Screening of antioxidant phenolic compounds produced by in vitro shoots of Brassica oleracea L. var. costata DC.

The phenolic compounds produced by in vitro shoots of Brassica oleracea L. var. costata DC were screened by HPLC-DAD-MS/MS. Thirty seven compounds were characterized, which included chlorogenic acids, flavonoids (the majority of them were hydroxycinnamic acid esters of kaempferol and quercetin glycosides) and hydroxycinnamic acyl glycosides (with predominance of synapoyl gentiobiosides). The antioxidant capacity of the shoots was assessed against DPPH radical and two reactive oxygen species (superoxide radical and hypochlorous acid). A strong concentration-dependent antioxidative capacity was verified in the DPPH and superoxide radicals assays, but a reduced effect was noticed against hypochlorous acid. The results obtained indicate that the in vitro production of B. oleracea var. costata shoots can become important in the obtention of a noticeable dietary source of compounds with health protective potential.

Oxygen and nitrogen reactive species are effectively scavenged by Eucalyptus globulus leaf water extract.

Eucalyptus globulus Labill. (Family Myrtaceae) is a plant of Australian origin, with a reported therapeutic use in airway inflammatory diseases. Considering that reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been implicated in the pathogenesis of airway inflammatory diseases such as asthma and chronic obstructive pulmonary disease, an effective scavenging activity against these reactive species may contribute for the therapeutic effect of this plant. In the present study, a water extract of E. globulus leaves was evaluated for its putative in vitro scavenging effects on ROS (HO(*), O(2)(*-), ROO(*), and H(2)O(2)) and RNS ((*)NO and ONOO(-)) and on 2,2-diphenyl-1-picrylhydrazyl radical (DPPH). Qualitative and quantitative analyses of the extract's phenolic composition were also performed. The Eucalyptus leaf water extract presented a remarkable capacity to scavenge all the reactive species tested, with all the 50% inhibitory concentrations being found at the mug/mL level. Phytochemical analysis showed the presence of polyphenols such as flavonoids (rutin and quercitrin) and phenolic acids (chlorogenic acid and ellagic acid), which may be partially responsible for the observed antioxidant activity. These observations provide further support, beyond the well-known antibacterial and antiviral activities of the Eucalyptus plant, for its reported use in traditional medicine such as in the treatment of airway inflammatory diseases, considering the important role of ROS and RNS in the inflammatory process, although further studies are needed to prove the bioavailability of the antioxidants/antibacterial compounds of the extract as well as the ability of the active compounds to reach specific tissues and to act in them.

Volatile composition of Catharanthus roseus (L.) G. Don using solid-phase microextraction and gas chromatography/mass spectrometry.

A total of 88 volatile and semi-volatile components were formally or tentatively identified in flowers, leaves and stems of Catharanthus roseus (L.) G. Don (cv. Little Bright Eye), by headspace solid-phase microextraction (HS-SPME) and by dichloromethane extraction, combined with gas chromatography-mass spectrometry (GC-MS). These include some diterpenic compounds (manool and manoyl oxides), a sesquiterpen (alpha-bisabolol), and some pyridine, pyrazine, indol and carotenoid derivatives. Applying multivariate analysis (principal component analysis and agglomerative hierarchic cluster analysis) to the HS-SPME-GC-MS data, it was possible to characterize each part of the vegetal material using a relative small number of compounds. Hence, flowers were richer in terpenic molecules (including limonene), alpha-bisabolol, methyljasmonate, cis-jasmone, 2-phenylethanol, phenylacetaldehyde, trans-2-octenal, benzylic alcohol and 2-isobutyl-3-methoxypyrazine. Leaves can be characterized by the methyl and propyl esters of fatty acids, mono- and disaturated, trans-phytol, carotenoid derivative compounds, hydrofarnesylacetone, methylanthranilate, manool and epi-manool oxide, while stems have high levels of volatile aldehydes, such as hexanal, octanal, cis-2-nonenal, cis-2-decenal, cis, trans-2,6-nonadienal, trans, trans-2,4-decadienal and cis, trans-2,4-decadienal. Dichloromethane extraction allowed also the identification of some alkaloid-like compounds that were not detected by HS-SPME.

Pieris brassicae inhibits xanthine oxidase.

The antioxidant potential of an aqueous extract obtained from Pieris brassicae larvae reared on Brassica oleracea L. var. costata DC was evaluated against 2,2-diphenyl-1-picrylhydrazyl radical and several reactive oxygen species. The results revealed an effective concentration-dependent protective activity against superoxide and hydroxyl radicals, being superior to that of the host plant. In addition, the larvae extract also exhibited a strong inhibitory effect on xanthine oxidase that was not observed for B. oleracea var. costata. A weak scavenging ability was noticed for hypochlorous acid. Several phenolic compounds with complex chemical structures that are hard to synthesize in the laboratory were found in P. brassicae extract. This is the first time that an insect has been tested for its xanthine oxidase inhibitory capacity, which proved to be very high. These findings are interesting considering that they can be used by food or pharmaceutical industries to prevent the oxidation of their products, to increase the dietary supply of antioxidants, or for prevention of free radical-mediated diseases, namely, gout.

New phenolic compounds and antioxidant potential of Catharanthus roseus.

Screening of the phenolic compounds from seeds, stems, leaves and petals of Catharanthus roseus (L.) G. Don (cv. Little Bright Eye) was achieved by HPLC-DAD-ESI-MS/MS. This is the first detailed study of noncolored phenolics in C. roseus, which allowed the characterization of three caffeoylquinic acids and fifteen flavonol glycosides (di- and trisaccharides of kaempferol, quercetin and isorhamnetin). Fifteen compounds are reported for the first time in this species. The scavenging ability of the different plant matrices was assessed against DPPH(*) radical and against reactive oxygen (superoxide radical) and a reactive nitrogen (nitric oxide) species. A concentration-dependent protective effect was observed for seeds and tissues, with petals shown to be the most active.

Comparative study on free amino acid composition of wild edible mushroom species.

A comparative study on the amino acid composition of 11 wild edible mushroom species (Suillus bellini, Suillus luteus, Suillus granulatus, Tricholomopsis rutilans, Hygrophorus agathosmus, Amanita rubescens, Russula cyanoxantha, Boletus edulis, Tricholoma equestre, Fistulina hepatica, and Cantharellus cibarius) was developed. To define the qualitative and quantitative profiles, a derivatization procedure with dabsyl chloride was performed, followed by HPLC-UV-vis analysis. Twenty free amino acids (aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, glycine, alanine, valine, proline, arginine, isoleucine, leucine, tryptophan, phenylalanine, cysteine, ornithine, lysine, histidine, and tyrosine) were determined. B. edulis and T. equestre were revealed to be the most nutritional species, whereas F. hepatica was the poorest. The different species exhibited distinct free amino acid profiles. The quantification of the identified compounds indicated that, in a general way, alanine was the major amino acid. The results show that the analyzed mushroom species possess moderate amino acid contents, which may be relevant from a nutritional point of view because these compounds are indispensable for human health. A combination of different mushroom species in the diet would offer good amounts of amino acids and a great diversity of palatable sensations.

In vivo skin irritation potential of a Castanea sativa (Chestnut) leaf extract, a putative natural antioxidant for topical application.

Topical application of natural antioxidants has proven to be effective in protecting the skin against ultraviolet-mediated oxidative damage and provides a straightforward way to strengthen the endogenous protection system. However, natural products can provoke skin adverse effects, such as allergic and irritant contact dermatitis. Skin irritation potential of Castanea sativa leaf ethanol:water (7:3) extract was investigated by performing an in vivo patch test in 20 volunteers. Before performing the irritation test, the selection of the solvent and extraction method was guided by the 1,1-diphenyl-2-picryl hydrazyl (DPPH) free radical scavenging test and polyphenols extraction (measured by the Folin Ciocalteu assay). Iron-chelating activity and the phenolic composition (high performance liquid chromatography/diode array detection) were evaluated for the extract obtained under optimized conditions. The extraction method adopted consisted in 5 short extractions (10 min.) with ethanol:water (7:3), performed at 40 degrees. The IC(50) found for the iron chelation and DPPH scavenging assays were 132.94 +/- 9.72 and 12.58 +/- 0.54 microg/ml (mean +/- S.E.M.), respectively. The total phenolic content was found to be 283.8 +/- 8.74 mg GAE/g extract (mean +/- S.E.M.). Five phenolic compounds were identified in the extract, namely, chlorogenic acid, ellagic acid, rutin, isoquercitrin and hyperoside. The patch test carried out showed that, with respect to irritant effects, this extract can be regarded as safe for topical application.

Targeted metabolite analysis and antioxidant potential of Rumex induratus.

Targeted metabolite analysis of aqueous extract of Rumex induratus leaves, in terms of phenolic compounds and organic acids, and the study of its antioxidant activity against the DPPH(*) radical, a reactive oxygen species, hypochlorous acid, and a reactive nitrogen species, nitric oxide ((*)NO), were performed. The samples were collected in several locations, spontaneously occurring or from greenhouse culture, at different stages of development and seasons. The phenolic composition was achieved by high-performance liquid chromatography (HPLC)-diode array detection, and four hydroxycinnamic acid derivatives and 10 flavonoid glycosides (C- and O-heterosides) were determined. Organic acids composition was established by HPLC-UV, revealing five compounds. The total amount of phenolic compounds and organic acids were affected by growing conditions and developmental phase. The aqueous extract exhibited a dose-related activity against all tested reactive species.

Do cultivar, geographical location and crop season influence phenolic profile of walnut leaves?

Walnut leaves from nine different cultivars (Arco, Franquette, Hartley, Lara, Marbot, Mayette, Meylannaise, Parisienne and Rego) were studied for their phenolic compounds. Samples were harvested along three consecutive years, at two different geographical locations, in order to evaluate if significant differences in the phenolics composition can be related with genetic, climatic or geographical factors. Nine compounds (3-caffeoylquinic, 3-p-coumaroylquinic and 4-p-coumaroylquinic acids, quercetin 3- galactoside, quercetin 3-arabinoside, quercetin 3-xyloside, quercetin 3-rhamnoside, a quercetin 3-pentoside derivative and a kaempferol 3-pentoside derivative) were quantified using an HPLC-DAD methodology. The qualitative profiles were identical for all samples, but differences were observed in terms of individual compounds' contents. Multivariate statistical analysis was carried out, showing that significant differences exist among production years, which can be related to climatic reasons.

Free amino acids of tronchuda cabbage (Brassica oleracea L. Var. costata DC): influence of leaf position (internal or external) and collection time.

The free amino acid profile of 18 samples of tronchuda cabbage ( Brassica oleracea L. var. costata DC) leaves, harvested at three different months, was determined by HPLC/UV-vis. The tronchuda cabbage leaves total free amino acid content varied from 3.3 to 14.4 g/kg fresh weight. Generally, arginine was the major compound, followed by proline, threonine, glutamine, cysteine, and glutamic acid. This study indicates that free amino acids are not similarly distributed: in external leaves, proline and arginine were the major free amino acids, while in internal ones, arginine was the main free amino acid, followed by threonine, glutamine, and cysteine. Significant differences were observed for valine, proline, arginine, leucine, cysteine, lysine, histidine, and tyrosine contents. The levels of some free amino acids were significantly affected by the collection period. In external leaves, this occurred with glutamic acid, serine, valine, leucine, cysteine, and ornithine contents, while in internal leaves, it occurred with aspartic acid, arginine, and total contents.

Leucopaxillus giganteus mycelium: effect of nitrogen source on organic acids and alkaloids.

The aim of this work was to find the most useful inorganic nitrogen source to enable Leucopaxillus giganteus to become a more nutritious mushroom, regarding organic acid and phenolic composition and total alkaloids content. For these, the influence of NH 4NO 3,NaNO 2, KNO 3,, and (NH 4) 2HPO 4 on the organic acid production was determined by HPLC-UV and total alkaloid content was assessed by a spectrophotometric method, after precipitation by Dragendorff's reagent. The results showed that L. giganteus presented an organic acid profile composed of oxalic, cis-aconitic, citric, and fumaric acids, citric acid being the major one. The quantitative organic acid profile and total alkaloid content were affected by the nitrogen source and depended on the developmental stage of mycelium and nitrogen availability. Despite being present in all samples, no phenolic compound could be identified.

Tronchuda cabbage (Brassica oleracea L. var. costata DC): scavenger of reactive nitrogen species.

The ability of tronchuda cabbage ( Brassica oleracea L. var. costata DC) to act as a scavenger of the reactive nitrogen species nitric oxide and peroxynitrite was investigated. The aqueous extracts obtained from tronchuda cabbage seeds and from its external and internal leaves exhibited a concentration dependent scavenging capacity. The antioxidant potential observed against the two reactive species was as follows: seeds > external leaves > internal leaves. In order to establish a possible correlation with the chemical composition of the extracts, the activity of ascorbic and sinapic acids and kaempferol 3- O-rutinoside was also studied. Among the compounds tested, sinapic acid showed the strongest antioxidant activity against both species.

Correlation between the pattern volatiles and the overall aroma of wild edible mushrooms.

Volatile and semivolatile components of 11 wild edible mushrooms, Suillus bellini, Suillus luteus, Suillus granulatus, Tricholomopsis rutilans, Hygrophorus agathosmus, Amanita rubescens, Russula cyanoxantha, Boletus edulis, Tricholoma equestre, Fistulina hepatica, and Cantharellus cibarius, were determined by headspace solid-phase microextraction (HS-SPME) and by liquid extraction combined with gas chromatography-mass spectrometry (GC-MS). Fifty volatiles and nonvolatiles components were formally identified and 13 others were tentatively identified. Using sensorial analysis, the descriptors "mushroomlike", "farm-feed", "floral", "honeylike", "hay-herb", and "nutty" were obtained. A correlation between sensory descriptors and volatiles was observed by applying multivariate analysis (principal component analysis and agglomerative hierarchic cluster analysis) to the sensorial and chemical data. The studied edible mushrooms can be divided in three groups. One of them is rich in C8 derivatives, such as 3-octanol, 1-octen-3-ol, trans-2-octen-1-ol, 3-octanone, and 1-octen-3-one; another one is rich in terpenic volatile compounds; and the last one is rich in methional. The presence and contents of these compounds give a considerable contribution to the sensory characteristics of the analyzed species.

Multivariate analysis of tronchuda cabbage (Brassica oleracea L. var. costata DC) phenolics: influence of fertilizers.

A field experiment was carried out to investigate the effect of fertilization level on the phenolic composition of tronchuda cabbage ( Brassica oleracea L. var. costata DC) external and internal leaves. Eight different plots were constituted: a control without fertilization, one with organic matter, and six experiments with conventional fertilizers (nitrogen, boron, and sulfur, two levels each). The phenolic compounds were analyzed by reversed-phase HPLC-DAD. External and internal leaves revealed distinct qualitative composition. In the internal leaves were found 15 phenolics (5 kaempferol and 10 cinnamic acid derivatives), whereas the external leaves presented 3- p-coumaroylquinic acid and 13 kaempferol derivatives. Principal component analysis (PCA) was applied to assess the relationships between phenolic compounds, agronomical practices, and harvesting time. Samples obtained with conventional practices were quite effectively separated from organic samples, for both types of leaves. In general, samples developed without any fertilization presented the highest phenolics amounts: external and internal leaves contained 1.4- and 4.6-fold more phenolic compounds than the ones that received conventional fertilizer, respectively, and the internal leaves presented 2.4 times more phenolics than the ones grown with organic amendment. Additionally, samples from organic production exhibited higher total phenolics content than those from conventional practices, collected at the same time. Samples harvested first were revealed to be distinct from the ones collected later. The results show that it is possible to grow tronchuda cabbage without excess fertilizers, with highest amounts of phenolics and reduced environment contamination.

HPLC-DAD-MS/MS-ESI screening of phenolic compounds in Pieris brassicae L. Reared on Brassica rapa var. rapa L.

The phenolic profiles of cabbage white butterfly ( Pieris brassicae L.; Lepidoptera: Pieridae) at different development stages (larvae, exuviae, and butterfly), its excrements, and its host plant Brassica rapa var. rapa L. were determined by high performance liquid chromatography- diode-array detector-mass spectrometry/mass spectrometry-electrospray ionization (HPLC-DAD-MS/MS-ESI). Twenty-five acylated and nonacylated flavonoid glycosides and ferulic and sinapic acids were identified in host plant, from which only 12 compounds were found in the excrements. In addition, the excrements showed the presence of sulfate flavonoids and other flavonoid glycosides that were not detected in the leaves. In the larvae kept without food for 12 h, only 3 compounds common to the plant material and 2 others, also present in the excrements, were characterized. The results indicate that deacylation, deglycosylation, and sulfating steps are involved in the metabolic process of P. brassicae and that its excrements may constitute a promising source of bioactive compounds, which could be used to take profit of this common pest of Brassica cultures.

Oak leaf extract as topical antioxidant: free radical scavenging and iron chelating activities and in vivo skin irritation potential.

The topical application of antioxidants may be beneficial for the protection of the skin against UV damage. An extract of Quercus robur leaves was prepared and evaluated considering its putative application as topical antioxidant. The solvent and extractive method selection was monitored by 1,1-diphenyl-2-picryl hydrazyl (DPPH) free radical scavenging activity and polyphenols extraction (measured by the Folin Ciocalteu assay). Iron chelating activity and the phenolic composition (HPLC/DAD) were assessed on the extract obtained under optimized conditions. Skin irritation potential was investigated by performing an in vivo patch test in 19 volunteers. The extraction solvent which resulted in the highest activity was ethanol:water (4:6) and thus it was selected for further preparation of this extract. The IC50s for the iron chelation and DPPH scavenging assays were 153.8 +/- 26.3 microg x mL-1 and 7.53 +/- 0.71 microg x mL-1 (mean +/- SD), respectively. The total phenolic content was found to be 346.3 +/- 6.7 mg gallic acid equivalents (GAE)/g extract (mean +/- SD). Three phenolic compounds were identified in the extract namely: ellagic acid, rutin and hyperoside. The major identified component was ellagic acid. The patch test carried out showed that the extract can be regarded as safe for topical application.

Inflorescences of Brassicacea species as source of bioactive compounds: A comparative study.

Two Brassica oleracea varieties (B. oleracea L. var. costata DC and B. oleracea L. var. acephala) and Brassica rapa L. var. rapa inflorescences were studied for their chemical composition and antioxidant capacity. Phenolic compounds and organic acids profiles were determined by HPLC-DAD and HPLC-UV, respectively. B. oleracea var. costata and B. oleracea L. var. acephala inflorescences presented a similar qualitative phenolic composition, exhibiting several complex kaempferol derivatives and 3-p-coumaroylquinic acid, while B. rapa var. rapa was characterized by kaempferol and isorhamnetin glycosides and several phenolic acids derivatives. B. oleracea L. var. costata and B. rapa var. rapa showed the highest phenolics content. The three Brassica exhibited the same six organic acids (aconitic, citric, pyruvic, malic, shikimic and fumaric acids), but B. oleracea L. var. acephala presented a considerably higher amount. Each inflorescence was investigated for its capacity to act as a scavenger of DPPH radical and reactive oxygen species (superoxide radical, hydroxyl radical and hypochlorous acid), exhibiting antioxidant capacity in a concentration dependent manner against all radicals. These samples were also studied for its antimicrobial potential against Gram-positive and Gram-negative bacteria and fungi, displaying antimicrobial capacity only against Gram-positive bacteria.

Organic acids composition of Cydonia oblonga Miller leaf.

Organic acid profiles of 36 Cydonia oblonga Miller leaf samples, from three different geographical origins of northern (Bragança and Carrazeda de Ansiães) and central Portugal (Covilhã), harvested in three collection months (June, August and October of 2006), were determined by HPLC/UV (214nm). Quince leaves presented a common organic acid profile, composed of six constituents: oxalic, citric, malic, quinic, shikimic and fumaric acids. C. oblonga leaves total organic acid content varied from 1.6 to 25.8g/kg dry matter (mean value of 10.5g/kg dry matter). Quinic acid was the major compound (72.2%), followed by citric acid (13.6%). Significant differences were found in malic and quinic acids relative abundances and total organic acid contents according to collection time, which indicates a possible use of these compounds as maturity markers. Between June and August seems to be the best period to harvest quince leaves for preparation of decoctions or infusions, since organic acids total content is higher in this season.

Comparative study of phytochemicals and antioxidant potential of wild edible mushroom caps and stipes.

A comparative study of the organic acids and phenolics composition and of the total alkaloids content of entire wild edible mushrooms (Russula cyanoxantha, Amanita rubescens, Suillus granulatus and Boletus edulis) and correspondent caps and stipes was performed. All species presented oxalic, citric, malic and fumaric acids, with A. rubescens exhibiting the highest total organic acids content. Organic acids were preferably fixed in the cap. Among phenolics, only p-hydroxybenzoic acid was found in A. rubescens and S. granulatus, in very low amounts. B. edulis was the species that presented the highest total alkaloid amounts. Except for this species, alkaloids mainly accumulated in the cap. All of the species exhibited a concentration-dependent scavenging ability against DPPH(·). B. edulis revealed the highest antioxidant capacity. The cap seemed to be the part with highest antioxidant potential. Some relationships between chemical composition and antioxidant capacity were considered.

New C-deoxyhexosyl flavones and antioxidant properties of Passiflora edulis leaf extract.

The flavonoids present in passion fruit (Passiflora edulis) leaves were identified by a high-performance liquid chromatography-diode array detection-tandem mass spectrometry (HPLC-DAD-MS/MS) method. Sixteen apigenin or luteolin derivatives were characterized, which included four mono-C-glycosyl, eight O-glycosyl- C-glycosyl, and four O-glycosyl derivatives. With the exceptions of C-hexosyl luteolin and C-hexosyl apigenin, all the compounds exhibited a deoxyhexose moiety. Moreover, the uncommon C-deoxyhexosyl derivatives of luteolin and apigenin have been identified for first time in P. edulis by HPLC-DAD-MS/MS. The antioxidative capacity of passion fruit leaves was checked against DPPH radical and several reactive oxygen species (superoxide radical, hydroxyl radical, and hypochlorous acid), revealing it to be concentration-dependent, although a pro-oxidant effect was noticed for hydroxyl radical.