New Insights on Primary and Secondary Metabolite Contents of Seven Italian Wild Food Plants with Medicinal Applications: A Comparative Study.

Wild food plants are widely consumed all over the world and many have both nutritional and therapeutic value due to the presence of biologically active compounds. The present research, for the first time, aims to compare primary and secondary metabolite levels among different plant organs (flower, leaf, stem, root, bark) of seven species (Borago officinalis L., Cynodon dactylon (L.) Pers., Foeniculum vulgare Mill., Hypericum perforatum L., Malva sylvestris L., Sambucus nigra L., Urtica dioica L.) collected in three different Italian regions (Liguria, Tuscany, Apulia). Plant organ samples were extracted with water or 95% (v/v) methanol and liquid fractions were analyzed using spectrophotometric assays. The best results were obtained for Hypericum perforatum L. samples, followed by Sambucus nigra L. and Borago officinalis L. As also confirmed via PCA analysis on normalized data, flower and leaf extracts of all species exhibited higher levels of polyphenols (up to 105.7 mg GA eq/gDW), reducing sugars (up to 389.2 mg GLUC eq/gDW), proteins (up to 675.7 mg BSA eq/gDW) and of antioxidant capacity (up to 263.5 mg AA eq/gDW). No differences among the regions of gathering were detected after spectrophotometric assays, which was confirmed via PCA analysis. These data contribute to further validate the traditionally reported healing effects of these species on human health.

Differences and similarities in selenium biopathways in Astragalus, Neptunia (Fabaceae) and Stanleya (Brassicaceae) hyperaccumulators.

BACKGROUND AND AIMS: Selenium hyperaccumulator species are of primary interest for studying the evolution of hyperaccumulation and for use in biofortification because selenium is an essential element in human nutrition. In this study, we aimed to determine whether the distributions of selenium in the three most studied hyperaccumulating taxa (Astragalus bisulcatus, Stanleya pinnata and Neptunia amplexicaulis) are similar or contrasting, in order to infer the underlying physiological mechanisms. METHODS: This study used synchrotron-based micro-X-ray fluorescence (µXRF) techniques to visualize the distribution of selenium and other elements in fresh hydrated plant tissues of A. racemosus, S. pinnata and N. amplexicaulis. KEY RESULTS: Selenium distribution differed widely in the three species: in the leaves of A. racemosus and N. amplexicaulis selenium was mainly concentrated in the pulvini, whereas in S. pinnata it was primarilylocalized in the leaf margins. In the roots and stems of all three species, selenium was absent in xylem cells, whereas it was particularly concentrated in the pith rays of S. pinnata and in the phloem cells of A. racemosus and N. amplexicaulis. CONCLUSIONS: This study shows that Astragalus, Stanleya and Neptunia have different selenium-handling physiologies, with different mechanisms for translocation and storage of excess selenium. Important dissimilarities among the three analysed species suggest that selenium hyperaccumulation has probably evolved multiple times over under similar environmental pressures in the US and Australia.

Foraging for selenium: a comparison between hyperaccumulator and non-accumulator plant species.

Selenium (Se) hyperaccumulators are a unique group of plants that can accumulate this element in their aerial parts at concentrations exceeding 100 mg kgDW-1. These plants actively search for Se in the soil, a phenomenon known as root foraging, reported to date only by few studies. In this study, the effect of localized Se enrichment, in the form of selenite and selenate, was investigated on the root architecture of two Se-hyperaccumulators (Stanleya pinnata and Astragalus bisulcatus) and two non-accumulators (Brassica juncea and Medicago sativa). Rhizoboxes were divided into two halves: one half was filled with control soil while the other with selenate or selenite (30 mg kgDW-1) spiked soil. Seedling were transferred into the interface of the two soils and allowed to grow for three weeks under controlled light and temperature conditions. Staneya pinnata exhibited equal root density in both halves of the rhizobox when grown in control/control and selenite/control soil treatments. However, in the presence of selenate, S. pinnata developed 76% of the roots towards the selenate-enriched half, indicating an active root foraging. In contrast, A. bisulcatus and the non-accumulators B. juncea and M. sativa did not show any preferential distribution of roots. This study revealed that only S. pinnata showed the ability to detect and forage for Se when provided as selenate. Non-accumulators did not show any morphological or Se-accumulation difference associated with the presence of Se in soil in either form.

Antioxidant and Sensory Properties of Raw and Cooked Pork Meat Burgers Formulated with Extract from Non-Compliant Green Coffee Beans.

The effects of polyphenol-rich extract obtained from non-compliant defatted green coffee beans (dGCBs) on physicochemical and antioxidant properties, as well as on the sensory profile of vacuum-packed pork burgers stored at 4 °C for 14 days and after cooking were assessed. The dGCB extract obtained by means of supercritical water extraction was analyzed for its polyphenol profile, total phenolic content, radical scavenging, and ferric-reducing antioxidant activities (DPPH and FRAP), Fe2+-chelating capacity, and total iron. The most abundant polyphenol component observed in the dGCB extract was chlorogenic acid, and the alkaloid caffeine was also present. This extract showed antioxidant properties. Thereafter, five formulations of pork meat burgers with added NaCl (1%) were prepared; one without the antioxidant (negative control, C) and one with the use of a synthetic antioxidant (0.05% ascorbic acid = positive control, A), while the other three were supplemented with a different amount of dGCB extract (P15 = 0.15%; P30 = 0.30%; P60 = 0.60%). The addition of dGCB extract increased the antioxidant activity of the raw and cooked burgers and reduced the lipid oxidation of the cooked burgers (0.47, 0.21, and 0.20 vs. 1.28 and 0.55 mg MDA eq./Kg, for P15, P30, and P60 vs. C and A, respectively). No negative effects were observed on the meat's color parameters and its stability during refrigerated storage and after cooking, nor on sensory attributes (color and aroma) for the lowest concentration of coffee extract. The results obtained indicate that 0.15% dGCB extract is a promising alternative to commercial synthetic antioxidants to improve the quality of refrigerated pork burgers.

Ethnobotanical Review and Dataset Compiling on Wild and Cultivated Plants Traditionally Used as Medicinal Remedies in Italy.

Over the centuries, wild plants have constituted the main food ingredients and traditional medicine in rural communities. In the last decades, thousands of ethnobotanical studies have been conducted, with the aim of documenting the traditional knowledge on wild and cultivated plants both for food and therapeutic purposes. In the present work, 75 published papers related to Italian ethnobotanical knowledge on wild and cultivated plants traditionally used for medical purposes were analyzed and data on 1117 different species organized in the first dataset to target medicinal applications only. For each plant species, the Italian region of use, plant organs, mode of preparation, specific pathological group of application, citation index, and use index were listed. The different therapeutic applications were subdivided into nine main pathological groups according to the targeted human apparatus. Overall, the cited species with highest number of uses were related to the treatment of the digestive system and skin-ears-eyes-hair diseases, followed by diseases of the genito-urinary and respiratory systems. The 13 most relevant species were identified on the basis of their citation and use indexes. The present review on Italian medicinal flora aims to provide valuable information on wild and cultivated species, which are potential sources of plant-based therapeutic remedies, to preserve and reevaluate endangered traditional folk knowledge.

Phytochemical characterization of raw and cooked traditionally consumed alimurgic plants.

In the past, wild edible alimurgic plants became an important alternative food source when poverty, wars or drought made it difficult to access crops. These plants were considered rich in highly nutritional compounds and also frequently used as food-medicine given their health-promoting properties. With the aim of improving our knowledge on the content of beneficial or detrimental compounds in relation with past local dietary and curative traditions, 12 wild food plant species were collected from two study areas selected for their very different degree of industrialization, urbanization, and conservation of local past traditions among the population: the Bologna province (Northern Italy) and the Middle Agri Valley (Southern Italy). Protein, polyphenol flavonoid and biogenic amine (both free and conjugated) contents and antioxidant activity of raw and boiled wild food plant extracts, and of cooking water were analyzed by means of spectrophotometric and high-performance liquid chromatography methods. The results demonstrated that most of the phenolic compounds were released in the cooking water which also showed the highest antioxidant activity. Seventeen different phenolic compounds were identified, of which the health-related luteolin, luteolin-7-glucoside and rutin were the most abundant (e.g., S. pratensis L. and C. intybus L.). On the other hand, biogenic amines were absent or present at very low levels in cooking water of those very same species (e.g., S. pratensis L., T. officinalis Weber, C. vesicaria subsp. taraxacifolia and C. intybus L.) of which traditionally a decoction is used for therapeutic purposes. Free and conjugated spermidine and spermine were generally the most abundant biogenic amines, while none of the known detrimental monoamines (e.g., histamine) was detected. In conclusion, the present results seem to support past local popular traditions which indicated beneficial medical properties of some wild edible plant, as well as of their cooking water.

Enzymatic Digestion of Calf Fleshing Meat By-Products: Antioxidant and Anti-Tyrosinase Activity of Protein Hydrolysates, and Identification of Fatty Acids.

The food waste reduction through an efficient recovery of its valuable building molecules has become an important topic with a positive effect on the economy and the environment. In this work, the revalorization of slaughterhouse calf fleshing meat through its enzymatic hydrolysis is proposed. The proteolytic activity of 11 enzymes was initially screened and the four most efficient enzymes (papain, trypsin, pancreatin, and bromelain) were selected. The molecular profiling of the different protein/peptide fractions by the Linear Trap Quadrupole-OrbiTrap technique showed compositional differences due to the specificity of the enzymes' cleavage sites. In order to find a potential reuse of these hydrolysates, the analysis of antioxidant and, for the first time on fleshing meat hydrolysates, of anti-tyrosinase activities, was performed. Papain-digested samples were those showing the highest inhibition activity of tyrosinase enzyme (55.6%) as well as the highest antioxidant activity (3.52 g TEAC/L). In addition, the composition analysis of the lipid fraction was performed. The mono-unsaturated fatty acids resulted to be the most abundant lipid in all the samples with the exception of pancreatin-treated hydrolysates in which poly-unsaturated fatty acids were predominant. The present results seemed to support a possible valorization of isolated fractions from calf fleshing by-products, as food or feed ingredients, by the implementation of fraction isolation within the meat-processing pipeline.

Cascade strategies for the full valorisation of Garganega white grape pomace towards bioactive extracts and bio-based materials.

Agro-waste reduction and reuse are among the current main social challenges. In this perspective, the present research was aimed at the complete valorisation of Garganega grape pomace by recovering bioactive phenol extracts and by testing the solid fibre extract residues in composite formulation for packaging applications. The pomace was derived from white wine production, therefore, respect to red pomace, it was promptly removed from must after pressing, and its exploitation can be particularly interesting and valuable as still rich in active compounds. Phenol extracts were obtained both via solvent-based and pressurised liquid extractions and their phytochemical compositions were compared in terms of total amount of phenols, flavonoids, flavanols, anthocyanins, hydroxycinnamic acids, and reducing sugars. Antioxidant activity and detailed phenol profiles were also achieved. The highest phenol yield was obtained via solvent-based extraction with 75% acetone (v/v), solid/liquid ratio 1:5, 2h incubation at 50°C (77.9 gGAeq/kgDW). The fibrous solid residue of the extraction was characterized via thermogravimetric analysis and used for composite preparation by melt mixing with the renewable and biodegradable PHBV polymer through a green approach (solvent-less process). The composites resulted thermally stable at high temperatures, showing initial degradation processes only at temperatures higher than 250°C. Differential scanning calorimetry analyses were carried out to study melting and crystallization phenomena, while mechanical properties were investigated by tensile tests. The materials finally showed properties similar to those of the matrix. The bio-composites can be considered as an alternative to plain PHBV, since they are less expensive and eco-friendlier thanks to a reduced polymeric content, and they could represent a suitable way for full agro-waste exploitation.

Microbial Fermentation of Industrial Rice-Starch Byproduct as Valuable Source of Peptide Fractions with Health-Related Activity.

The rice-starch processing industry produces large amounts of a protein-rich byproducts during the conversion of broken rice to powder and crystal starch. Given the poor protein solubility, this material is currently discarded or used as animal feed. To fully exploit rice's nutritional properties and reduce this waste, a biotechnological approach was adopted, inducing fermentation with selected microorganisms capable of converting the substrate into peptide fractions with health-related bioactivity. Lactic acid bacteria were preferred to other microorganisms for their safety, efficient proteolytic system, and adaptability to different environments. Peptide fractions with different molecular weight ranges were recovered from the fermented substrate by means of cross-flow membrane filtration. The fractions displayed in vitro antioxidant, antihypertensive, and anti-tyrosinase activities as well as cell-based anti-inflammatory and anti-aging effects. In the future, the peptide fractions isolated from this rice byproduct could be directly exploited as health-promoting functional foods, dietary supplements, and pharmaceutical preparations. The suggested biotechnological process harnessing microbial bioconversion may represent a potential solution for many different protein-containing substrates currently treated as byproducts (or worse, waste) by the food industry.

Prêt-à-porter nanoYESα and nanoYESß bioluminescent cell biosensors for ultrarapid and sensitive screening of endocrine-disrupting chemicals.

Cell-based assays utilizing reporter gene technology have been widely exploited for biosensing, as they provide useful information about the bioavailability and cell toxicity of target analytes. The long assay time due to gene transcription and translation is one of the main drawbacks of cell biosensors. We report the development of two yeast biosensors stably expressing human estrogen receptors α and ß and employing NanoLuc as the reporter protein to upgrade the widely used yeast estrogen screening (YES) assays. A viability control strain was also developed based on a chimeric green-emitting luciferase, PLG2, expressed for the first time in Saccharomycescerevisiae. Thanks to their brightness, NanoLuc and PLG2 provided excellent sensitivity, enabling the implementation of these biosensors into low-cost smartphone-based devices. The developed biosensors had a rapid (1 h) response and reported on (anti)estrogenic activity via human estrogen receptors α and ß as well as general sample toxicity. Under optimized conditions, we obtained LODs of 7.1 ± 0.4 nM and 0.38 ± 0.08 nM for E2 with nanoYESα and nanoYESß, respectively. As a proof of concept, we analyzed real samples from plants showing significant estrogenic activity or known to contain significant amounts of phytoestrogens. Graphical abstract.

Ethnobotanical survey of wild food plants traditionally collected and consumed in the Middle Agri Valley (Basilicata region, southern Italy).

BACKGROUND: This research was carried out in a scarcely populated area of the Middle Agri Valley (Basilicata region, southern Italy). The aim of the study was to record local knowledge on the traditional uses of wild food plants, as well as to collect information regarding the practices (gathering, processing and cooking) and the medicinal uses related to these plants. METHODS: Fifty-eight people still possessing traditional local knowledge (TLK), 74% women and 26% men, were interviewed between May-August 2012 and January 2013, using open and semi-structured ethnobotanical interviews. For each described plant species, the botanical family, the Italian common and folk names, the plant parts used, the culinary preparation and, when present, the medicinal use, were recorded and the relative frequency of citation index (RFC) was determined. RESULTS: The 52 plant species mentioned by the respondents belong to 23 botanical families, with Asteraceae (12 plants) and Rosaceae (7 plants) being most frequently cited. The species with the highest RFC index is Cichorium intybus L. (0.95), followed by Sonchus spp. (S. oleraceus L., S. asper L. and S. arvensis L.) (0.76). The plant parts preferably used are leaves (22 plants), fruits (12) and stems (7). Only six wild plants were indicated as having both food use and therapeutic effect. CONCLUSIONS: The survey conducted on the traditional use of wild food plants in the Middle Agri Valley revealed that this cultural heritage is only partially retained by the population. Over the last few decades, this knowledge has been in fact quickly disappearing along with the people and, even in the rural context of the study area, is less and less handed down to younger generations. Nevertheless, data also revealed that the use of wild plants is recently being revaluated in a way closely related to local habits and traditions.

White grape pomace extracts, obtained by a sequential enzymatic plus ethanol-based extraction, exert antioxidant, anti-tyrosinase and anti-inflammatory activities.

The present work aimed at optimizing a two-step enzymatic plus solvent-based process for the recovery of bioactive compounds from white grape (Vitis vinifera L., mix of Trebbiano and Verdicchio cultivars) pomace, the winemaking primary by-product. Phenolic compounds solubilised by water enzyme-assisted and ethanol-based extractions of wet (WP) and dried (DP) pomace were characterised for composition and tested for antioxidant, anti-tyrosinase and anti-inflammatory bioactivities. Ethanol treatment led to higher phenol yields than water extraction, while DP samples showed the highest capacity of releasing polyphenols, most probably as a positive consequence of the pomace drying process. Different compositions and bioactivities were observed between water and ethanol extracts and among different treatments and for the first time the anti-tyrosinase activity of V. vinifera pomace extracts, was here reported. Enzymatic treatments did not significantly improve the total amount of solubilised compounds; Celluclast in DP led to the recovery of extracts enriched in specific compounds, when compared to control. The best extracts (enzymatic plus ethanol treatment total levels) were obtained from DP showing significantly higher amounts of polyphenols, flavonoids, flavanols and tannins and exerted higher antioxidant and anti-tyrosinase activities than WP total extracts. Conversely, anti-inflammatory capacity was only detected in water (with and without enzyme) extracts, with WP samples showing on average a higher activity than DP. The present findings demonstrate that white grape pomace constitute a sustainable source for the extraction of phytochemicals that might be exploited as functional ingredients in the food, nutraceutical, pharmaceutical or cosmetic industries.

Traditional knowledge on wild and cultivated plants in the Kilombero Valley (Morogoro Region, Tanzania).

BACKGROUND: This research was performed in four villages adjacent the boundary of Udzungwa Mountains National Park in the Kilombero River plain of Tanzania. The area adjacent the villages is characterized by self-consumption agriculture, with a population that is on average poor, still very tied to traditions and almost entirely unaffected by modernization and technology. The aim of the present study was to investigate and record local knowledge regarding the use of wild and traditionally cultivated plants used for traditional medicine and for other everyday purposes (e.g., food, fibers and timber). METHODS: Ten traditional local healers, with solid botanical knowledge, were interviewed between June and August 2014 by means of semi-structured questionnaires. For each mentioned plant species, the Swahili folk name and, when possible, the classification by family, genus and species was recorded as well as the part of the plant used, the preparation method and the main uses (medicine, food or others). RESULTS: In total 196 species were mentioned of which 118 could be botanically classified. The identified species belong to 44 different botanical families, with that of the Leguminosae being the most representative (24 species). The plants were mostly used as medical treatments (33.3% of the species) and foods (36.8%), and to produce wood and fibers (19.4%). CONCLUSION: The present study revealed that numerous plant species are still essential in the everyday life of the tribes living in Kilombero Valley. Most of the plants were usually harvested in the wild, however, after the creation of the Udzungwa Mountains National Park, the harvesting pressure has become concentrated on a few unprotected forest patches. Consequently, many useful species are becoming increasingly rare with the risk of losing the connected botanical and traditional knowledge. The present study may, therefore, contribute to record the ethnobotanical knowledge held by these populations, in order to preserve this valuable richness for future generations.

Exploiting NanoLuc luciferase for smartphone-based bioluminescence cell biosensor for (anti)-inflammatory activity and toxicity.

The availability of smartphones with high-performance digital image sensors and processing power has completely reshaped the landscape of point-of-need analysis. Thanks to the high maturity level of reporter gene technology and the availability of several bioluminescent proteins with improved features, we were able to develop a bioluminescence smartphone-based biosensing platform exploiting the highly sensitive NanoLuc luciferase as reporter. A 3D-printed smartphone-integrated cell biosensor based on genetically engineered Hek293T cells was developed. Quantitative assessment of (anti)-inflammatory activity and toxicity of liquid samples was performed with a simple and rapid add-and-measure procedure. White grape pomace extracts, known to contain several bioactive compounds, were analyzed, confirming the suitability of the smartphone biosensing platform for analysis of untreated complex biological matrices. Such approach could meet the needs of small medium enterprises lacking fully equipped laboratories for first-level safety tests and rapid screening of new bioactive products. Graphical abstract Smartphone-based bioluminescence cell biosensor.

Recovery of polyphenols from red grape pomace and assessment of their antioxidant and anti-cholesterol activities.

The present work aimed at the recovery and characterization of polyphenolic compounds extracted from red grape pomace (Vitis vinifera L.), a winemaking by-product. Polyphenolic compounds of wet (WP) and dried (DP) red pomace were recovered by enzymatic digestions and ethanol-based extractions. Fungamyl and Celluclast enzymes were found to be the most effective in enhancing polyphenol release from WP. WP samples showed the highest capacity of releasing polyphenols with 2h control 24°C and 2h 1% Celluclast resulting as the best treatments. A significantly lower amount of polyphenols was recovered from DP most probably as a consequence of the pomace drying. The best extracts contained high amounts of total polyphenols, flavonoids, tannins and anthocyanins and exerted antioxidant and cholesterol-lowering activities. The results support the possibility of exploiting the extracts coming from grape processing by-products as ingredients for functional and innovative products in the nutraceutical, pharmaceutical or cosmetic fields.

Wild food plants traditionally consumed in the area of Bologna (Emilia Romagna region, Italy).

BACKGROUND: This research was performed in an area belonging to the province of the city of Bologna (Emilia-Romagna region, Northern Italy). The purpose of the present survey was to record the local knowledge concerning traditional uses of wild food plants and related practices, such as gathering, processing, cooking, therapeutic uses, with the aim of preserving an important part of the local cultural heritage. METHODS: Thirty-nine people still retaining Traditional Local Knowledge (TLK) were interviewed between March-April 2012 and September-October 2013 by means of open and semi-structured ethnobotanical interviews. For each plant species mentioned, we recorded the botanical family, the English common name, the Italian common and/or folk names, the parts of the plant used, the culinary preparation, and the medicinal usage. The relative frequency of citation index (RFC), a tool that measures the local cultural importance of a plant species, was also included. RESULTS: The folk plants mentioned by the respondents belonged to 33 botanical families, of which the Rosaceae (14 plants) and the Asteraceae (9 plants) were the most representative. The species with the highest RFC index (0.77) were Crepis vesicaria subsp. taraxacifolia (Thuill) Thell and Taraxacum officinale Weber. Eleven folk plants were indicated as having therapeutic effects. T. officinale Weber, C. vesicaria subsp. taraxacifolia (Thuill) Thell and Sonchus spp., which are used as food, were reported to be depurative, blood cleaning, refreshing, diuretic and laxative. The most commonly used species was Urtica spp, which was also the most frequently cited for medicinal uses. CONCLUSIONS: The present survey documented the wild food plant traditional knowledge of an area belonging to the province of the city of Bologna (Emilia-Romagna region, Northern Italy). The general perception obtained is that on one side the TLK related to wild food plants has strongly been eroded, mainly due to immigration and urbanization phenomena, whereas on the other side these plants are revaluated today because they are perceived as healthy and also because they represent the preservation of biodiversity and a way of getting back to nature.

Polyamines and salt stress response and tolerance in Arabidopsis thaliana flowers.

In the present study we analysed polyamine metabolism in Arabidopsis thaliana (ecotype Columbia) flowers and stalks collected from plants germinated and grown under increasing salt-stress conditions (0-75 mM NaCl). The expression level of the different isoforms of polyamine biosynthetic enzymes was analysed by reverse transcriptase-polymerase chain reaction (RT-PCR). Spermidine synthase enzyme activity determined both in supernatant and pellet fractions, together with RT-PCR results, led us to hypothesize a different intracellular compartmentation of the isoforms of these enzymes. Free and conjugated polyamines (perchloric acid-soluble and -insoluble) were measured. Free spermidine was the most abundant polyamine and its levels, such as those of free spermine, increased with salt concentration, supporting the hypothesis for a specific role of those polyamines in the response and tolerance to salt stress of Arabidopsis thaliana flowers.