'Sorrento' and 'Tulare' Walnut Cultivars: Morphological Traits and Phytochemical Enhancement of Their Shell Waste.

Walnut processing generates considerable quantities of by-products that could be reprocessed into value-added products that have food and non-food applications. In this context, the aim of this study is to characterize the 'Sorrento' and 'Tulare' walnut cultivars using the UPOV guidelines and analyze the chemical composition and antioxidant activity of their shells. Insight into the chemical composition of the different granulometric fractions of walnut shell, obtained by sieving, was obtained following ultrasound-assisted extraction by Ultra-High-Performance Liquid Chromatography-High-Resolution Mass Spectrometry (UHPLC-HRMS). The total phenolic, flavonoid, and tannin content and antiradical capacity, obtained by DPPH and ABTS assays, and the Fe(III) reducing power of the extracts were also evaluated. The UHPLC-HRMS analysis indicated the presence of thirty-two compounds ascribable to four major classes of specialized metabolites. Furthermore, the extraction efficiency of gallic acid, ellagic acid derivatives, as well as glansreginin A, increased with the decrease in shell matrix particle size in contrast to chlorogenic acids and flavonoid glycosides. This is the first study to highlight new knowledge on the chemical composition of walnut shells. The results obtained demonstrate the feasibility of recovering valuable bioactive components from agro-waste that may be further valorized.

Galdieria sulphuraria: An Extremophilic Alga as a Source of Antiviral Bioactive Compounds.

In the last decades, the interest in bioactive compounds derived from natural sources including bacteria, fungi, plants, and algae has significantly increased. It is well-known that aquatic or terrestrial organisms can produce, in special conditions, secondary metabolites with a wide range of biological properties, such as anticancer, antioxidant, anti-inflammatory, and antimicrobial activities. In this study, we focused on the extremophilic microalga Galdieria sulphuraria as a possible producer of bioactive compounds with antiviral activity. The algal culture was subjected to organic extraction with acetone. The cytotoxicity effect of the extract was evaluated by the 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The antiviral activity was assessed through a plaque assay against herpesviruses and coronaviruses as enveloped viruses and poliovirus as a naked one. The monolayer was treated with different concentrations of extract, ranging from 1 µg/mL to 200 µg/mL, and infected with viruses. The algal extract displayed strong antiviral activity at non-toxic concentrations against all tested enveloped viruses, in particular in the virus pre-treatment against HSV-2 and HCoV-229E, with IC50 values of 1.7 µg/mL and IC90 of 1.8 µg/mL, respectively. However, no activity against the non-enveloped poliovirus has been detected. The inhibitory effect of the algal extract was confirmed by the quantitative RT-PCR of viral genes. Preliminary chemical profiling of the extract was performed using ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS), revealing the enrichment in primary fatty acid amides (PFAA), such as oleamide, palmitamide, and pheophorbide A. These promising results pave the way for the further purification of the mixture to explore its potential role as an antiviral agent.

Lavandula austroapennina (Lamiaceae): Getting Insights into Bioactive Polyphenols of a Rare Italian Endemic Vascular Plant.

Lavandula austroapennina N.G. Passal., Tundis and Upon has recently been described as a new species endemic to the southern Apennines (Italy). Locally, this species has a long ethnobotanical tradition of use for curative and decoration purposes and has been the protagonist of a flourishing essential oil production chain. Currently, while this tradition has long since ended, attention to the species is necessary, with a view to enhancing marginal and rural areas, as a recovery of a precious resource to (i) get insights into its (poly)phenolic fraction and (ii) address new and innovative uses of all its organs in various application fields (e.g., cosmeceutical sector). Therefore, after field sampling and dissection of its organs (i.e., corolla, calyx, leaf, stem and root), the latter, previously deterpenated and defatted, were subjected to accelerated ultrasound extraction and the related alcoholic extracts were obtained. Chemical composition, explored by UHPLC-QqTOF-MS/MS, and the following multivariate data analysis showed that the hydroxycinnamoyl derivatives are abundant in the leaf, stem and root, while flavonoids are more present in corolla and calyx. In particular, coumaroyl flavonoids with glyconic portion containing also hexuronyl moieties differentiated corolla organ, while yunnaneic acid D isomers and esculin distinguished root. When antiradical and reducing properties were evaluated (by means of ABTS, DPPH and PFRAP tests), a similar clustering of organs was achieved and the marked antioxidant efficacy of leaf, stem and root extracts was found. Thus, following cytotoxicity screening by MTT test on HaCaT keratinocytes, the protective effects of the organ extracts were assessed by wound closure observed after the scratch test. In addition, the extracts from corolla, leaf and stem were particularly active at low doses inducing rapid wound closure on HaCaT cells at a concentration of 1 µg/mL. The diversity in (poly)phenols of each organ and the promising bioactivity preliminarily assessed suggest further investigation to be carried out to fully recover and valorize this precious endemic vascular plant.

Polyphenols for Livestock Feed: Sustainable Perspectives for Animal Husbandry?

There is growing interest in specialized metabolites for fortification strategies in feed and/or as an antioxidant, anti-inflammatory and antimicrobial alternative for the containment of disorders/pathologies that can also badly impact human nutrition. In this context, the improvement of the diet of ruminant species with polyphenols and the influence of these compounds on animal performance, biohydrogenation processes, methanogenesis, and quality and quantity of milk have been extensively investigated through in vitro and in vivo studies. Often conflicting results emerge from a review of the literature of recent years. However, the data suggest pursuing a deepening of the role of phenols and polyphenols in ruminant feeding, paying greater attention to the chemistry of the single compound or to that of the mixture of compounds more commonly used for investigative purposes.

Encapsulating Calendula arvensis (Vaill.) L. Florets: UHPLC-HRMS Insights into Bioactive Compounds Preservation and Oral Bioaccessibility.

Wild edible plants, once consumed in times of famine or for health purposes, today represent an interesting dietary supplement, aimed at enriching local dishes and/or formulating healthy nutraceutical products. In fact, the broad content of different, and diversely bioactive, specialized metabolites therein suggests new scenarios of use which, in order to be as functional as possible, must maximize the bioactivity of these compounds while preserving their chemistry. In this context, based on a recent investigation on the metabolic profile of the organs of Calendula arvensis that highlighted that florets are abundant in flavonol glycosides and triterpene saponins, the freeze-drying encapsulation of their alcoholic extract (FE) into maltodextrin (MD) was investigated. FE-MD chemical composition was evaluated using Fourier Transform InfraRed spectroscopy (FTIR), while ultra-high performance liquid chromatography coupled with high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) techniques were employed to unravel FE compound preservation also during in vitro simulated digestion. The establishment of H-bonds between FE compounds and MD hydroxyl groups was in line with FE-MD biocompatibility in Caco-2 cells, while in vitro digestion mostly affected structural integrity and/or diversity. Flavonol compounds underwent deglycosylation and demethylation, while deacylation, beyond oxidation, involved triterpene saponins, which massively preserve their aglycone core.

UHPLC-ESI-QqTOF Analysis and In Vitro Rumen Fermentation for Exploiting Fagus sylvatica Leaf in Ruminant Diet.

In recent years, animal husbandry has aimed at improving the conditions of livestock animals useful for humans to solve environmental and health problems. The formulation of animal feeds or supplements based on antioxidant plant compounds is considered a valuable approach and an alternative for livestock productivity. Forest biomass materials are an underestimated source of polyphenolic compounds whose sustainable recovery could provide direct benefits to animals and, indirectly, human nutrition. In this context, an alcohol extract from leaves of Fagus sylvatica L. was first investigated through an untargeted ultra-high-performance liquid chromatography-high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) approach. Then, it was fractionated into a fatty acid-rich and a polyphenolic fraction, as evidenced by total lipid, phenol, and flavonoid content assays, with antiradical and reducing activity positively correlated to the latter. When tested in vitro with rumen liquor to evaluate changes in the fermentative parameters, a significant detrimental effect was exerted by the lipid-rich fraction, whereas the flavonoid-rich one positively modulated the production of volatile fatty acids (i.e., acetate, butyrate, propionate, etc.).

Castanea sativa Mill. Leaf: UHPLC-HR MS/MS Analysis and Effects on In Vitro Rumen Fermentation and Methanogenesis.

Castanea sativa Mill. (Fagaceae) is a deciduous tree grown for its wood and edible fruits. Chestnut processing produces residues (burs, shells, and leaves) exploitable for their diversity in bioactive compounds in animal nutrition. In fact, plant-specialized metabolites likely act as rumen modifiers. Thus, the recovery of residual plant parts as feed ingredients is an evaluable strategy. In this context, European chestnut leaves from northern Germany have been investigated, proving to be a good source of flavonoids as well as gallo- and ellagitannins. To this purpose, an alcoholic extract was obtained and an untargeted profiling carried out, mainly by means of ultra-high-performance liquid chromatography/high-resolution tandem mass spectrometry (UHPLC-HR MS/MS) techniques. To better unravel the polyphenol constituents, fractionation strategies were employed to obtain a lipophilic fraction and a polar one. This latter was highly responsive to total phenolic and flavonoid content analyses, as well as to antiradical (DPPH● and ABTS+●) and reducing activity (PFRAP) assays. The effect of the alcoholic extract and its fractions on rumen liquor was also evaluated in vitro in terms of fermentative parameter changes and impact on methanogenesis. The data acquired confirm that chestnut leaf extract and the fractions therefrom promote an increase in total volatile fatty acids, while decreasing acetate/propionate ratio and CH4 production.

LC-HR/MS Analysis of Lipophilic Extracts from Calendula arvensis (Vaill.) L. Organs: An Unexplored Source in Cosmeceuticals.

As part of a project aimed at promoting the use of Calendula arvensis (Vaill.) L. (field marigold, Asteraceae) phytocomplexes in cosmeceutical formulations, the chemical composition in apolar specialized metabolites is herein elucidated. Furthermore, the screening of the cytotoxicity of the apolar extracts was evaluated in order to underline their safety as functional ingredients for cosmetics. After dissection of Calendula organs (florets, fruits, leaves, bracts, stems, and roots), ultrasound-assisted maceration in n-hexane as an extracting solvent allowed us to obtain oil-like mixtures, whose chemical composition has been highlighted through a UHPLC-ESI-QqTOF-MS/MS approach. Twenty-nine metabolites were tentatively identified; different compounds, among which the well-known poly-unsaturated fatty acids, and oxylipins and phosphatides were detected for the first time in Calendula genus. The screening of the dose-response cytotoxicity of the apolar extracts of C. arvensis highlighted the concentration of 10 µg/mL as the most suitable for the formulation of cosmeceutical preparations. Sera enriched with leaf and fruit apolar extracts turned out to have the best activity, suggesting it can be used as a new source in skin care thanks to their higher content in fatty acids.

A Joint Approach of Morphological and UHPLC-HRMS Analyses to Throw Light on the Autochthonous 'Verdole' Chestnut for Nutraceutical Innovation of Its Waste.

Nowadays, chestnut by-products are gaining a lot of interest as a low-cost raw material, exploitable for developing added-value products. This is in line with suitable chestnut by-products' management, aimed at reducing the environmental impact, thus improving the chestnut industry's competitiveness and economic sustainability. In this context, with the aim of valorizing local cultivars of European chestnuts (Castanea sativa Mill.), our attention focused on the Verdole cultivar, which has been characterized by using the UPOV guidelines for its distinctness, homogeneity, and stability. After harvesting, Verdole chestnuts were properly dissected to collect the outer and inner shells, and episperm. Each chestnut part, previously crushed, shredded, and passed through diverse sieves, underwent ultrasound-assisted extraction. The extracts obtained were evaluated for their total phenolic, flavonoid, and tannin content. The antiradical capacity by DPPH and ABTS assays, and the Fe(III) reducing power, were also evaluated. Although all the samples showed dose-dependent antioxidant efficacy, plant matrix size strongly impacted on extraction efficiency. LC-HRMS-based metabolic profiling highlighted the occurrence of different polyphenol subclasses, whose quantitative ratio varied among the chestnut parts investigated. The outer shell was more chemically rich than inner shell and episperm, according to its pronounced antioxidant activity. The polyphenol diversity of Verdole by-products is a resource not intended for disposal, appliable in the nutraceutical sector, thus realizing a new scenario in processing chestnut waste.

Cannabidiolic acid in Hemp Seed Oil Table Spoon and Beyond.

Cannabidiolic acid (CBDA) is the main precannabinoid in industrial hemp. It represents a common constituent of hemp seed oil, but mainly abundant in the aerial parts of the plant (including their processing waste). Thus, the optimization of fast and low-cost purification strategies is mandatory, as well as a deep investigation on its nutraceutical and cosmeceutical properties. To this purpose, CBDA content in hemp seed oil is evaluated, and its recovery from wasted leaves is favorably achieved. The cytotoxicity screening towards HaCaT cells, by means of MTT, SRB and LDH release assays, suggested it was not able to decrease cell viability or perturb cell integrity up to 10 µM concentration. Thus, the ability of CBDA to differentially modulate the release of proinflammatory cytokines and chemokines mediators has been evaluated, finding that CBDA decreased IFN-γ, CXCL8, CXCL10, CCL2, CCL4 and CCL5, mostly in a dose-dependent manner, with 10 µM tested concentration exerting the highest activity. These data, together with those from assessing antimicrobial activity against Gram(+) and Gram(-) bacteria and the antibiofilm formation, suggest that CBDA is able to counteract the inflammatory response, also preventing bacteria colonization.

Editorial to the Special Issue "Food Bioactives: Chemical Challenges and Bio-Opportunities".

This Special Issue, entitled "Food Bioactives: Chemical Challenges and Bio-Opportunities", was born with the aim of attracting contributions on analytical challenges in food bioactives' chemistry and bioactivity, which form the basis of proper bio-opportunities [...].

Could Polyphenols Really Be a Good Radioprotective Strategy?

Currently, radiotherapy is one of the most effective strategies to treat cancer. However, deleterious toxicity against normal cells indicate for the need to selectively protect them. Reactive oxygen and nitrogen species reinforce ionizing radiation cytotoxicity, and compounds able to scavenge these species or enhance antioxidant enzymes (e.g., superoxide dismutase, catalase, and glutathione peroxidase) should be properly investigated. Antioxidant plant-derived compounds, such as phenols and polyphenols, could represent a valuable alternative to synthetic compounds to be used as radio-protective agents. In fact, their dose-dependent antioxidant/pro-oxidant efficacy could provide a high degree of protection to normal tissues, with little or no protection to tumor cells. The present review provides an update of the current scientific knowledge of polyphenols in pure forms or in plant extracts with good evidence concerning their possible radiomodulating action. Indeed, with few exceptions, to date, the fragmentary data available mostly derive from in vitro studies, which do not find comfort in preclinical and/or clinical studies. On the contrary, when preclinical studies are reported, especially regarding the bioactivity of a plant extract, its chemical composition is not taken into account, avoiding any standardization and compromising data reproducibility.

Antiproliferative and antioxidant effect of polar hemp extracts (Cannabis sativa L., Fedora cv.) in human colorectal cell lines.

Total phenolic content and antioxidant activity of polar extracts of edible resources from Fedora hemp cultivar (Cannabis sativa L.), namely seed, flour and oil, were evaluated. The main components in the polar extracts were identified using HPLC-DAD and HPLC-ESI-MS/MS. As expected, the molecular profile of components from seeds and flour was strictly similar, dominated by N-trans-caffeoyltyramine. The profile of oil polar extracts contained hydroxycinnamic acid derivatives and cannabinoids at lower extent. While the extracts from hemp seed and flour did not interfere with growth of Caco-2 and HT-29 cell, the one from oil (150 µg/mL) significantly reduced cell viability after 24 h of treatment. This effect was associated with the activation of apoptotic cell death and was independent from the antioxidant capacity of the oil polar extract. Notably, HT-29 cells differentiated with sodium butyrate were not sensitive to the cytotoxic effect of the oil extract.

Coumaroyl Flavonol Glycosides and More in Marketed Green Teas: An Intrinsic Value beyond Much-Lauded Catechins.

Marketed green teas (GTs) can highly vary in their chemical composition, due to different origins, processing methods, and a lack of standardization of GT-based products. Consequently, biological activities become difficult to correlate to the presence/content of certain constituents. Herein, ultra-high-performance liquid chromatography (UHPLC) combined with high-resolution tandem mass spectrometry (HR MS/MS) was successfully applied to six commercial GT products, extracted by ethanol sonication, to disclose their polyphenol profile beyond the well-known catechins. The relative abundance of each class of metabolites was correlated to antiradical and antilipoperoxidant data through hierarchical clustering analysis, since it reasonably affects the beneficial properties of the product that reaches the consumer. The thiobarbituric acid reactive substances (TBARS) assay demonstrated that GT extracts effectively counteracted the UV-induced lipoperoxidation of hemp oil, which is highly rich in Polyunsaturated Fatty Acids (PUFAs), and therefore highly unstable. The Relative Antioxidant Capacity Index (RACI) comprehensively emphasized that gunpower and blend in filter GTs appeared to be the less active matrices, and except for a GT-based supplement, the Sencha GT, which was particularly rich in flavonol glycosides, was the most active, followed by Bancha GT.

Polyphenols vs. Coronaviruses: How Far Has Research Moved Forward?

The epidemic, caused by SARS-CoV-2 at the beginning of 2020, led us to a serious change in our lifestyle that for about three months has confined us to our homes, far from our laboratory routine. In this period, the belief that the work of a researcher should never stop has been the driving force in writing the present paper. It aims at reviewing the recent scientific knowledge about in vitro experimental data that focused on the antiviral role of phenols and polyphenols against different species of coronaviruses (CoVs), pointing up the viral targets potentially involved. In the current literature scenario, the papain-like and the 3-chymotrypsin-like proteases seem to be the most deeply investigated and a number of isolated natural (poly)phenols has been screened for their efficacy.

Chemical Analysis of Minor Bioactive Components and Cannabidiolic Acid in Commercial Hemp Seed Oil.

Although hemp seed (HS) oil is characterized by more than 80% polyunsaturated fatty acids (PUFAs), a very high ω-6-to-ω-3 ratio is not a popular commodity. The aim of this work was to provide useful data about the bioactive components and cannabidiolic acid content in thirteen different commercial hemp seed oils. The investigated HS oils showed a good ω-6/ω-3 ratio, ranging from 1.71 to 2.27, massively differed in their chlorophylls (0.041-2.64 µg/g) and carotenoids contents (0.29-1.73 µg/g), as well as in total phenols (22.1-160.8 mg Gallic Acid Equivalents (GAE)/g) and tocopherols (3.47-13.25 mg/100 g). Since the high content of PUFAs in HS oils, photo-oxidative stability was investigated by determining the Thiobarbituric Acid Reactive Substances (TBARS) assay and extinction coefficient K232 and K270 after the photo-oxidative test. The percentage of increase in K232 and K270 ranged from 1.2 to 8.5% and from 3.7 to 26.0%, respectively, indicating good oxidative stability, but TBARS showed a 1.5- to 2.5-fold increase in oxidative behavior when compared to the initial values. Therefore, the diversity in bioactive compounds in HS oils, and their high nutritional value, suggest the need for a disciplinary booklet that well defines agronomic and post-harvest management conditions for achieving a good food objective.

Hempseed Lignanamides Rich-Fraction: Chemical Investigation and Cytotoxicity towards U-87 Glioblastoma Cells.

The weak but noteworthy presence of (poly)phenols in hemp seeds has been long overshadowed by the essential polyunsaturated fatty acids and digestible proteins, considered responsible for their high nutritional benefits. Instead, lignanamides and their biosynthetic precursors, phenylamides, seem to display interesting and diverse biological activities only partially clarified in the last decades. Herein, negative mode HR-MS/MS techniques were applied to the chemical investigation of a (poly)phenol-rich fraction, obtained from hemp seeds after extraction/fractionation steps. This extract contained phenylpropanoid amides and their random oxidative coupling derivatives, lignanamides, which were the most abundant compounds and showed a high chemical diversity, deeply unraveled through high resolution tandem mass spectrometry (HR-MS/MS) tools. The effect of different doses of the lignanamides-rich extract (LnHS) on U-87 glioblastoma cell line and non-tumorigenic human fibroblasts was evaluated. Thus, cell proliferation, genomic DNA damage, colony forming and wound repair capabilities were assessed, as well as LnHS outcome on the expression levels of pro-inflammatory cytokines. LnHS significantly inhibited U-87 cancer cell proliferation, but not that of fibroblasts, and was able to reduce U-87 cell migration, inducing further DNA damage. No modification in cytokines' expression level was found. Data acquired suggested that LnHS acted in U-87 cells by inducing the apoptosis machinery and suppressing the autophagic cell death.

(‒)-Cannabidiolic Acid, a Still Overlooked Bioactive Compound: An Introductory Review and Preliminary Research.

Cannabidiolic acid (CBDA) is the main phytocannabinoid in fiber and seed-oil hemp (Cannabis sativa L.) plants, but its potential health-related capabilities have been masked for years by a greater scientific interest towards its neutral derivative cannabidiol (CBD). This review aims to collect from the literature and critically discuss all the information about this molecule, starting from its biosynthesis, and focusing on its bioactivity, as an anti-inflammatory, anti-emetic, anti-convulsant, and anti-cancerogenic drug. Furthermore, in the awareness that, despite its multiple bioactive effects, currently poor efforts have been made to achieve its reliable purification, herein, we propose a relatively simple, fast, and inexpensive procedure for its recovery from pollen of industrial hemp cultivars. Spectroscopic and spectrometric techniques allowed us to unequivocally identify pure isolated CBDA and to distinguish it from the constitutional isomer tetrahydrocannabinolic acid (THCA-A).

UHPLC-HR-MS/MS-Guided Recovery of Bioactive Flavonol Compounds from Greco di Tufo Vine Leaves.

Leaves of Vitis vinifera cv. Greco di Tufo, a precious waste made in the Campania Region (Italy), after vintage harvest, underwent reduction, lyophilization, and ultrasound-assisted maceration in ethanol. The alcoholic extract, as evidenced by a preliminary UHPLC-HR-MS analysis, showed a high metabolic complexity. Thus, the extract was fractionated, obtaining, among others, a fraction enriched in flavonol glycosides and glycuronides. Myricetin, quercetin, kaempferol, and isorhamnetin derivatives were tentatively identified based on their relative retention time and TOF-MS2 data. As the localization of saccharidic moiety in glycuronide compounds proved to be difficult due to the lack of well-established fragmentation pattern and/or the absence of characteristic key fragments, to obtain useful MS information and to eliminate matrix effect redundancies, the isolation of the most abundant extract's compound was achieved. HR-MS/MS spectra of the compound, quercetin-3-O-glucuronide, allowed us to thoroughly rationalize its fragmentation pattern, and to unravel the main differences between MS/MS behavior of flavonol glycosides and glycuronides. Furthermore, cytotoxicity assessment on the (poly)phenol rich fraction and the pure isolated compound was carried out using central nervous system cell lines. The chemoprotective effect of both the (poly)phenol fraction and quercetin-3-O-glucuronide was evaluated.

Recovering Cucurbita pepo cv. 'Lungo Fiorentino' Wastes: UHPLC-HRMS/MS Metabolic Profile, the Basis for Establishing Their Nutra- and Cosmeceutical Valorisation.

Food-waste is produced throughout all the food supply chain, with a large part already achieved at farm level. In fact, fruits and vegetables, which do not satisfy aesthetic demands, cannot be marketed, but their recovery could favour their valorisation for the obtainment of highly qualified goods. In this context, faulty zucchini fruits (cultivar 'Lungo Fiorentino'), intended for disposal, were rescued as effective, inexpensive and bio-sustainable source for cosmeceutical purposes. Zucchini fruits underwent extraction and fractionation to obtain ZLF-O and ZLF-A extracts, which were chemically characterized by UHPLC-HRMS. ZLF-A extract, rich in flavonols and flavones, scavenged massively DPPH• and ABTS•+, and was not cytotoxic at doses up to 200 µ g/mL. Thus, ZLF-A was incorporated into a base cream formula. Zucchini-based emulsion was deeply screened for its antiradical properties and cytotoxicity towards human keratinocytes and fibroblasts. ZLF-A-enriched cream, whose chemical stability was assessed over time and mimicking different storage conditions, was further tested on reconstructed epidermis disks (EpiskinTM). The recovery of valuable chemical substances from zucchini agro-food waste, complying with the principles of valorisation and sustainable development, can represent a new market force for local farmers. Data acquired were eager to convey a suitable reuse of nutraceuticals rich zucchini waste.