Antioxidant Activity and Phenolic Composition of a Red Bean (Phasoelus vulgaris) Extract and its Fractions.

- The activities of the crude acetonic extract of red bean and its two fractions were determined using a 0-carotene-linoleate model system as well as the total antioxidant activity (TAA), the total phenolics content (TPC), the DPPH radical-scavenging activity, and the reducing power assays. Results from the in vitro assays showed the highest values when tannins (fraction II) were tested. Specifically, the TAA of the tannins fraction was 4.37 mmol Trolox eq./g fraction; whereas, the crude extract and fraction I were 0.481 and 0.093 µmol Trolox eqi/mg extract or fraction, respectively. The content of total phenolics in fraction II was the utmost (612 mg/g); the tannins content, assayed by the vanillin method and expressed as absorbance units at 500 nm per I g, was 938. RP-HPLC- PAD-MS profiling revealed the presence of 33 compounds: quercetin arabinoglucoside, quercetin rutinoside, quercetin, p-hydroxybenzoic acid, and kaempferol rutinoside were the most abundant phenolics in the extract.

A comparison of the bioactivity and phytochemical profile of three different cultivars of globe amaranth: red, white, and pink.

The phytochemical profiles and bioactivities of red, white and pink globe amaranth (Gomphrena haageana K., Gomphrena globosa var. albiflora and Gomphrena sp., respectively), much less studied than the purple species (G. globosa L.), were compared. The chemical characterization of the samples included the analysis of macronutrients and individual profiles of sugars, organic acids, fatty acids, tocopherols, and phenolic compounds. Their bioactivity was evaluated by determining the antioxidant and anti-inflammatory activities; the absence of cytotoxicity was also determined. Red and pink samples showed the highest sugar content. Otherwise, the white sample gave the highest level of organic acids, and together with the pink one showed the highest tocopherol and PUFA levels. Quercetin-3-O-rutinoside was the major flavonol in white and pink samples, whereas a tetrahydroxy-methylenedioxyflavone was the major compound in the red variety, which revealed a different phenolic profile. The pink globe amaranth hydromethanolic extract revealed the highest antioxidant activity, followed by those of red and white samples. The anti-inflammatory activity was more relevant in red and pink varieties. None of the samples presented toxicity in liver cells. Overall, these samples can be used in bioactive formulations against inflammatory processes and in free radical production.

Fermentation enhances the content of bioactive compounds in kidney bean extracts.

The influence of solid (SSF) or liquid state fermentation (LSF) for 48 and 96 h on the production of water soluble extracts from kidney beans was investigated. SSF was carried out by Bacillus subtilis, whilst LSF was performed either by natural fermentation (NF) or by Lactobacillus plantarum strain (LPF). SSF extracts showed high soluble phenolic compound content (31-36 mg/g) and antioxidant activity (508-541 µg trolox equivalents/g), whilst LSF extracts exhibited potential antihypertensive activity due to their large γ-aminobutyric acid (GABA) content (6.8-10.6 mg/g) and angiotensin converting enzyme inhibitory (ACEI) activity (>90%). Therefore, fermentation can be considered as a valuable process to obtain bioactive ingredients from kidney beans, which could encourage their utilisation in the formulation of added-value functional foods.

Chemical characterisation and bioactive properties of Prunus avium L.: the widely studied fruits and the unexplored stems.

The aim of this study was to characterise sweet cherry regarding nutritional composition of the fruits, and individual phytochemicals and bioactive properties of fruits and stems. The chromatographic profiles in sugars, organic acids, fatty acids, tocopherols and phenolic compounds were established. All the preparations (extracts, infusions and decoctions) obtained using stems revealed higher antioxidant potential than the fruits extract, which is certainly related with its higher phenolic compounds (phenolic acids and flavonoids) concentration. The fruits extract was the only one showing antitumor potential, revealing selectivity against HCT-15 (colon carcinoma) (GI50∼74 µg/mL). This could be related with anthocyanins that were only found in fruits and not in stems. None of the preparations have shown hepatotoxicity against normal primary cells. Overall, this study reports innovative results regarding chemical and bioactive properties of sweet cherry stems, and confirmed the nutritional and antioxidant characteristics of their fruits.

Bioactivity and phytochemical characterization of Arenaria montana L.

The bioactivity (antioxidant and cytotoxic activities) of the aqueous and methanolic extracts of Arenaria montana L., a plant commonly used in Portuguese folk medicine, was evaluated and compared. Furthermore, the phytochemical composition was determined based on hydrophilic (sugars, organic acids and phenolic compounds) and lipophilic (fatty acids and tocopherols) compounds, in order to valorize this plant material as a functional food/nutraceutical. Fructose, oxalic acid, methyl-luteolin 2''-O-feruloylhexosyl-C-hexoside, α-tocopherol, and linoleic acid were the main individual compounds found in A. montana. In general, the aqueous extract showed higher antioxidant and cytotoxic activities than the methanolic extract; the latter showed activity only against HeLa and HepG2 cell lines. Both aqueous and methanolic extracts showed some hepatotoxicity but at higher doses than the ones active for tumor cell lines. Moreover, the aqueous extract of A. montana may be used as a functional food or nutraceutical due to the high antioxidant and cytotoxic activities, and due to the presence of bioactive compounds. As far as we know, this is the first report on the phytochemical composition and bioactivity of A. montana.

Nutritional and antioxidant contributions of Laurus nobilis L. leaves: would be more suitable a wild or a cultivated sample?

Medicinal and aromatic plants are used since ancient times in folk medicine and traditional food, but also in novel pharmaceutical preparations. The controversy lies in the use of cultivated and/or wild plants presenting both advantages and disadvantages in biological, ecological but also economic terms. Herein, cultivated and wild samples of Laurus nobilis L. were chemically characterized regarding nutritional value, free sugars, organic acids, fatty acids and tocopherols. Furthermore, the antioxidant activity (scavenging activity, reducing power and lipid peroxidation inhibition) and individual phenolic profile of L. nobilis extracts and infusions were evaluated. Data showed that the wild sample gave higher nutritional contribution related to a higher content of proteins, free sugars, organic acids, PUFA and tocopherols. It also gave better PUFA/SFA and n-6/n-3 ratios. Regarding antioxidant activity and phenolic compounds, it was the cultivated sample (mostly the infusion) that showed the highest values. The present study supports the arguments defending the use of wild and cultivated medicinal and aromatic plants as both present very interesting features, whether nutritional or antioxidant, that can be an assessed by their consumption. In vitro culture could be applied to L. nobilis as a production methodology that allows combination of the benefits of wild and cultivated samples.

Chemical composition of wild and commercial Achillea millefolium L. and bioactivity of the methanolic extract, infusion and decoction.

Medicinal plants used in folk medicine are being increasingly studied and used on pharmaceutical, food and nutraceutical fields. Herein, wild and commercial samples of Achillea millefolium L. (yarrow) were chemically characterized with respect to their macronutrients, free sugars, organic acids, fatty acids and tocopherols. Furthermore, in vitro antioxidant properties (free radicals scavenging activity, reducing power and lipid peroxidation inhibition) and antitumour potential (against breast, lung, cervical and hepatocellular carcinoma cell lines) of their methanolic extract, infusion and decoction (the most consumed forms) was evaluated and compared to the corresponding phenolic profile obtained by high performance liquid chromatography and mass spectrometry. Data obtained showed that the chemical profiles of wild and commercial samples, and also their methanolic extract, infusion and decoction were similar, varying only in the quantities found. Commercial yarrow have higher content of fat and saturated fatty acids, proteins, ash, energy value, sugars and flavonoids, while the wild sample revealed higher levels of carbohydrates, organic acids, unsaturated fatty acids, tocopherols and phenolic acids. The heterogeneity among the antioxidant and antitumour results of the samples and some low correlations with total phenolic compounds indicates that specific compounds, rather than the totality of them, are involved in the bioactive properties of samples.

Characterisation of phenolic compounds in wild fruits from Northeastern Portugal.

This study aimed to analyse the phenolic composition of wild fruits of Arbutus unedo (strawberry-tree), Prunus spinosa (blackthorn), Rosa canina and Rosa micrantha (wild roses). Analyses were performed by HPLC-DAD-ESI/MS. P spinosa fruits presented the highest concentration in phenolic acids (29.78 mg/100 g dry weight), being 3-O-caffeoylquinic acid the most abundant one, and flavone/ols (57.48 mg/100 g), among which quercetin3-O-rutinoside (15.63 mg/100 g) was the majority compound. (+)-Catechin was the most abundant compound in A. unedo (13.51 mg/100 g) and R. canina (3.59 mg/100 g) fruits. A. unedo fruits presented the highest concentration in flavan-3-ols (36.30 mg/100 g). Cyanidin 3-O-glucoside was found in all the studied fruits, being the major anthocyanin in most of them, with the exception of P. spinosa samples, in which cyaniding 3-O-rutinoside and peonidin 3-O-rutinoside predominated; P. spinosa fruit presented the more complex anthocyanin profile among the analysed fruits and also the highest anthocyanin concentrations, which was coherent with its greater pigmentation. All in all, P. spinosa presented the highest levels of phenolic acids and flavonoids, including anthocyanins, flavonols and flavones, although no flavan-3-ols could be identified in its fruits. The present study represents a contribution to the chemical characterisation of phenolic compounds from wild fruits with acknowledged antioxidant activity and traditionally used for several folk medicinal applications.

Study of zalema grape pomace: phenolic composition and biological effects in Caenorhabditis elegans.

The phenolic composition of the extractable fraction of Zalema grape pomace has been analyzed by HPLC-DAD-MS and consisted of mainly flavanols and flavonols (122.75 and 23.11 mg/100 g dry pomace, respectively). The antioxidant activity has been determined by in vitro FRAP, ABTS, and ORAC assays (11.7, 34.9, and 63.6 mmol of Trolox equivalents (TE) per 100 g of dry pomace, respectively) and in vivo using the model organism Caenorhabditis elegans . Cultivation of C. elegans in media containing 100 µg/mL dry pomace extract increased the survival of worms submitted to thermally induced oxidative stress, whereas a decrease in the rate of worm survival was found for 300 µg/mL extract. Interestingly, the levels of reactive oxygen species (ROS) were significantly decreased in stressed worms treated with the pomace extract at the two concentration levels. Further studies are required to explain this unexpected behavior, as well as to determine the compounds and mechanisms involved in the observed effects.

Chemical composition and antioxidant activity of dried powder formulations of Agaricus blazei and Lentinus edodes.

Several mushroom species have been pointed out as sources of antioxidant compounds, in addition to their important nutritional value. Agaricus blazei and Lentinus edodes are among the most studied species all over the world, but those studies focused on their fruiting bodies instead of other presentations, such as powdered preparations, used as supplements. In the present work the chemical composition (nutrients and bioactive compounds) and antioxidant activity (free radical scavenging activity, reducing power and lipid peroxidation inhibition) of dried powder formulations of the mentioned mushroom species (APF and LPF, respectively) were evaluated. Powder formulations of both species revealed the presence of essential nutrients, such as proteins, carbohydrates and unsaturated fatty acids. Furthermore, they present a low fat content (<2g/100g) and can be used in low-calorie diets, just like the mushrooms fruiting bodies. APF showed higher antioxidant activity and higher content of tocopherols and phenolic compounds (124 and 770 µg/100g, respectively) than LPF (32 and 690 µg/100g). Both formulations could be used as antioxidant sources to prevent diseases related to oxidative stress.

Nutrients, phytochemicals and bioactivity of wild Roman chamomile: a comparison between the herb and its preparations.

Roman chamomile, Chamaemelum nobile L. (Asteraceae), has been used for medicinal applications, mainly through oral dosage forms (decoctions and infusions). Herein, the nutritional characterisation of C. nobile was performed, and herbal material and its decoction and infusion were submitted to an analysis of phytochemicals and bioactivity evaluation. The antioxidant activity was determined by free radicals scavenging activity, reducing power and inhibition of lipid peroxidation, the antitumour potential was tested in human tumour cell lines (breast, lung, colon, cervical and hepatocellular carcinomas), and the hepatotoxicity was evaluated using a porcine liver primary cell culture. C. nobile proved to be an equilibrated valuable herb rich in carbohydrates and proteins, and poor in fat, providing tocopherols, carotenoids and essential fatty acids (C18:2n6 and C18:3n3). Moreover, the herb and its infusion are a source of phenolic compounds (flavonoids such as flavonols and flavones, phenolic acids and derivatives) and organic acids (oxalic, quinic, malic, citric and fumaric acids) that showed antioxidant and antitumour activities, without hepatotoxicity. The most abundant compounds in the plant extract and infusion were 5-O-caffeoylquinic acid and an apigenin derivative. These, as well as other bioactive compounds, are affected in C. nobile decoction, leading to a lower antioxidant potential and absence of antitumour potential. The plant bioactivity could be explored in the medicine, food, and cosmetic industries.

Infusion and decoction of wild German chamomile: bioactivity and characterization of organic acids and phenolic compounds.

Natural products represent a rich source of biologically active compounds and are an example of molecular diversity, with recognised potential in drug discovery. Herein, the methanol extract of Matricaria recutita L. (German chamomile) and its decoction and infusion (the most consumed preparations of this herb) were submitted to an analysis of phytochemicals and bioactivity evaluation. The antioxidant activity was determined by free radicals scavenging activity, reducing power and inhibition of lipid peroxidation; the antitumour potential was tested in human tumour cell lines (breast, lung, colon, cervical and hepatocellular carcinomas), and the hepatotoxicity was evaluated using a porcine liver primary cell culture (non-tumour cells). All the samples revealed antioxidant properties. The decoction exhibited no antitumour activity (GI(50)>400 µg/mL) which could indicate that this bioactivity might be related to compounds (including phenolic compounds) that were not extracted or that were affected by the decoction procedure. Both plant methanol extract and infusion showed inhibitory activity to the growth of HCT-15 (GI(50) 250.24 and 298.23 µg/mL, respectively) and HeLa (GI(50) 259.36 and 277.67 µg/mL, respectively) cell lines, without hepatotoxicity (GI(50)>400 µg/mL). Infusion and decoction gave higher contents of organic acids (24.42 and 23.35 g/100g dw). Otherwise, the plant methanol extract contained the highest amounts of both phenolic acids (3.99 g/100g dw) and flavonoids (2.59 g/100g dw). The major compound found in all the preparations was luteolin O-acylhexoside. Overall, German chamomile contains important phytochemicals with bioactive properties (mainly antitumour potential selective to colon and cervical carcinoma cell lines) to be explored in the pharmaceutical, food and cosmetics industries.

Phenolic profiles of cultivated, in vitro cultured and commercial samples of Melissa officinalis L. infusions.

Melissa officinalis L. (lemon balm) is normally consumed as an infusion and presents therapeutic properties, such as sedative, carminative and antispasmodic, also being included in some pharmaceutical preparations. The phenolic profiles of different samples of lemon balm, prepared as infusions, were evaluated by HPLC-DAD-ESI/MS. The profiles were compared in order to understand the differences between cultivated, in vitro cultured and commercial (bags and granulated) samples. All the samples showed a similar phenolic profile, presenting differences only in the quantities found of each compound. Rosmarinic acid was the most abundant compound, being higher in commercial samples, especially in tea bag sample (55.68mg/g of infusion) and lower in in vitro cultured sample (15.46mg/g). Moreover, dimers, trimers and tetramers of caffeic acid were identified and quantified for the first time in lemon balm. Only one flavonoid, luteolin-3'-O-glucuronide was found in all the samples, ranging from 8.43mg/g in commercial granulate sample to 1.22mg/g in in vitro cultured sample. Overall, cultivated and in vitro cultured samples presented the lowest amounts of phenolic compounds (59.59 and 30.21mg/g, respectively); otherwise, commercial samples showed the highest contents (109.24mg/g for tea bag and 101.03mg/g for granulate sample). The present study shows that infusion of lemon balm can be a source of phenolic compounds, known for their bioactive effects.

Effect of cooking and germination on phenolic composition and biological properties of dark beans (Phaseolus vulgaris L.).

Legumes are the basés diet in several countries. They hold a high nutritional value, but other properties related to human health are nowadays being studied. The aim of this work was to study the influence of processes (boiling or germination) on the phenolic composition of dark beans (Phaseolus vulgaris L. c.v. Tolosana) and their effect on their antioxidant, neuroprotective and anticancer ability. Phenolic composition of raw and processed dark beans was analysed by HPLC-PAD and HPLC-ESI/MS. The antioxidant activity was evaluated by ORAC. Astrocytes cultures (U-373) have been used to test their neuroprotective effect. Anticancer activities were evaluated on three different cell lines (renal adenocarcinoma (TK-10), breast adenocarcinoma (MCF-7) and melanoma (UACC-62)) by sulphorhodamine B method. Qualitative and quantitative differences in phenolic composition have been observed between raw and processed dark beans that influence the antioxidant activity, mainly for germinated samples which show a decrease of antioxidant capacity. Although every assayed extracts decreased reactive oxygen species release and exhibited cytotoxicity activities on cancer cell lines, raw beans proved to be the most active in neuroprotective and antitumoral effects; this sample is especially rich in phenolic compounds, mainly anthocyanins. This study further demonstrated that phenolic composition of dark beans is related with cooking process and so with their neuroprotective and anticancer activity; cooking of dark beans improves their digestion and absorption at intestinal level, while maintaining its protective ability on oxidative process at cellular level.

Antioxidant activity, ascorbic acid, phenolic compounds and sugars of wild and commercial Tuberaria lignosa samples: effects of drying and oral preparation methods.

The antioxidant activity and phytochemical composition (ascorbic acid, free sugars and phenolic compounds) of decoctions and infusions of wild and commercial samples of Tuberaria lignosa (Sweet) Samp. Aerial parts were evaluated and compared. Among wild samples, the effects of the drying method (freeze or shade-drying) on those parameters were studied. Infusion of the freeze-dried wild sample gave the highest levels of sugars, while infusion of shade-dried wild sample and decoction of the freeze-dried sample presented higher ascorbic acid and phenolic compounds content (including ellagitannins and flavonoids) than the other samples. The last two samples also revealed higher antioxidant activity, in some cases even higher than Trolox. Decoctions gave lower amounts of disaccharides than infusions, which seemed to be hydrolysed, increasing the content of monosaccharides. Commercial samples showed the lowest content in phenolic compounds, mainly in ellagitannins and flavonoids, and also the lowest antioxidant activity. This work gives scientific evidence to the traditional medicinal uses of wild Tuberaria lignosa, highlighting the interest of its decoctions and infusions as a source of bioactive compounds and functional beverages.

Characterization of phenolic compounds in flowers of wild medicinal plants from Northeastern Portugal.

Crataegus monogyna, Cytisus multiflorus, Malva sylvestris and Sambucus nigra have been used as important medicinal plants in the Iberian Peninsula since a long time ago, and are claimed to have various health benefits. This study aimed to determine the phenolic profile and composition of wild medicinal flowers of those species. The analysis was performed by HPLC-DAD-ESI/MS. Flavonoids, and particularly flavonols and flavones, were the main groups in almost all the studied samples. C. multiflorus sample gave the highest levels of total flavonoids (54.5 mg/gdw), being a chrysin derivative the most abundant flavone found (22.3 mg/gdw). C. monogyna revealed the highest concentration in phenolic acids (5.5 mg/gdw) that were not found in C. multiflorus sample; 5-O-caffeoylquinic acid was the most abundant phenolic acid found in the first species, being a procyanidin trimer also found (1.4 mg/gdw). Kaempferol-3-O-rutinoside (0.84 mg/gdw) and quercetin-3-O-rutinoside (14.9 mg/gdw) were the main flavonols present in M. sylvestris and S. nigra, respectively. Due to the well established antioxidant activity of phenolic compounds, the studied wild medicinal flowers could be selected for processing extracts with health-promoting properties or to be incorporate into functional beverages or products with bioactive properties related to oxidative stress.

Extraction and isolation of phenolic compounds.

Phenolic compounds constitute a major class of plant secondary metabolites that are widely distributed in the plant kingdom and show a large structural diversity. These compounds occur as aglycones or glycosides, as monomers or constituting highly polymerized structures, or as free or matrix-bound compounds. Furthermore, they are not uniformly distributed in the plant and their stability varies significantly. This greatly complicates their extraction and isolation processes, which means that a single standardized procedure cannot be recommended for all phenolics and/or plant materials; procedures have to be optimized depending on the nature of the sample and the target analytes, and also on the object of the study. In this chapter, the main techniques for sample preparation, and extraction and isolation of phenolic compounds have been reviewed-from classical solvent extraction procedures to more modern approaches, such as the use of molecularly imprinted polymers or counter-current chromatography.

Antioxidant activity of a red lentil extract and its fractions.

Phenolic compounds were extracted from red lentil seeds using 80% (v/v) aqueous acetone. The crude extract was applied to a Sephadex LH-20 column. Fraction 1, consisting of sugars and low-molecular-weight phenolics, was eluted from the column by ethanol. Fraction 2, consisting of tannins, was obtained using acetone-water (1:1; v/v) as the mobile phase. Phenolic compounds present in the crude extract and its fractions demonstrated antioxidant and antiradical activities as revealed from studies using a beta-carotene-linoleate model system, the total antioxidant activity (TAA) method, the DPPH radical-scavenging activity assay, and a reducing power evaluation. Results of these assays showed the highest values when tannins (fraction 2) were tested. For instance, the TAA of the tannin fraction was 5.85 micromol Trolox eq./mg, whereas the crude extract and fraction 1 showed 0.68 and 0.33 micromol Trolox eq./mg, respectively. The content of total phenolics in fraction 2 was the highest (290 mg/g); the tannin content, determined using the vanillin method and expressed as absorbance units at 500 nm per 1 g, was 129. There were 24 compounds identified in the crude extract using an HPLC-ESI-MS method: quercetin diglycoside, catechin, digallate procyanidin, and p-hydroxybenzoic were the dominant phenolics in the extract.