Paula B. Andrade

Phenolics: from chemistry to biology

In recent years, few classes of natural products have received as much attention as phenolics and polyphenols. This special issue of Molecules, “Phenolics and Polyphenolics”, is a remarkable confirmation of this trend. Several aspects related to phenolics chemistry, comprising the several classes, will be discussed. In addition, the increasing interest in phenolics’ biological activities is covered, and several works addressing this matter are referred.

Analysis of Honey Phenolic Acids by HPLC, Its Application to Honey Botanical Characterization

In this work we described an analytical technique that allowed the identification of 12 phenolic acids in honey samples, with emphasis on those phenolics acids which are markers of the botanical origin. After optimization of the HPLC conditions, this was applied to the phenolic acids analysis of 20 Erica sp. (heather) and 20 Lavandula stoechas (lavander) portuguese honeys. A close correlation between the phenolic acids patterns and the botanical origin of honey has been found. Erica sp. honeys are characterized by ellagic, p-hydroxybenzoic, syringic and o-coumaric acids, and Lavandula stoechas honeys by gallic acid.

Identification of phenolic compounds in isolated vacuoles of the medicinal plant Catharanthus roseus and their interaction with vacuolar class III peroxidase: an H2O2 affair?

Class III peroxidases (Prxs) are plant enzymes capable of using H(2)O(2) to oxidize a range of plant secondary metabolites, notably phenolic compounds. These enzymes are localized in the cell wall or in the vacuole, which is a target for secondary metabolite accumulation, but very little is known about the function of vacuolar Prxs. Here, the physiological role of the main leaf vacuolar Prx of the medicinal plant Catharanthus roseus, CrPrx1, was further investigated namely by studying its capacity to oxidize co-localized phenolic substrates at the expense of H(2)O(2). LC-PAD-MS analysis of the phenols from isolated leaf vacuoles detected the presence of three caffeoylquinic acids and four flavonoids in this organelle. These phenols or similar compounds were shown to be good CrPrx1 substrates, and the CrPrx1-mediated oxidation of 5-O-caffeoylquinic acid was shown to form a co-operative regenerating cycle with ascorbic acid. Interestingly, more than 90% of total leaf Prx activity was localized in the vacuoles, associated to discrete spots of the tonoplast. Prx activity inside the vacuoles was estimated to be 1809 nkat ml(-1), which, together with the determined concentrations for the putative vacuolar phenolic substrates, indicate a very high H(2)O(2) scavenging capacity, up to 9 mM s(-1). Accordingly, high light conditions, known to increase H(2)O(2) production, induced both phenols and Prx levels. Therefore, it is proposed that the vacuolar couple Prx/secondary metabolites represent an important sink/buffer of H(2)O(2) in green plant cells.

Determination of phenolic compounds in honeys with different floral origin by capillary zone electrophoresis

Abstract Twenty-six phenolic compounds from honey samples with different floral origin were analysed by capillary zone electrophoresis. All the phenolics were separated on a fused-silica column (50 cm × 50 μm) using 100 mM sodium borate buffer (pH 9.5)−20% methanol. This technique was applied to the separation of phenolic compounds from heather, lavender, acacia, rape, sunflower, rosemary, citrus, rhododendron, thyme, chestnut-tree and calluna honey samples, to establish correlations between the phenolics profiles and the botanical origin of the honey. Some individual honey samples showed potential floral markers. Thus, thyme honey was characterised by the presence of rosmarinic acid, heather honey by ellagic acid, citrus honey by hesperetin and lavender honey by naringenin.

Phenolic fingerprint of peppermint leaves

A reversed-phase high-performance liquid chromatography procedure is proposed for the determination of 10 phenolic compounds (eriodictyol 7-O-rutinoside, eriodictyol 7-O-glucoside, luteolin 7-O-rutinoside, luteolin 7-O-glucoside, hesperetin 7-O-rutinoside, apigenin 7-O-rutinoside, rosmarinic acid, 5,6-dihydroxy-7,8,3′,4′-tetramethoxyflavone, pebrellin and gardenin B) in peppermint. The chromatographic separation was achieved using a reversed-phase Spherisorb ODS 2 (5 μm particle size; 25.0×0.46 cm) column. Of the several extractive solvents tried, ethanol was the best for qualitative and quantitative analysis. Best resolution was obtained using a gradient of water-phosphoric acid (999:1) and acetonitrile. Fourteen samples were subjected to quantification and showed a common composition pattern.

Floral nectar phenolics as biochemical markers for the botanical origin of heather honey

In order to find out biochemical markers for the botanical origin of heather (Erica) honey, the phenolic metabolites present in heather floral nectar, collected from the honey-stomach of bees gathering nectar from these flowers, were analysed. The flavonoid fraction of nectar contained four main flavonoids. Two of them were quercetin and kaempferol 3-rhamnosides, and the other two were tentatively identified as myricetin 3′-methyl ether and isorhamnetin 3-rhamnosides. Since the natural glycosides are hydrolysed by bee enzymes to render the corresponding aglycones, which are the metabolites detected in honey, acid hydrolysis of the nectar glycosides was achieved. The aglycones quercetin, myricetin 3′-methyl ether, kaempferol and isorhamnetin were identified, as well as the gallic acid derivative ellagic acid. The analysis of Portuguese heather honey samples showed that ellagic acid was present in all the samples in significant amounts ranging between 100 μg and 600 μg per 100 g honey. The other nectar-derived flavonoids were also present, although some of them in very variable amounts. Ellagic acid and myricetin 3′-methyl ether, which have not been detected in any of the monofloral honey samples investigated so far, with the only exception being a French honey sample of the botanically relatedCalluna (Ericaceae) which also contained ellagic acid, seem to be the most useful potential markers for the floral origin of heather honey. However, more detailed and extensive investigations are needed to prove the utility of these markers.

Phenolic antioxidant compounds produced by in vitro shoots of sage (Salvia officinalis L.)

In vitro shoots of sage (Salvia officinalis L.) were established under four different cytokinin supplementations by culturing nodal segments excised from aseptically germinated seedlings. The highest rates of shoot proliferation and linear shoot growth occurred with the supplementation of 1.5 mg/l benzyladenine and 0.05 mg/l dichlorophenoxyacetic acid. However, under these conditions, the specific production of total antioxidant phenolics was the lowest. Variation in kinetin (KIN) concentration (1.5; 2.0; 4.0 mg/l), in the presence of 0.05 mg/l 2,4-D, did not influence significantly the rates of shoot proliferation and linear shoot growth but influenced the production of antioxidant phenolics and biomass. Seventeen compounds were identified in the antioxidant phenolic extracts from shoots: gallic acid, 3-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, caffeic acid, and rosmarinic acid, as phenolic acids; hesperetin, apigenin, hispidulin, cirsimaritin, and genkwanin, as flavonoids; epirosmanol, epirosmanol methyl ether, carnosol, epiisorosmanol ethyl ether, rosmadial, carnosic acid, and methyl carnosate, as phenolic diterpenes. With exception of carnosic acid and methyl carnosate, all the other phenolic compounds (15) were also identified in a commercial sample of this species. Rosmarinic acid and carnosol were the main compounds in all the antioxidant phenolic extracts. The increase in concentration of KIN decreased the accumulation of the most of phenolic diterpenes, particularly that of carnosol.

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.

Can Phlorotannins Purified Extracts Constitute a Novel Pharmacological Alternative for Microbial Infections with Associated Inflammatory Conditions

Bacterial and fungal infections and the emerging multidrug resistance are driving interest in fighting these microorganisms with natural products, which have generally been considered complementary to pharmacological therapies. Phlorotannins are polyphenols restricted to brown seaweeds, recognized for their biological capacity. This study represents the first research on the antibacterial, antifungal, anti-inflammatory and antioxidant activity of phlorotannins purified extracts, which were obtained from ten dominant brown seaweeds of the occidental Portuguese coast. Phlorotannins content was determined by the specific dimethoxybenzaldehyde (DMBA) method and a yield between 75 and 969 mg/Kg phloroglucinol units (dry matter) was obtained. Fucus spiralis ranked first, followed by three Cystoseira species. The anti-inflammatory potential of the purified extracts was assessed via inhibitory effect on nitric oxide (NO) production by lipopolysaccharide-stimulated RAW 264.7 macrophage cells, Cystoseira tamariscifolia being the one showing promising activity for the treatment of inflammation. NO scavenging ability was also addressed in cell free systems, F. spiralis being the species with highest capacity. The antimicrobial potential of the extracts was checked against five Gram-positive and four Gram-negative bacteria and three fungi strains, that commonly colonize skin and mucosa and are responsible for food contamination. The different extracts were more effective against Gram-positive bacteria, Staphylococcus epidermidis being the most susceptible species. Concerning antifungal activity, Trichophyton rubrum was the most sensitive species. Although the molecular mechanisms underlying these properties remain poorly understood, the results obtained turn phlorotannins purified extracts a novel and potent pharmacological alternative for the treatment of a wide range of microbial infections, which usually also present an inflammatory component. In addition to the biological properties demonstrated herein, phlorotannins extracts may also be preferred, in order to avoid side effects and allergic reactions commonly associated with synthetic drugs.

Analysis and quantification of flavonoidic compounds from Portuguese olive (Olea Europaea L.) leaf cultivars

Twenty three samples of 18 Portuguese olive leaf cultivars were analysed by a reversed-phase HPLC/DAD procedure and eight flavonoidic compounds were identified and quantified (luteolin 7,4′-O-diglucoside, luteolin 7-O-glucoside, rutin, apigenin 7-O-rutinoside, luteolin 4′-O-glucoside, luteolin, apigenin and diosmetin). Luteolin 7,4′-O-diglucoside and luteolin 4′-O-glucoside were identified by HPLC/DAD/MS/MS – ESI. The studied olive leaf samples showed a common phenolic pattern, in which luteolin 4′-O-glucoside was almost always the major compound.