Improving bioactive compounds extractability of Amorphophallus paeoniifolius (Dennst.) Nicolson

Elephant foot yam (Amorphophallus paeoniifolius (Dennst.) Nicolson) is an underground, unbranched deciduous plant that produces a large tubercle (rhizome) with recognized health effects. In this study, the influence of solvent nature (water, water/etanol (1:1) and absolute ethanol) and processing type (fresh, lyophilized and boiled) on the antioxidant activity and bioactive compounds extractability of elephant foot yam was evaluated. Extracts were compared for their contents in total phenolics, flavonoids and tannins. Moreover, their antioxidant capacity was assessed by the ferric reducing antioxidant power (FRAP) and the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH▪) scavenging capacity assays. Phenolics (154 mg GAE/L) and tannins (109 mg GAE/L) were maximized in lyophilized samples extracted with the hydroalcoholic solvent, which attained also the highest FRAP value (711 mg FSE/L). In turn, flavonoids reached the highest yields in lyophilized samples (95 mg ECE/L) extracted with pure ethanol, as well as the highest DPPH▪ scavenging activity. These findings might have practical applications to define the best processing methodology regarding the enhancement of elephant foot yam, either for prompt consumption, as well as to develop food supplements or pharmaceutical related products.

Wild mushrooms and their mycelia as sources of bioactive compounds: antioxidant, anti-inflammatory and cytotoxic properties

Mushrooms are an important source of natural compounds with acknowledged bioactivity. Pleurotus eryngii (DC.) Quél., in particular, is widely recognized for its organoleptic quality and favorable health effects, being commercially produced in great extent. On the other hand, Suillus bellinii (Inzenga) Watling is an ectomycorrhizal symbiont, whose main properties were only reported in a scarce number of publications. Some current trends point toward using the mycelia and the culture media as potential sources of bioactive compounds, in addition to the fruiting bodies. Accordingly, P. eryngii and S. bellinii were studied for their composition in phenolic acids and sterols, antioxidant capacity (scavenging DPPH radicals, reducing power, β-carotene bleaching inhibition and TBARS formation inhibition), anti-inflammatory effect (by down-regulating LPS-stimulated NO in RAW264.7 cells) and anti-proliferative activity (using MCF-7, NCI-H460, HeLa, HepG2 and PLP2 cell lines). Overall, S. bellinii mycelia showed higher contents of ergosterol and phenolic compounds (which were also detected in higher quantity in its fruiting body) and stronger antioxidant activity than P. eryngii. On the other hand, P. eryngii mycelia showed anti-inflammatory (absent in S. bellinii mycelia) and a cytotoxicity similar (sometimes superior) to its fruiting bodies, in opposition to S. bellinii, whose mycelia presented a decreased anti-proliferative activity. Furthermore, the assayed species showed differences in the growth rate and yielded biomass of their mycelia, which should also be considered in further applications.

Nutritional and chemical characterization of food products enriched with biological produced fruits of Vaccinum myrtillus L

Vaccinum myrtillus L. belongs to Ericaceae family, being commonly known for its sweet small fruits: the blueberries. Widely consumed in fresh, these fruits are also used in jams and marmalades due to their digestive and hypoglycemic properties and also due to the presence of several bioactive compounds [!]. Therefore, it has become a very appealing matrix in the development of functional products that, beyond their nutritional properties, will add a long-term beneficial physiological/health effect [2]. In the present work, three novel blueberry based products developed by RBR Foods Company (Portugal), were characterized in terms of their nutritional and chemical properties: carbohydrates, ash, proteins, fat and energetic value (following official methods of food analysis), fatty acids profile (by CG-FID), soluble sugars (by HPLCRI), organic acids (by HPLC-DAD) and tocopherols (by HPLC-fluorescence). The products result from a mixture of the fruits with rose petals (PI), marigold petals (P2) and apple and goji berries (P3). The blueberry fruits were used as control sample. The nutritional profile of the novel products was very similar to the control sample: the carbohydrates were the most abundant macronutrient, followed by proteins and total fat. Regarding sugars, fructose, glucose and sucrose were identified in all the samples. P 1 and P2 didn't show significant differences in comparison to the control, however, P3 revealed a lower concentration of sugars. In terms of fatty acids composition, all the studied samples presented higher contents in polyunsaturated fatty acids, especially due to the contribution of linoleic and alinolenic acids. The results of tocopherols revealed that the control sample only presented two isoforrns of tocopherols, a- and y-tocopherol, being the same observed in P3. However, P 1 revealed the presence of all the isoforrns of tocopherols, while P2 was lacking otocopherol; which is related with the contribution of rose and marigold petals, respectively. The a-tocopherol isoforrn was the most abundant in all the studied samples. Overall, this work contributed to the nutritional characterization of novel blueberry based products and is a part of a wider project that aims the detailed study of these products, namely their potential to be used as functional foods.

Chromatographic analysis of antioxidant and related compounds in Polyporus squamosus from different origins

The antioxidant potential of mushrooms is mainly attributed to their composition in polysaccharides, phenolic compounds, tocopherols and some organic acids [1]. Phenolic compounds contribute directly to the antioxidative action and play an important role in stabilizing lipid peroxidation [2]; exhibit a wide range of bioactive properties such as anti-allergenic, anti-inflammatory and antimicrobial, which have been in part related to their antioxidant activity [3]. Tocopherols are important fatsoluble antioxidants, acting in the cellular membrane; due to their role as scavenger of free radicals protecting human cells against degenerative malfunctions [4]. Some organic acids are very common in natural matrices; malic acid contributes to a pleasantly sour taste and is often used as a food additive; citric acid is known due to its antibacterial and antioxidant properties and fumaric acid is important because of its antioxidant, anti-inflammatory, antimicrobial and acidifying properties [5]. The purpose of the present study was to analyze antioxidant and related compounds (phenolic compounds, tocopherols and organic acids) of Polyporus squamosus (Huds.) Fr. samples originated from two different origins (Portugal and Serbia). Specimens of P. squamosus were collected in Bragança (Northeast Portugal) and Jabučki rit (Northern Serbia) during April 2015 and 2012, respectively. Phenolic compounds, organic acids and tocopherols were determined by high performance liquid chromatograph (HPLC) coupled to a diode array detector (DAD), in the two first cases, and a fluorescence detector in the last one. With respect to phenolic and related compounds, p-hydroxybenzoic and cinnamic acids were identified in both samples; the first one predominates in the sample from Portugal, while cinnamic acid was more abundant in the sample from Serbia. Tocopherols (α-, β and γ-isoforms) were found in the sample from Serbia, but in the sample from Portugal, γ-tocopherol was not identified. This sample showed the highest total tocopherols content, and revealed the highest level of β-tocopherol; γ- tocopherol predominated in the sample from Serbia. Among organic acids, it was possible to quantify oxalic, malic and fumaric acids in both samples. Malic acid was found in higher amounts in the sample from Serbia. Overall, the present study shows that mushroom samples from different origins have dissimilar results, but are both rich in bioactive compounds, being a valuable source for the development of natural medicines and nutraceuticals.

Extraction, identification, fractionation and isolation of phenolic compounds in plants with hepatoprotective effects

The liver is one of the most important organs of human body, being involved in several vital functions and regulation of physiological processes. Given its pivotal role in the excretion of waste metabolites and drugs detoxification, the liver is often subjected to oxidative stress that leads to lipid peroxidation and severe cellular damage. The conventional treatments of liver diseases such as cirrhosis, fatty liver and chronic hepatitis are frequently inadequate due to side effects caused by hepatotoxic chemical drugs. To overcome this problematic paradox, medicinal plants, owing to their natural richness in phenolic compounds, have been intensively exploited concerning their extracts and fraction composition in order to find bioactive compounds that could be isolated and applied in the treatment of liver ailments. The present review aimed to collect the main results of recent studies carried out in this field and systematize the information for a better understanding of the hepatoprotective capacity of medicinal plants in in vitro and in vivo systems. Generally, the assessed plant extracts revealed good hepatoprotective properties, justifying the fractionation and further isolation of phenolic compounds from different parts of the plant. Twenty-five phenolic compounds, including flavonoids, lignan compounds, phenolic acids and other phenolic compounds, have been isolated and identified, and proved to be effective in the prevention and/or treatment of chemically induced liver damage. In this perspective, the use of medicinal plant extracts, fractions and phenolic compounds seems to be a promising strategy to avoid side effects caused by hepatotoxic chemicals.

The broad spectrum of bioactive properties of phenolic extracts: a prospective study in three different plants

Natural resources like plants are currently used all over developed and under developed countries of the world as traditional home remedies and are promising agents for drug discovery as they play crucial role in traditional medicine. The use of plants for medicinal purpose usually varies from country to country and region to region because their use depends on the history, culture, philosophy and personal attitudes of the users (Ahmad et al., 2015). The use of plants and plant products as drugs predates the written human history (Hayta et al., 2014). Plants are a very important resource for traditional drugs and around 80% of the population of the planet use plants for the treatment of many diseases and traditional herbal medicine accounts for 30-50% of the total medicinal consumption in China. In North America, Europe and other well-developed regions over 50% of the population have used traditional preparations at least once (Dos Santos Reinaldo et al., 2015). Medicinal plants have been used over years for multiple purposes, and have increasingly attract the interest of researchers in order to evaluate their contribution to health maintenance and disease’s prevention (Murray, 2004). Recently between 50,000 and 70,000 species of plants are known and are being used in the development of modern drugs. Plants were the main therapeutic agents used by humans from the 19th century, and their role in medicine is always topical (Hayta et al., 2014). The studies of medicinal plants are rapidly increasing due to the search for new active molecules, and to improve the production of plants or bioactive molecules for the pharmaceutical industries (Rates, 2001). Several studies have been reported, but numerous active compounds directly responsible for the observed bioactive properties remain unknown, while in other cases the mechanism of action is not fully understood. According to the WHO 25% of all modern medicines including both western and traditional medicine have been extracted from plants, while 75% of new drugs against infective diseases that have arrived between 1981 and 2002 originated from natural sources, it was reported that the world market for herbal medicines stood at over US $60 billion per year and is growing steadily (Bedoya et al., 2009). Traditional medicine has an important economic impact in the 21st century as it is used worldwide, taking advantage on the low cost, accessibility, flexibility and diversity of medicinal plants (Balunas & Kinghorn, 2005).

A prospective study on bioactive properties of wild mushrooms mycelium grown in vitro under different conditions

Wild mushrooms have been extensively studied for their value as sources of high quality nutrients and of powerful physiologically bioactive compounds [1,2]. The present study was designed to evaluate the in vitro development of two wild edible mushroom species: Pleurotus eryngii (DC.) Quél. and Suillus belinii (Inzenga) Watling, by testing different solid (Potato Dextrose Agar medium –PDA and Melin-Norkans medium- MMN) and liquid culture media (Potato dextrose broth- PDB and Melin-Norkans medium- MMN). Each strain of mushroom produces a special type of mycelium and this range of characteristics varies in form, color and growth rate. S. bellinii presents a pigmented and rhizomorphic mycelia, whereas, P. eryngii has depigmented and cottony mycelia. The mycelium isolated and grown in PDA showed a faster radial growth compared to the mycelium isolated and grown in both solid and liquid incomplete MMN medium. P. eryngii exhibited a rapid growth and a higher mycelia biomass in both medium compared to S. belinii. Moreover, the obtained mycelia will be characterized in terms of well-recognized bioactive compounds namely, phenolic acids and mycosterols (mainly ergosterol), by using high performance liquid chromatography coupled to diode array and ultraviolet detectors, respectively. These compounds will be correlated to mycelia bioactivity: i) antioxidant activity, evaluated through free radicals scavenging activity, reducing power and lipid peroxidation inhibition in vitro assays; ii) anti-inflammatory activity, assessed through nitric oxide production inhibition in murine macrophages (RAW 264.7 cell line); iii) cytotoxic activity, evaluated either in human tumor cell lines (MCF-7- breast adenocarcinoma, NCIH460- non-small cell lung cancer, HeLa- cervical carcinoma and HepG2- hepatocellular carcinoma) as also in a non-tumor porcine primary liver cells culture established in-house (PLP2). Overall, our expectation is that the bioactive formulations obtained by in vitro culture can be applied as nutraceuticals or incorporated in functional foods.

Utilization of free and microencapsulated Rosmarinus oficinalis L. extracts as bioactives ingredientes for new functional foods developmen

Currently, many consumers search for food with functional characteristics beyond their nutritional properties. Thus, the concept of functional food becomes a hot topic, allowing the obtaining of health benefits, including disease prevention. In this context, plants are recognized as sources of a wide range of bioactives, mainly phenolic compounds. In particular, the Rosmarinus officina/is L., commonly referred as rosemary, has several phenolic compounds with different bioactive properties such as antioxidant, antiinflammatory and antimicrobial activities, among others [!]. Hence, this plant has great potential for incorporation into foods in order to confer bioactivity to the final products. However, it should be highlighted that the bioactive compounds if exposed to adverse environments, for example: light, moisture, extreme pH, storage, food processing conditions, can be degraded leading to the consequent loss of bioactivity [2]. The microencapsulation is an alternative to overcome this problematic of bioactive compounds, as also to ensure controlled release, or target deliver to a specific site [3]. In this work, lyophilized rosemary aqueous extract prepared by in:'usion was used as a functional ingredient for cottage cheeses, after proving that it possesses, both higher content in phenolic compounds and higher antioxidant activity, comparatively with the corresponding hydroethanolic extract. The rosemary aqueous extract revealed, for example, a DPPH scavenging activity with an EC50 value of 73.44±0.54j!g/mL and presented as main phenolic compound the caffeic acid dimer, commonly named as rosmarinic acid. For the functionalized cottage cheeses, a decrease of bioactivity was observed after seven days under storage in fridge, when the extracts were incorporated in its free form. Therefore, to preserve the antioxidant activity, the rosemary aqueous extract was efficiently microencapsulated by using an atomization/coagulation technique and alginate as the matrix material and thereafter incorporated into the cottage cheeses. The final microspheres showed a size, estimated by OM using a magnification of I OOx, ranging between 51.1 and 122.6 J!m and an encapsulation efficiency, estimated through an indirect method, approaching 100%. Overall, the introduction of both free and microencapsulated extracts did not change the nutritional value of cottage cheeses, providing bioactivity that was more preserved with microencapsulated extracts putting in evidence the importance of using microencapsulation to develop effective functional foods.

Antiangiogenic potential of yogurts added with extracts rich in apigenin derivatives

Angiogenesis is a biological process through which there is the formation of new blood vessels from preexisting ones [I]. However, in pathological cases, the abnormal growth of new blood vessels promotes the development of various diseases including cancer [2) through the production of atypically large amounts of angiogenesis factors, e.g. the vascular endothelial growth factor (VEGF) [3]. The plant secondary metabolites have been the subject of several studies to evaluate their benefits to human health. In particular, the phenolic compounds have high potential for use in the food industry, including the development of functional foods. Among these, apigenin has been associated with chemopreventive effects related to cancer [4]. In fact, chemoprevention is a present-day concept and contemplates the use of medicines, biological compounds or nutrients as an intervention strategy of cancer prevention. In this work, an Arenaria montana L hydroethanolic extract was prepared and after characterization by HPLC-DAD-ESI/MS showed to be rich in apigenin derivatives. Furthermore, it exhibited ability to inhibit the phosphorylation of VEGFR-2 (vascular endothelium growth factor receptor) through an enzymatic assay. However, for the major protection of bioactive compounds, the extract was microencapsulated by an atomization/coagulation technique with alginate as the matrix material. Posteriorly, the hydroethanolic extract, in free and microencapsulated forms, was incorporated in yogurts in order to develop a novel chemopreventer food in relation to the angiogenesis process. The functionalized yogurts with A. montana extracts (free and microencapsulated) showed a nutritional value similar to the used control (yogurt without extract); however, the samples enriched with extracts revealed added-value regarding the VEGFR-2 phosphorylation inhibition ability. This effect was more effectively preserved over time in the samples functionalized with the protected extract. Overall, this work contributes to the valorization of plants rich in flavonoids, exploring its antiangiogenic potential with VEGFR-2 as target. Moreover, the atomization/coagulation technique allowed the production of viable microspheres enriched with the plant extract. The microspheres were effectively incorporated into yogurts, protecting the extract thus envisaging the development of novel functional foods with chemopreventive effects.

Micoencapsulation of bioactive plant extracts: a tool to functionalize cottage cheeses

Foeniculum vulgare Mill. (fennel) and Matricaria recutita L. (chamomile) are two examples of plants with reported antioxidant and antimicrobial properties, which can be related with their composition in phenolic compounds [1,2]. Furthermore, according to previous results of our research group, the direct incorporation of the aqueous extracts showed capacity to maintain the nutritional properties of the cottage cheeses, up to 7 days of storage, while improving the antioxidant potential. However, after 14 days, a decrease in the antioxidant properties was observed [1,2], which can be related with factors such as light, moisture, temperature and pH, that can cause bioactive compounds degradation. Therefore, the aim of the present study was to prepare microcapsules with the aqueous extracts of fennel and chamomile for incorporation in cottage cheese samples, in order to protect the bioactive molecules present in the extracts, such as phenolic compounds, and prevent the decrease of the antioxidant activity observed after the 14 days period. The microspheres were prepared using an atomization/coagulation technique. Sodium alginate was used as the matrix material to produce the microspheres that were characterized through optical microscopy (OM), during and after atomization, for inspecting morphology. The encapsulation efficiency (EE) was determined by HPLC-DAD by an indirect method by analysing the coagulation solution. FTIR was also used to attest the presence of the extract inside of the alginate matrix. These microencapsulated extracts were incorporated in cottage cheese samples that were further characterized in terms of nutritional properties and antioxidant potential right after incorporation, and after 7 and 14 days of storage at 4•c. The EE was estimated as -100% and the FTIR analysis confirmed the presence of the extracts inside the microspheres. The results showed that the incorporation of the microencapsulated extracts did not cause changes in the nutritional value of cottage cheeses (through a comparison with control samples without extracts). The predominant fatty acids were palmitic (C16:0) and oleic (CI8:0) acids. The order of abundance of fatty acids was as follows: saturated fatty acids (SF A)> monounsaturatcd fatty acids (MUF A)> polyunsaturated fatty acids (PUF A). Regarding free sugars, lactose was the only sugar identified and quantified in all samples. Regarding the antioxidant activity, the samples functionalized with the microencapsulated extracts showed a higher preservation of this property even after the 7th day of storage. Overall, the incorporation of the protected plant extracts in dairy foods can be a strategy to provide health benefits to consumers.

The use of wild fruits of Fragaria vesca L. in preparations with bioactive properties: chemical characterization, antioxidant, antibacterial and antibiofilm activities

Wild strawberry, Fragaria vesca L., belongs to Rosaceae family and is commonly found in roadsides and slopes [1]. The most consumed parts of this plant are its sweet small fruits, which constitute a source of vitamins and phenolic compounds, being also used in infusions due to their organoleptic properties and for the treatment of some intestinal disorders [2, 3]. In the present work, F. vesca fruits were evaluated for their nutritional value and further used in the preparation of infusions. The chemical composition of the fruits and corresponding infusions was determined in terms of soluble sugars, organic acids, tocopherols, folates (by HPLC coupled to different detectors), phenolic compounds (by HPLC-DAD/ESI-MS) and mineral elements (atomic absorption spectroscopy). Some of these bioactive compounds were correlated with antioxidant and antibacterial properties evaluated either in infusions as also in hydromethanolic extracts. Carbohydrates were the main macronutrients in the fruits, followed by fat and proteins. Regarding the fatty acids, polyunsaturated fatty acids showed higher prevalence, mainly due to the presence of D-linolenic (Cl8:3n3) and y-linolenic (Cl8:3n6) acids. Sucrose and citric acid were, respectively, the main sugar and organic acid found in the fruits and in its infusions. The microelement found in higher amounts in both samples was manganese, while potassium and calcium were the macroelements present in higher levels in the fruits and infusions, respectively. Both samples presented folates and tocopherols, being ytocopherol the main isoform detected in the fruits, while a-tocopherol was the only isoform quantified in the infusion. The hydromethanolic extract prepared from the fruits gave higher antioxidant and antibacterial activities, namely against Escherichia coli and Pseudomonas aeruginosa, than the infusion; it also showed capacity to inhibit the formation of bacterial biofilm. Both bioactivities are highly correlated with the presence of phenolic compounds, in which the major are ellagic acid derivatives (sanguiin hlO) followed by tlavan 3-ols ((+)catechin) and anthocyanin compounds (pelargonidin-3-glucoside). Although fruits of wild F. vesca are mainly consumed in fresh, this study also proves the potentiality of their infusions as a source of bioactive molecules and properties.

Chromatographic techniques to obtain the biomolecules profile of wild Suillus granulates

In recent years the interest in naturally occurring compounds has been increasing worldwide. Indeed, many of the bioactive compounds currently used as medicines have been synthesized based on the structure of natural compounds [1]. In order to obtain bioactive fractions and subsequently isolated compounds derived from natural matrices, several procedures have been carried out. One of these is to separate and assess the concentration of the active compound(s) present in the samples, a step in which the chromatographic techniques stand out [2]. In the present work the mushroom Sui/Ius granulatus (L.) Roussel was chemically characterized by chromatographic techniques coupled to different detectors, in order to evaluate the presence of nutritional and/or bioactive molecules. Some hydrophilic compounds, namely free sugars, were identified by high performance liquid chromatography coupled to a refraction index detector (HPLC-RI), and organic and phenolic acids were assessed by HPLC coupled to a photodiode array detector (HPLC-PDA). Regarding lipophilic compounds, fatty acids weredetermined by gas chromatography with a flame ionization detector (GC-FID) and tocopherols by HPLC-fluorescence detection. Mannitol and trehalose were the main free sugars detected. Different organic acids were also identified (i.e. oxalic, quinic and fumaric acids), as well as phenolic acids (i.e. gallic and p-hydroxybenzoic acids) and the related compound cinnamic acid. Mono- and polyunsaturated fatty acids were the prevailing fatty acids and a-, ~- and ~-tocopherol were the isoforms of vitamin E detected in the samples. Since this species proved to be a source of biologically active compounds, the antioxidant and antimicrobial properties were evaluated. The antioxidant activity was measured through the reducing power, free radical's scavenging activity and lipid peroxidation inhibition of its methanolic extract, and the antimicrobial activity was also tested in Gram positive and Gram negative bacteria and iri different fungi. S. granulatus presented antioxidant properties in all the performed assays, and proved to inhibit the growth of different bacterial and fungal strains. This study is a first step for classifying S. granulatus as a functional food, highlighting the potential of mushrooms as a source of nutraceutical and biologically active compounds.

Bioactivity and chromatographic analysis of nutrients and non-nutrients of two Leccinum species

Nowadays the rising cost of health care and pharmaceutical products, the increase in life expectancy as well as the demand for an improved quality of life, has led to an increased concern about food intake and an emergence of new concepts of nutrition [1]. Mushrooms have been pointed out as an excellent option to include in a healthy diet, due to their nutritional value [2] associated with their bioactive properties [3]. The current study presents the chemical profile of two edible species, Leccinum molle (Ban) Ban and Leccinum vulpinum Watling, harvested in the outskirts of Bragan9a (Northeastern Portugal), regarding their content in nutrients and nonnutrients. Individual profiles of sugars and fatty acids were obtained by HPLC-RI and GC-FID, respectively. Tocopherols were analysed by HPLC-fluorescence, and the non-nutrients (i.e., phenolic and other organic acids) by HPLC-PDA. The antioxidant activity of the methanolic extracts obtained from both species was assessed with different assays (e.g. reducing power, radical scavenging activity and lipid peroxidation inhibition) and their hepatotoxicity was evaluated in primary cell cultures obtained from porcine liver, PLP2. Generally, both Leccinum species revealed similar nutrient profiles, with low fat levels, fructose, mannitol and trehalose as the foremost free sugars, and higher percentages of mono- and polyunsaturated fatty acids in comparison with saturated fatty acids. The presence of bioactive compounds was also detected, namely phenolic (e.g., gallic, protocatechuic and p-hydroxybenzoic acids) and organic acids (e.g., citric and fumaric acids). Both species presented antioxidant properties, being L. vulpinum the species which showed the most promising results (higher contents of total phenolic acids and lower ECso values in all the performed assays). Neither of the extracts presented toxicity against the liver primary cells PLP2, up to maximal concentration tested (Giso > 400 μg/ml).

Phenolic compounds in Coleostephus myconis (L.) Rchb.f.: characterization by HPLC-DAD-ESI/MS and phenology effects

The Asteraceae family is spread worldwide. In Portugal, there are more than 300 species, standing out as one of the botanical families with largest representation in the Portuguese flora. Coleostephus myconis (L.) Rchb.f. is a scarcely studied Asteraceae species, characterized as having ruderal growth and persistence in abandoned soils (an expanding problem due to the desertification phenomena in rural areas). In this work, the flowers of C. myconis were collected in three different flowering stages (i: flower bud; ii: flower in anthesis; iii: senescent flower) from the Northwestern area of the Portuguese territory. Powdered samples (1 g) were extracted twice with ethanol:water 50:50 (v/v). After removing solvents, the combined extracts were re-dissolved, filtered through 0.22-μm disposable LC filter disks and analyzed by high performance liquid chromatography coupled to a diode array detector and electrospray ionization-mass spectrometry (HPLC-DAD/ESI-MS). The phenolic compounds were characterized according to their UV and mass spectra, and retention times. For the quantitative analysis, calibration curves of standard compounds were used. According to the UV spectra (λmax = 314-330 nm) and pseudomolecular ions ([M-H]-) at m/z 353 and 515, all producing an m/z 191 ion, four compounds derived from quinic acid were detected: 3-O-caffeoylquinic acid (Figure 1A), 5-O-caffeoylquinic acid (Figure 1B), 3,5-O-dicaffeoylquinic acid (Figure 1C) and 4,5-O-dicaffeoylquinic acid (Figure 1D), as also supported by the literature [1,2]. A fifth phenolic acid was identified as protocatechuic acid. The detected flavonoid were quercetin-O-glucuronide, quercetin-3-Oglucoside, myricetin-O-methyl-hexoside and a second glycosylated myricetin (not possible to identify completely). Some statistically significant changes were detected among the different assayed flowering stages; nevertheless, 3,5-O-dicaffeoylquinic acid was the major compound, independently of the phenologic stage. According to the previous results, C. myconis might be considered as a potential natural source of these valuable bioactive compounds, especially considering the high botanical representativeness of this plant and its inexpensiveness.

Effects of gamma and electron-beam irradiation on the individual phenolics of Viola tricolor L. edible flowers.

Edible flowers are being used in culinary preparations to improve the sensorial and nutritional qualities of food, besides improving human health due to the profusion in bioactive compounds [1]. Nevertheless, edible flowers are highly perishable and must be free of insects, which is difficult because they are usually cultivated without using pesticides [2]. Food irradiation is an economically viable technology to extend shelf life of foods, improving their hygiene and quality, while disinfesting insects [3]. The efficiency and safety of radiation processing (using Co-60 or electronaccelerators) have been approved by legal authorities (FDA, USDA, WHO, FAO), as also by the scientific community, based on extensive research [4]. Viola tricolor L. (heartseases), from Violaceae family, is one of the most popular edible flowers. Apart from being used as food, it has also been applied for its medicinal properties, mainly due to their biological activity and phenolic composition [5]. Herein, the phenolic compounds were analyzed by HPLC-DAD-ESI/MS and linear discriminant analysis (LDA) was performed to compare the results from flowers submitted to different irradiation doses and technologies (Co-60 and electron-beam). Quercetin-3-O-(6-O-rhamnosylglucoside)-7-O-rhamnoside (Figure 1) was the most abundant compound, followed by quercetin-3-O-rutinoside and acetyl-quercetin-3-O (6-O-rhamnosylglucoside)-7-O-rhamnoside. In general, irradiated samples (mostly with 1 kGy) showed the highest phenolic compounds content. The LDA outcomes indicated that differences among phenolic compounds effectively discriminate the assayed doses and technologies, defining which variables contributed mostly to that separation. This information might be useful to define which dose and/or technology optimizes the content in a specific phenolic compound. Overall, irradiation did not negatively affect the levels of phenolic compounds, providing the possibility of its application to expand the shelf life of V. tricolor and highlighting new commercial solutions for this functional food.