, chlorogenic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid and 3,4,5-tri-O-caffeoylquinic acid, to caffeic acid. Furthermore, the koji extract also converted the major polyphenolic components from sweetpotato, burdock (Arctium lappa L.), and mugwort (Artemisia indica var. maximowiczii) leaves to caffeic acid. These results suggest that the production of caffeic acid from plant resources containing caffeoylquinic acid derivatives is possible.     

Variability in the composition of anti-oxidant compounds in Echinacea species by HPLC

A fast and reliable HPLC method for the determination of caffeic acid derivatives (caftaric acid, chlorogenic acid, caffeic acid, cynarin, echinacoside and cichoric acid) in various species of the genus Echinacea has been developed. Extraction of root samples by magnetic stirring with 80% methanol aqueous solution at room temperature allowed the complete recovery of all compounds of interest. Root extracts were analysed on a reversed-phase column with gradient elution and photodiode array detection. Caffeic acid derivatives showed differential qualitative and quantitative distributions in Echinacea species. The total amount of phenolic compounds ranged from 33.95 to 0.32 mg/g. The highest contents of caffeic acid derivatives were found in E. paradoxa var. paradoxa, E. paradoxa var. neglecta and E. purpurea, followed by E. angustifolia var. angustifolia, E. simulata, E. pallida and E. laevigata, whilst E. tennesseensis, E. sanguinea and E. atrorubens had low amounts of phenolic compounds. The radical scavenging activities of methanolic extracts of roots of Echinacea species was evaluated in vitro using the DPPH* radical scavenging method. The EC50 values of the samples ranged from 122 to 1223 microg/mL. The radical scavenging activities of the root extracts were correlated with the content of phenolic compounds, with a correlation coefficient (r2) of 0.923.     

Comparison of antioxidant capacity and phenolic compounds of berries, chokecherry and seabuckthorn

Antioxidant capacity and phenolic compounds (phenolic acids and anthocyanins) of four berry fruits (strawberry, Saskatoon berry, raspberry and wild blueberry), chokecherry and seabuckthorn were compared in the present study. Total phenolic content and total anthocyanin content ranged from 22.83 to 131.88 g/kg and 3.51 to 13.13 g/kg, respectively. 2,2-Diphenyl-1-picryhydrazyl free radical scavenging activity ranged from 29.97 to 78.86%. Chokecherry had the highest antioxidant capacity when compared with berry fruits and seabuckthorn. The highest caffeic acid, gallic acid and trans-cinnamic acid levels were found in chokecherry (6455 mg/kg), raspberry (1129 mg/kg) and strawberry (566 mg/kg), respectively. Caffeic acid was also the major phenolic acid in Saskatoon berry (2088 mg/kg) and wild blueberry (1473 mg/kg). The findings that chokecherry has very high antioxidant capacity and caffeic acid levels, are useful for developing novel value-added antioxidant products and also provide evidence essential for breeding novel cultivars of fruit plants with strong natural antioxidants.     

Production and characterization of functional substances from a by-product of rice bran oil and protein production by a compressed hot water treatment

A by-product of rice bran oil and protein production was treated with water and compressed hot water at 20 degrees C to 260 degrees C for 5 min, and at 200 degrees C and 260 degrees C for 5 to 120 min. Each extract was evaluated for its yield, radical scavenging activity, carbohydrate, protein, total phenolic and furfural contents, molecular-mass distribution and antioxidative activity. The maximum yield was obtained at 200 degrees C. The radical scavenging activity and the protein, total phenolic and furfural contents of the extract increased with increasing temperature. However, the carbohydrate content abruptly decreased when treated at above 200 degrees C. The extract treated at 260 degrees C for 5 min exhibited suppressive activity toward the autoxidation of linoleic acid. Each extract obtained at temperatures lower than or equal to 200 degrees C exhibited emulsifying ability.     

Free radical scavengers from the Mexican herbal tea "poleo" (Hedeoma drummondii)

The aerial parts of the Lamiaceae Hedeoma drummondii (Benth.) are used in Mexico to prepare a herbal tea and by North American Amerindians as a spice. The methanolic extract of the aerial parts exhibited a strong antioxidant effect measured by the scavenging of the free diphenyl picrylhydrazyl (DPPH) radical. Assay-guided fractionation of the crude methanolic extract allowed the identification of three major active constituents, chlorogenic, caffeic and rosmarinic acid, as well as sideritoflavone derivatives and simple phenolics. The TEAC, FRAP, total phenolic and flavonoid content were determined. The high content of caffeic acid and rosmarinic acid relates to the antioxidant activity of H. drummondii.     

In vitro antioxidant profile of phenolic acid derivatives

Several caffeic acid esters isolated from propolis exhibit interesting antioxidant properties, but their in vivo use is compromised by hydrolysis of the ester bond in the gastrointestinal tract. Therefore, a series of caffeic acid amides were synthesized and their in vitro antioxidant profile was determined. A series of hydroxybenzoic acids, hydroxycinnamic acids, and the synthesized caffeic acid amides were tested for both their 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging and microsomal lipid peroxidation-inhibiting activity. Some of the highly active antioxidants were further tested by means of electron paramagnetic resonance for their hydroxyl radical scavenging activity. Since a promising antioxidant compound should show a lipid peroxidation-inhibiting activity at micromolar level and a low cytotoxicity, the cytotoxicity of the phenolic compounds was also studied. In all the assays used, the caffeic acid anilides and the caffeic acid dopamine amide showed an interesting antioxidant activity.     

In Vitro Antioxidant Profile of Phenolic Acid Derivatives

Several caffeic acid esters isolated from propolis exhibit interesting antioxidant properties, but their in vivo use is compromised by hydrolysis of the ester bond in the gastrointestinal tract. Therefore, a series of caffeic acid amides were synthesized and their in vitro antioxidant profile was determined. A series of hydroxybenzoic acids, hydroxycinnamic acids, and the synthesized caffeic acid amides were tested for both their 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging and microsomal lipid peroxidation-inhibiting activity. Some of the highly active antioxidants were further tested by means of electron paramagnetic resonance for their hydroxyl radical scavenging activity. Since a promising antioxidant compound should show a lipid peroxidation-inhibiting activity at micromolar level and a low cytotoxicity, the cytotoxicity of the phenolic compounds was also studied. In all the assays used, the caffeic acid anilides and the caffeic acid dopamine amide showed an interesting antioxidant activity.     

Caffeic acid attenuates neuronal damage, astrogliosis and glial scar formation in mouse brain with cryoinjury

Traumatic brain injury induces neuron damage in early phase, and astrogliosis and the formation of the glial scar in late phase. Caffeic acid (3, 4-dihydroxycinnamic acid), one of the natural phenolic compounds, exerts neuroprotective effects against ischemic brain injuries with anti-oxidant and anti-inflammatory properties, and by scavenging reactive species. However, whether caffeic acid has protective effects against traumatic brain injury is unknown. Therefore, we determined the effect of caffeic acid on the lesion in the early (1 day) and late phases (7 to 28 days) of cryoinjury in mice. We found that caffeic acid (10 and 50 mg/kg, i.p., for 7 days after cryoinjury) reduced the lesion area and attenuated the neuron loss around the lesion core 1 to 28 days, but attenuated the neuron loss in the lesion core only 1 day after cryoinjury. Moreover, caffeic acid attenuated astrocyte proliferation, glial scar wall formation and glial fibrillary acidic protein (GFAP) protein expression in the late phase of cryoinjury (7 to 28 days). Caffeic acid also inhibited the reduction of superoxide dismutase activity and the increase in malondialdehyde content in the brain 1 day after cryoinjury. These results indicate that caffeic acid exerts a protective effect in traumatic brain injury, especially on glial scar formation in the late phase, which at least is associated with its anti-oxidant ability.     

Antiradical and anti-H2O2 properties of polyphenolic compounds from an aqueous peppermint extract

Polyphenolic compounds such as eriocitrin, luteolin-7-O-rutinoside, diosmin, hesperidin, narirutin, isorhoifolin, rosmarinic and caffeic acids were identified in an aqueous extract (Ex) obtained from peppermint leaves (Menthae x piperitae folium). The content of polyphenols in Ex was as follows: eriocitrin 38%, luteolin-7-O-rutinoside 3.5%, hesperidin 2.9%, diosmin 0.8%, isorhoifolin 0.6%, narirutin 0.3%, rosmarinic acid 3.7% and caffeic acid 0.05%. The strongest antiradical activity (determined as DPPH* scavenging features) was observed for luteolin-7-O-rutinoside, eriocitrin and rosmarinic acid. Caffeic acid and hesperidin revealed a lower antiradical activity while isorhoifolin, narirutin and diosmin showed the lowest activity. The strongest anti-H2O2 activity was observed for eriocitrin, a little lower for rosmarinic acid. The rate of hydrogen peroxide scavenging activity displayed by luteolin-7-O-rutinoside and caffeic acid was lower than that of rosmarinic acids. Hesperidin appeared to be a very weak scavenger of hydrogen peroxide. Almost no anti-H2O2 activity was demonstrated for diosmin, narirutin and isorhoifolin. Among examined flavonoids, the strongest antiradical and anti-H2O2 activity was shown for compounds with two hydroxy groups bound to the Bring in ortho position in relation to each other. Replacement of one hydroxy group in the Bring with a methoxy group or removing one hydroxy group leads to decrease of antiradical and anti-H2O2 activity of flavonoids. Our results suggest that eriocitrin is a powerful peppermint antioxidant and a free radical scavenger.     

Effect of caffeic acid on tert-butyl hydroperoxide-induced oxidative stress in U937

Nonvitamin phenolic compounds are ubiquitous in food plants and therefore potentially present in human plasma in a diet-dependent concentration. The aim of this study was to evaluate the ability of caffeic acid, a phenolic acid with antioxidant activity, to affect cellular response in U937 human monocytic cells to t-butyl hydroperoxide-induced oxidative stress. In our experimental conditions caffeic acid was incorporated into cells without any cytotoxic effect. Caffeic acid-treated cells showed an increased resistance to oxidative challenge, as revealed by an higher percent of survival and the maintenance of an higher proliferative capacity in respect to control cells. This effect seems to be due to the ability of caffeic acid to reduce glutathione depletion and to inhibit lipid peroxidation during tBOOH treatment. It can be concluded that caffeic acid exerts an antioxidant action inside the cell, responsible for the observed modulation of the cellular response to oxidative challenge. Due to its presence in the diet, therefore, caffeic acid may play a role in the modulation of oxidative processes in vivo.     

Antioxidant mechanisms of polyphenolic caffeic acid oligomers, constituents of Salvia officinalis

Caffeic acid, rosmarinic acid and oligomers of caffeic acid with multiple catechol groups are all constituents of Salvia officinalis. Their antioxidant potential was investigated with regard to their radical scavenging activity and the stability and structure of the intermediate radicals. Pulse-radiolytic studies revealed very high rate constants with hydroxyl radicals. Evidence from kinetic modeling calculations suggested an unusual complex behavior due to the presence of both O4- and O3-semiquinones and formation and decay of a hydroxyl radical adduct at the vinyl side chain. The radical structures observed by EPR spectroscopy after autoxidation in slightly alkaline solutions were only partially identified due to their instability and generally represented dissociated O4-semiquinones. Hybrid density-functional calculations of the potential radical structures showed distinct differences between the resonance stabilization of the O4- and O3-semiquinones of caffeic and dihydrocaffeic acids, reflected also in the considerably faster decay of the O3-semiquinone observed by pulse radiolysis. No evidence was found for dimerization reactions via Cbeta radicals typical for lignin biosynthesis.     

Free radical scavengers from Cymbopogon citratus (DC.) stapf plants cultivated in bioreactors by the temporary immersion (TIS) principle

The biomass production of Cymbopogon citratus shoots cultivated in bioreactors according to the temporary immersion (TIS) principle was assessed under different growth conditions. The effect of gassing with CO2-enriched air, reduced immersion frequency, vessel size and culture time on total phenolic and flavonoid content and free radical scavenging effect of the methanolic extracts was measured. From the TIS-culture of C. citratus, seven compounds were isolated and identified as caffeic acid (1), chlorogenic acid (2), neochlorogenic acid (3), p-hydroxybenzoic acid (4), p-hydroxybenzoic acid 3-O-beta-D-glucoside (5), glutamic acid (6) and luteolin 6-C-fucopyranoside (7). The occurrence of compounds 1-7 and their variability in C. citratus grown under different TIS conditions was determined by HPLC. The free radical scavenging effect of the methanolic extract and compounds was measured by the discoloration of the free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH). The main metabolites in 6- and 8-week-old cultures, both in 5 and 10 1 vessels, were chlorogenic acid (2) (100-113 mg%) and neochlorogenic acid (3) (80-119 mg%), while in the cultures with CO2-enriched air and reduced immersion frequency the main compound detected in the extracts was glutamic acid (6) (400 and 670 mg% for the green and white biomass and 619 and 630 mg% for the green and white biomass, respectively). The most active compounds, as free radical scavengers, in the DPPH discoloration assay were caffeic acid (1), chlorogenic acid (2), neochlorogenic acid (3) and the flavonoid luteolin 6-C-fucopyranoside (7).     

THE STIGMATIC SECRETION OF THE SWEETPOTATO

The stigmatic exudate of sweetpotato, when removed with organic solvents, consisted chiefly of lipid and phenolic compounds. Only traces of sugar were obtained. The esterified lipids were similar in chain length to capric and lauric acids. The two prinicpal phenolic compounds have UV absorption peaks, and bathochromic shifts on ionization with NaOH similar to those of esters of caffeic acid. On acid hydrolysis, caffeic acid was obtained from these and from three minor phenolic compounds. From two of the minor phenolic compounds, glucose was released by hydrolysis. The phenolic content of stigmas increased up to 20–24 hr before anthesis, and then gradually decreased. Neither compatible nor incompatible pollination affects the amount or composition of the stigmatic exudate.     

Phytochemical and Antiproliferative Activity of Proso Millet

The phytochemical content, antioxidant activity and antiproliferative properties of three diverse varieties of proso millet are reported. The free phenolic content ranged from 27.48 (Gumi 20) to 151.14 (Mi2504-6) mg gallic acid equiv/100 g DW. The bound phenolic content ranged from 55.95 (Gumi20) to 305.81 (Mi2504-6) mg gallic acid equiv/100 g DW. The percentage contribution of bound phenolic to the total phenolic content of genotype samples analyzed ranged between 62.08% and 67.05%. Ferulic acid and chlorogenic acid are the predominant phenolic acid found in bound fraction. Caffeic acid and p-coumaric acid were also detected. Syringic acid was detected only in the free fraction. The antioxidant activity was assessed using the hydrophilic peroxyl radical scavenging capacity (PSC) assay. The PSC antioxidant activity of the free fraction ranged from 57.68 (Mi2504-6) to 147.32 (Gumi20) µmol of vitamin C equiv/100 g DW. The PSC antioxidant activity of the bound fraction ranged from 95.38 (Mizao 52) to 136.48 (Gumi 20) µmol of vitamin C equiv/100 g DW. The cellular antioxidant activity (CAA) of the extract was assessed using the HepG2 model. CAA value ranged from 2.51 to 6.10 µmol equiv quercetin/100 g DW. Antiproliferative activities were also studied in vitro against MDA human breast cancer and HepG2 human liver cancer cells. Results exhibited a differential and possible selective antiproliferative property of the proso millet. These results may be used to direct the consumption of proso millet with improved health properties.     

Anti-plasmodial and antioxidant activities of constituents of the seed shells of Symphonia globulifera Linn f

A xanthone derivative, named gaboxanthone (1), has been isolated from the seed shells of Symphonia globulifera, together with known compounds, symphonin (2), globuliferin (3), guttiferone A (4), sistosterol, oleanolic acid and methyl citrate. The structure of the compound was assigned as 5,10-dihydroxy-8,9-dimethoxy-2,2-dimethyl-12-(3-methylbut-2-enyl) pyrano [3,2-b]xanthen-6(2H)-one, by means of spectroscopic analysis. The anti-plasmodial and antioxidant activities of the phenolic compounds were evaluated, respectively, in culture against W2 strain of Plasmodium falciparum and using the free radical scavenging activity of the DPPH radical, respectively. Compounds 1-4 were found to be active against the Plasmodium parasites (IC(50) of 3.53, 1.29, 3.86 and 3.17 microM, respectively). Guttiferone A (4) showed a potent free radical scavenging activity compared to the well-known antioxidant caffeic acid.     

Changes in the composition of phenolic compounds and antioxidant properties of grapevine roots and leaves (Vitis vinifera L.) under continuous of long-term drought stress

Grapevine seedlings Vitis vinifera L. were grown in a greenhouse under optimum conditions (soil moisture ca 70 %) and under drought stress (soil moisture ca 30 %). Drought stress caused reduction in total phenolic compounds in grapevine leaves and roots, where were identified tree phenolic acids: caffeic acid, p-coumaric acid and ferulic acid. All acids found in leaves and roots occurred in the ester-bound form. Only caffeic acid in leaves appeared in the free and ester-bound form. Caffeic acid was present in the highest concentrations. The content of ferulic acid was the lowest in both tissues. The levels of all phenolic acids in leaves and roots decreased significantly under the drought stress. All the extracts from grapevine leaves and roots had antioxidative properties, but the antiradical activity of the extracts obtained from roots subjected to drought stress was lower to the control. The results of the analysis revealed that long-term drought stress caused a decrease in selected elements of secondary metabolism in such a different plant tissues that are the leaves and roots of the grapevine.     

Caffeic acid production by simultaneous saccharification and fermentation of kraft pulp using recombinant Escherichia coli

Caffeic acid (3,4-dihydroxycinnamic acid) serves as a building block for thermoplastics and a precursor for biologically active compounds and was recently produced from glucose by microbial fermentation. To produce caffeic acid from inedible cellulose, separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) reactions were compared using kraft pulp as lignocellulosic feedstock. Here, a tyrosine-overproducing Escherichia coli strain was metabolically engineered to produce caffeic acid from glucose by introducing the genes encoding a 4-hydroxyphenyllactate 3-hydroxylase (hpaBC) from Pseudomonas aeruginosa and tyrosine ammonia lyase (fevV) from Streptomyces sp. WK-5344. Using the resulting recombinant strain, the maximum yield of caffeic acid in SSF (233 mg/L) far exceeded that by SHF (37.9 mg/L). In the SSF with low cellulase loads (≤2.5 filter paper unit/g glucan), caffeic acid production was markedly increased, while almost no glucose accumulation was detected, indicating that the E. coli cells experienced glucose limitation in this culture condition. Caffeic acid yield was also negatively correlated with the glucose concentration in the fermentation medium. In SHF, the formation of by-product acetate and the accumulation of potential fermentation inhibitors increased significantly with kraft pulp hydrolysate than filter paper hydrolysate. The combination of these inhibitors had synergistic effects on caffeic acid fermentation at low concentrations. With lower loads of cellulase in SSF, less potential fermentation inhibitors (furfural, 5-hydroxymethyfurfural, and 4-hydroxylbenzoic acid) accumulated in the medium. These observations suggest that glucose limitation in SSF is crucial for improving caffeic acid yield, owing to reduced by-product formation and fermentation inhibitor accumulation.

     

Properties of rice straw extract after subcritical water treatment

Rice straw was separated into four parts: the upper, middle, and lower parts of the stem, and the leaf. They were treated with subcritical water at 140 to 260 °C. The yield, carbohydrate, protein, and phenolic contents were obtained as well as the UV-Vis absorption spectra and the radical scavenging activity of the extracts. The extracts obtained from the stem parts had almost the same characteristics and were different from those of the leaf part. The extracts, prepared at higher temperature, exhibited higher radical scavenging ability. The radical scavenging ability and the phenolic content showed a correlation (R2=0.92), suggesting that the phenolic substances in the extract cause its antioxidant ability.     

Caffeic acid phenethyl ester protects mice from lethal endotoxin shock and inhibits lipopolysaccharide-induced cyclooxygenase-2 and inducible nitric oxide synthase expression in RAW 264.7 macrophages via the p38/ERK and NF-kappaB pathways

Caffeic acid phenethyl ester has been shown to have anti-inflammatory and anti-cancer effects. We examined the effects of caffeic acid phenethyl ester on lipopolysaccharide-induced production of nitric oxide and prostaglandin E(2), and expression of inducible nitric oxide synthase and cyclooxygenase-2 in RAW 264.7 macrophages. We also investigated the effects of caffeic acid phenethyl ester on lipopolysaccharide-induced septic shock in mice. Our results indicate that caffeic acid phenethyl ester inhibits lipopolysaccharide-induced nitric oxide and prostaglandin E(2) production in a concentration-dependent manner and inhibits inducible nitric oxide synthase and cyclooxygenase-2 in RAW 264.7 cells, without significant cytotoxicity. To further examine the mechanism responsible for the inhibition of inducible nitric oxide synthase and cyclooxygenase-2 expression by caffeic acid phenethyl ester, we examined the effect of caffeic acid phenethyl ester on lipopolysaccharide-induced nuclear factor-kappaB activation and the phosphorylation of mitogen-activated protein kinases. Caffeic acid phenethyl ester treatment significantly reduced nuclear factor-kappaB translocation and DNA-binding in lipopolysaccharide-stimulated RAW 264.7 cells. This effect was mediated through the inhibition of the degradation of inhibitor kappaB and by inhibition of both p38 mitogen-activated protein kinase and extracellular signal-regulated kinase phosphorylation, at least in part by inhibiting the generation of reactive oxygen species. Furthermore, caffeic acid phenethyl ester rescued C57BL/6 mice from lethal lipopolysaccharide-induced septic shock, while decreasing serum levels of tumor necrosis factor-alpha and interleukin-1beta. Collectively, these results suggest that caffeic acid phenethyl ester suppresses the induction of cytokines by lipopolysaccharide, as well as inducible nitric oxide synthase and cyclooxygenase-2 expression, by blocking nuclear factor-kappaB and p38/ERK activation. These findings provide mechanistic insights into the anti-inflammatory and chemopreventive actions of caffeic acid phenethyl ester in macrophages.