In vitro and in vivo stability of caffeic acid phenethyl ester, a bioactive compound of propolis

The in vitro biochemical stability of caffeic acid phenethyl ester in rat and human plasma was investigated and compared with the stability of other caffeic acid esters (chlorogenic acid and rosmarinic acid). The incubation of the compounds in rat plasma for up to 6 h showed that caffeic acid phenethyl ester, but not the other compounds, was hydrolyzed, whereas human plasma did not affect the stability of all the assayed compounds. The products in rat plasma were caffeic acid and an unknown compound, which was identified by mass spectrometry as caffeic acid ethyl ester, produced by transesterification in the presence of ethanol used as vehicle for standard compounds. Specific inhibitors of different plasma esterases allowed the identification of a carboxylesterase as the enzyme involved in the metabolism of caffeic acid phenethyl ester. The oral administration in rats of caffeic acid phenethyl ester in the presence of both ethanol and 2-(2-ethoxyethoxy)ethanol gave rise to a dramatic increase of caffeic acid, as well as low levels of caffeic acid phenethyl ester, caffeic acid ethyl ester, and caffeic acid 2-(2-ethoxyethoxy)ethyl ester, in urine collected within 24 h after treatment. These results suggest that caffeic acid phenethyl ester is hydrolyzed also in vivo to caffeic acid as the major metabolite and that its biological activities should be more properly assayed and compared with those of caffeic acid, its bioactive hydrolysis product. Moreover, alcohols should be carefully used in vivo as solvents for caffeic acid phenethyl ester, since they can give rise to new bioactive caffeic acid esters.     

Enhanced carnosic acid levels in two rosemary accessions exposed to cold stress conditions

Two rosemary accessions were subjected to chilling temperatures in control environmental cabins analyzing their variations in rosmarinic and carnosic acids together with their adaptability to these stress conditions. Cold stressed plants of both accessions showed increases in caffeic acid and carnosic acid concentration levels, while other secondary metabolites such as rosmarinic acid, naringin, cirsimaritin, hispidulin, and carnosol showed different responses in both accessions. In addition, cold stressed plants exhibited significant reductions in chlorophylls, beta-carotene, and violaxanthin levels as well as the maximum quantum yield of PSII in both accessions. Hydrogen peroxide and lipid peroxidation levels showed similar responses in both accessions, which were positively and negatively correlated with rosmarinic and carnosic acids. From these results it is therefore suggested that carnosic acid biosynthesis in rosemary plants is induced by chilling periods. On the other hand, we demonstrate that not all rosemary accessions are equally well adapted to chilling temperatures. In fact, for (one) accession cold treated plants severe losses in chlorophyll, beta-carotene, and even xanthophylls (including zeaxanthin and antheraxanthin) were observed, despite no visual symptoms of leaf injury. More research is needed to understand rosmarinic acid variations in rosemary plants under stress conditions.     

Synergistic antioxidative effects of alkamides, caffeic acid derivatives, and polysaccharide fractions from Echinacea purpurea on in vitro oxidation of human low-density lipoproteins

Preparations of Echinacea are widely used as alternative remedies to prevent the common cold and infections in the upper respiratory tract. After extraction, fractionation, and isolation, the antioxidant activity of three extracts, one alkamide fraction, four polysaccharide-containing fractions, and three caffeic acid derivatives from Echinacea purpurea root was evaluated by measuring their inhibition of in vitro Cu(II)-catalyzed oxidation of human low-density lipoprotein (LDL). The antioxidant activities of the isolated caffeic acid derivatives were compared to those of echinacoside, caffeic acid, and rosmarinic acid for reference. The order of antioxidant activity of the tested substances was cichoric acid > echinacoside > or = derivative II > or = caffeic acid > or = rosmarinic acid > derivative I. Among the extracts the 80% aqueous ethanolic extract exhibited a 10 times longer lag phase prolongation (LPP) than the 50% ethanolic extract, which in turn exhibited a longer LPP than the water extract. Following ion-exchange chromatography of the water extract, the majority of its antioxidant activity was found in the latest eluted fraction (H2O-acidic 3). The antioxidant activity of the tested Echinacea extracts, fractions, and isolated compounds was dose dependent. Synergistic antioxidant effects of Echinacea constituents were found when cichoric acid (major caffeic acid derivative in E. purpurea) or echinacoside (major caffeic acid derivative in Echinacea pallida and Echinacea angustifolia) were combined with a natural mixture of alkamides and/or a water extract containing the high molecular weight compounds. This contributes to the hypothesis that the physiologically beneficial effects of Echinacea are exerted by the multitude of constituents present in the preparations.     

外源抗坏血酸与谷胱甘肽对打顶后烟草氧化 还原平衡及烟碱的影响

烟碱含量偏高是我国烤烟的现状。如何有效降低烟叶烟碱含量,提高烤烟工业可用性是烟叶生产中的一个难题。根据烟草打顶导致烟碱含量急剧上升和机械损伤造成细胞氧化迸发的的现象,本试验从打顶创伤引起的一系列生理变化入手,采取打顶后在伤口涂抹抗氧化剂抗坏血酸+谷胱甘肽(As A+GSH)和抗坏血酸(As A)两种方法来抑制活性氧含量的上升,探究茉莉酸刺激烟碱含量上升和活性氧含量之间的关系,并比较两种方法在抑制活性氧及烟碱上升的效果。结果发现,涂抹As A+GSH和As A处理对烟草叶片超氧阴离子、过氧化氢、丙二醛的上升有抑制效果,过氧化氢降解速度慢于超氧阴离子,在烟草内有积累现象。涂抹As A+GSH和As A处理在打顶6 h时茉莉酸含量低于常规打顶,对茉莉酸的产生有抑制效果。其中处理96 h后,打顶后涂抹As A+GSH的处理叶片烟碱含量比常规打顶低21.5%,打顶后涂抹As A的处理叶片烟碱含量比常规打顶低17.5%。且各检测指标之间存在显著或极显著的相关性。另外,打顶后24 h,各处理的活性氧含量回到对照(不打顶)的水平。试验表明,抗氧化型物质(As A+GSH)涂抹打顶后烟草的伤口能有效抑制活性氧、茉莉酸和烟碱含量的上升,且活性氧、茉莉酸、烟碱之间存在着密切联系。As A+GSH比As A有更强的抗氧化性,能更好地抑制打顶后烟碱的上升。     

Effects of nitrogen fertilization on the phenolic composition and antioxidant properties of basil (Ocimum basilicum L.)

Many herbs and spices have been shown to contain high levels of polyphenolic compounds with potent antioxidant properties. In the present study, we explore how nutrient availability, specifically nitrogen fertilization, affects the production of polyphenolic compounds in three cultivars (Dark Opal, Genovese, and Sweet Thai) of the culinary herb, basil ( Ocimum basilicum L.). Nitrogen fertilization was found to have a significant effect on total phenolic levels in Dark Opal ( p < 0.001) and Genovese ( p < 0.001) basil with statistically higher phenolic contents observed when nutrient availability was limited at the lowest (0.1 mM) applied nitrogen treatment. Similarly, basil treated at the lowest nitrogen fertilization level generally contained significantly higher rosmarinic ( p = 0.001) and caffeic ( p = 0.001) acid concentrations than basil treated at other nitrogen levels. Nitrogen fertilization also affected antioxidant activity ( p = 0.002) with basil treated at the highest applied nitrogen level, 5.0 mM, exhibiting lower antioxidant activity than all other nitrogen treatments. The anthocyanin content of Dark Opal basil was not affected by applied nitrogen level, but anthocyanin concentrations were significantly impacted by growing season ( p = 0.001). Basil cultivar was also determined to have a statistically significant effect on total phenolic levels, rosmarinic and caffeic acid concentrations, and antioxidant activities.     

Flavonoids and phenolic acids of sage: influence of some agricultural factors

A reversed-phase high-performance liquid chromatography/diode-array detector procedure is proposed for the determination of six phenolic compounds (caffeic acid, luteolin 7-O-glucoside, rosmarinic acid, apigenin, hispidulin, and cirsimaritin) in sage. The chromatographic separation was achieved using a reversed-phase Spherisorb ODS2 (5-microm particle size, 25.0 x 0.46 cm) column. Of the several extractive solvents assayed only ethyl ether, ethyl acetate, and acetone were able to extract all the compounds mentioned. Best resolution was obtained using a gradient of water/phosphoric acid (999:1) and acetonitrile. Ten samples cultivated in two experimental fields (1997-1999) were analyzed and the individual compounds quantified. Four commercially available samples were also analyzed and the results are discussed.     

Identification and quantification of caffeic and rosmarinic acid in complex plant extracts by the use of variable-temperature two-dimensional nuclear magnetic resonance spectroscopy

A combination of advanced nuclear magnetic resonance (NMR) methodologies for the analysis of complex phenolic mixtures that occur in natural products is described, with particular emphasis on caffeic acid and its ester derivative, rosmarinic acid. The combination of variable-temperature two-dimensional proton-proton double quantum filter correlation spectroscopy (1H-1H DQF COSY) and proton-carbon heteronuclear multiple quantum coherence (1H-13C HMQC) gradient NMR spectroscopy allows the identification and tentative quantification of caffeic and rosmarinic acids at 243 K in extracts from plants of the Lamiaceae family, without resorting to previous chromatographic separation of the components. The use of proton-carbon heteronuclear multiple bond correlation (1H-13C HMBC) gradient NMR spectroscopy leads to the complete assignment of the correlations of the spins of H2a and H3a with the ester and carboxyl carbons of rosmarinic and caffeic acid, even at room temperature, and confirms the results of the above methodology Quantitative results are in reasonable agreement with reverse phase HPLC measurements.     

Quantification of jasmonic acid by SPME in tomato plants stressed by ozone

Jasmonates are signalling molecules induced in plants as a response to various biotic and/or abiotic stresses. As ozone is known to activate defense responses in plants, we have monitored the concentration of jasmonic acid in tomato leaves during and after an acute exposure to this abiotic elicitor. In this experiment, we observed that the maximum induction of jasmonic acid in O3-fumigated plants occurred 9 h after the end of treatment and the concentration of jasmonic acid in stressed plants increased 13-fold. However, the level of endogenous methyl-jasmonate was constant during the observed period. The extraction and quantification of jasmonic acid as its methyl ester was performed by headspace-solid-phase microextraction (or HS-SPME) in combination with GC-FID and GC-MS. The sensitivity (LOD = 2 ng/g) of this method permitted the detection and quantification of jasmonic acid present in plant tissues at very low concentrations.     

Anthocyanin color behavior and stability during storage: effect of intermolecular copigmentation

Intermolecular copigmentation reactions are significantly responsible for the manifold color expression of fruits, berries, and their products. These reactions were investigated with five anthocyanins and five phenolic acids acting as copigments. The stability of the pigment-copigment complexes formed was studied during a storage period of 6 months. The study was conducted using a UV-visible spectrophotometer to monitor the hyperchromic effect and the bathochromic shift of the complexes. The greatest copigmentation reactions took place in malvidin 3-glucoside solutions. The strongest copigments for all anthocyanins were ferulic and rosmarinic acids. The immediate reaction of rosmarinic acid with malvidin 3-glucoside resulted in the biggest bathochromic shift (19 nm) and the strongest hyperchromic effect, increasing the color intensity by 260%. The color induced by rosmarinic acid was not very stable. The color intensity of pelargonidin 3-glucoside increased greatly throughout the storage period with the addition of ferulic and caffeic acids.     

Free radical scavenging and antioxidative activity of caffeic acid amide and ester analogues: structure-activity relationship.

The structure-activity relationships of synthetic caffeic acid amide and ester analogues as potential antioxidants and free radical scavengers have been investigated. The 2,2-diphenyl-1-picrylhydrazyl radical (DPPH.) scavenging activity of the test compounds was N-trans-caffeoyl-L-cysteine methyl ester (5) > N-trans-caffeoyldopamine (4) > N-trans-caffeoyltyramine (3) > N-trans-caffeoyl-beta-phenethylamine (2) > Trolox C (8) > caffeic acid phenethyl ester (1) > caffeic acid (6) > ferulic acid (7). This established that the radical scavenging activity of the compounds increased with increasing numbers of hydroxyl groups or catechol moieties and also with the presence of other hydrogen-donating groups (-NH, -SH). The antioxidative activity of the compounds was also investigated in an emulsified linoleic acid oxidation system accelerated by 2,2'-azobis(2-amidinopropane) dihydrochloride. The order was 1 > 2 > 4 > 3 > or = 5 > 6 > 8 > 7. Therefore, in the emulsion system, the antioxidative activity of the test compounds depends not only on the hydroxyl groups or catechol rings but also on the partition coefficient (log P) or hydrophobicity of the compounds. This supports the concept that hydrophobic antioxidants tend to exhibit better antioxidative activity in an emulsion system.     

Antioxidant Potentials and Phenolic Composition of Tef Varieties: An Indigenous Ethiopian Cereal

Tef, Eragrostis tef (Zucc.) Trotter, is a cereal crop originated and diversified in Ethiopia, where it is used to produce a range of food products. This study aimed to profile and quantify the phenolic composition and antioxidant potential of seven tef grain varieties. Soluble and bound phenolics ranged from 37 to 71 and from 226 to 376 mg of gallic acid equivalent/100 g dry basis (db), and soluble and bound flavonoid contents varied between 36 and 64 and between 113 and 258 mg of catechin equivalent/100 g db, respectively. Protocatechuic, vanillic, syringic, p‐coumaric, sinapic, ferulic, and rosmarinic acids, catechin, and naringenin were detected at least in three of the varieties studied. The dominant phenolic compounds were catechin, rosmarinic acid, and ferulic acid in the soluble extracts, whereas ferulic, rosmarinic, and p‐coumaric acids were the dominant ones in the bound extract. Gallic, caffeic, and salicylic acids were not detected in any of the varieties studied. The majority (>84%) of tef grain phenolics were found in bound form, contributing >84% of total 2,2‐diphenyl‐1‐picrylhydrazyl antioxidative capacity and >80% of total ferric reducing antioxidant power. These results clearly demonstrated the differences in phenolic profile among tef grain varieties. These results are relevant for developing healthy and nutritious tef‐based food products.     

Pharmacokinetic study of caffeic and rosmarinic acids in rats after oral administration

p-Coumaric and ferulic acid are actively taken up by monocarboxylic acid transporter (MCT), whereas gallic acid, caffeic acid (CA), and rosmarinic acid (RA) are absorbed by paracellular diffusion in human intestinal Caco-2 cells, although CA has low affinity for MCT. We previously demonstrated that p-coumaric acid has a much higher absorption efficiency than gallic acid in rats, owing to the MCT-mediated absorption of p-coumaric acid in vivo (J. Agric. Food Chem. 2004, 52, 2527-2532). Here, absorption of orally administered CA and RA in rats has been studied to investigate their intestinal absorption characteristics and pharmacokinetics in vivo and to compare the results with those of p-coumaric and gallic acids obtained under identical conditions. Rats were given 100 micromol/kg body weight of CA and RA, and blood was collected from the portal vein and abdominal artery after administration. CA, RA, and their metabolites were quantified by a coulometric detection method using HPLC-ECD. The serum concentration of intact CA and RA in the portal vein peaked at 10 min after administration, with a C(max) of 11.24 micromol/L for CA and 1.36 micromol/L for RA. The area under the curve (AUC) for intact CA and RA in the portal vein was calculated from the serum concentration-time profile to be 585.0 and 60.4 micromol min L-1, respectively. The absorption efficiency of CA was about 9.7-fold higher than that of RA. Overall, the absorption efficiency of these compounds in vivo increases in the order: gallic acid = RA < CA < p-coumaric acid, which is in good agreement with results obtained in Caco-2 cells in vitro.     

Influence of Salicylic Acid and Jasmonic Acid on Wheat Under Drought Stress

Salicylic acid and jasmonic acid play an important role in plants coping with abiotic stresses. An experiment was conducted to examine the effect of salicylic acid and jasmonic acid on wheat under drought. Seeds were primed with jasmonic acid (100µM) and salicylic acid (10 Mm). Water stress was applied by withholding water and each treatment was replicated three times with a factorial block design. Application of Salicylic acid and Jasmonic acid mitigated drought effects in wheat. Results revealed that 100µM Jasmonic acid was more effective than 10 mM SA. Drought decreased germination by 26%, whereas application of Jasmonic acid and Salicylic acid ameliorated stress with the increase of germination by 27% and 21%, respectively. An increase in the shoot length of 23% and 20% was observed with Jasmonic acid and Salicylic acid, under drought conditions. The increase in water potential was 60% and 47% with JA and SA while the increase in proline and soluble sugar was 14% and 25% respectively. The application of Jasmonic acid and Salicylic acid has a potential to enhance the growth of wheat plants under drought.     

Effect of methyl jasmonate on secondary metabolites of sweet basil (Ocimum basilicum L.)

The effect of methyl jasmonate (MeJA) in terms of its induction of inherent bioactive chemicals in sweet basil (Ocimum basilicum L.) was evaluated after MeJA was sprayed on healthy basil plants. The total phenolic content of the sweet basil significantly increased after 0.1 and 0.5 mM MeJA treatments compared with the control not subjected to MeJA. Two phenolic compounds, rosmarinic acid (RA) and caffeic acid (CA), were identified as strong antioxidant constituents of the sweet basil. Their amounts also significantly increased after the MeJA treatment. In addition, eugenol and linalool increased 56 and 43%, respectively, by the 0.5 mM MeJA treatment. Due to the accumulation of RA, CA, and eugenol, which possess strong 2,2-diphenyl-1-picrylhydrazyl (DPPH*) free radical scavenging activities, the antioxidant activity of the sweet basil extract was 2.3-fold greater than that of the control after the 0.5 mM MeJA treatment. In the DPPH* assay, the EC50 values of RA, CA, and eugenol were determined as 23, 46, and 59 microM, respectively, which indicated they were 6-, 3-, and 2.4-fold more efficient than BHT (140 microM). Besides, an unidentified HPLC peak in the methanolic extract of the sweet basil was 4.3-fold higher than that of the control after the 0.5 mM MeJA treatment.     

Distribution of a new rosmarinic acid derivative in Eryngium alpinum L. and other Apiaceae

The roots of Eryngium alpinum L. (Apiaceae) demonstrated radical scavenging properties toward the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical in a TLC autographic assay. Isolation of the bioactive compounds allowed the identification of R-(+)-3'-O-beta-D-glucopyranosyl rosmarinic acid, a new rosmarinic acid derivative. The quantitative determination of the antioxidant capacity of the compound by chemoluminescence demonstrated half the activity of R-(+)-rosmarinic acid. To find another source richer in this compound, a chemotaxonomic study was conducted. The higher content was found in the aerial parts of Sanicula europeae L., also belonging to the Saniculoideae subfamily. Although present in most of the Eryngium species, this compound was not detected in Imperatoria ostruthium L., Pimpinella peregrina L., and Levisticum officinalis L. species from the Apioideae subfamily and Hydrocotyle asiatica L. from the Hydrocotyloideae subfamily. The results indicate that the new derivative R-(+)-3'-O-beta-D-glucopyranosyl rosmarinic acid is a potential chemotaxonomic marker of the Saniculoideae subfamily.     

Effect of added caffeic acid and tyrosol on the fatty acid and volatile profiles of camellia oil following heating

Camellia oil is widely used in some parts of the world partly because of its high oxidative stability. The effect of heating a refined camellia oil for 1 h at 120 degrees C or 2 h at 170 degrees C with exogenous antioxidant, namely, caffeic acid and tyrosol, was studied. Parameters used to assess the effect of heating were peroxide and K values, volatile formation, and fatty acid profile. Of these, volatile formation was the most sensitive index of change as seen in the number of volatiles and the total area count of volatiles in gas chromatograms. Hexanal was generally the dominant volatile in treated and untreated samples with a concentration of 2.13 and 5.34 mg kg(-1) in untreated oils heated at 120 and 170 degrees C, respectively. The hexanal content was significantly reduced in heated oils to which tyrosol and/or caffeic acid had been added. Using volatile formation as an index of oxidation, tyrosol was the more effective antioxidant of these compounds. This is contradictory to generally accepted antioxidant structure-activity relationships. Changes in fatty acid profiles after heating for up to 24 h at 180 degrees C were not significant.     

HPLC analysis of rosmarinic acid in feed enriched with aerial parts of Prunella vulgaris and its metabolites in pig plasma using dual-channel coulometric detection

This paper describes a sensitive isocratic HPLC/ECD method developed for the determination of rosmarinic acid (RA) in plant material, animal feed, and pig plasma. The plasma sample preparation only includes protein precipitation and adjustment of the pH. The applicability of the method was tested on plasma samples of pigs that were exposed to a 91-day oral intake of RA via feed enriched by aerial parts of Prunella vulgaris. The plasma was directly analyzed using the method described as well as after enzymatic hydrolysis. When no hydrolysis step was included, RA and caffeic acid (CA) were quantified in the plasma. In hydrolyzed plasma samples, several other metabolites were determined, including dihydrocaffeic, ferulic, and dihydroferulic acid. The dual-channel coulometric detection employed, as an alternative to mass spectrometry, offers good selectivity and sensitivity owing to the electrochemical properties of the phenolic constituents.     

Effect of cysteinyl caffeic acid, caffeic acid, and L-dopa on the oxidative cross-linking of feruloylated arabinoxylans by a fungal laccase

To study a way to covalently link arabinoxylans and proteins using a fungal laccase from the fungus Pycnoporus cinnabarinus, the effect of cysteinyl caffeic acid on the cross-linking of wheat arabinoxylans was investigated by means of capillary viscometry and RP-HPLC of alkali labile phenolic compounds. Cysteinyl caffeic acid provoked a delay in gelation and in the consumption of the esterified ferulic acid on arabinoxylans. When reacting free ferulic acid and cysteinyl caffeic acid with laccase, the ferulic acid consumption and the dehydrodimers production were also diminished. These results suggest that cysteinyl caffeic acid is oxidized while reducing the semiquinones of ferulic acid produced by laccase. Thus, ferulic acid could not be oxidized into dimers until all cysteinyl caffeic acid was consumed, preventing the cross-linking of feruloylated arabinoxylan chains. A similar mechanism is proposed in the case of caffeic acid and of L-Dopa.     

Induction of antioxidant flavonol biosynthesis in fresh-cut potatoes. Effect of domestic cooking

The effect of fresh-cutting and subsequent cold storage on phenolic compounds from five long-term-stored potato cultivars (Agria, Cara, Liseta, Monalisa, and Spunta) was studied. Fresh-cutting induced the biosynthesis of three flavonols, which were identified by HPLC-DAD-ESIMS as quercetin 3-rutinoside, quercetin 3-diglucoside, and quercetin 3-glucosylrutinoside. The flavonols were detected after a lag period of 3 days of cold storage. The content ranged from 6 to 14 mg/100 g of fresh weight depending on the cultivar after 6 days of storage. Chlorogenic acid as the main caffeic acid derivative and the amino acids tyrosine and tryptophan were also quantified. The effect of cold storage under light or in dark was studied with new-season-harvested Monalisa potatoes. The flavonol induction was higher in fresh-cut potatoes stored under light than in the dark. However, caffeic acid derivatives were not affected. Domestic cooking such as boiling, microwaving, and frying provoked a partial loss of the flavonols, which were retained in the range of 4-16 mg per serving (213 g). Steam-cooking resulted in the highest retention of caffeic acid derivatives and aromatic amino acids compared with the other cooking methods studied. This means that due to the large amount of potatoes consumed in the Western diet, fresh-cut potatoes can be a significant source of health-promoting phenolics.