Carotenoid-Enriched Nanoemulsions and γ-Rays Synergistically Induce Cell Death in a Novel Radioresistant Osteosarcoma Cell Line.

We previously demonstrated that SAOS human osteosarcoma cells, incubated with carotenoid-enriched nanoemulsions (CEN), activated a nonprotective form of autophagy and delayed cell proliferation. The present work focuses on the biological effects of CEN on a derivative of SAOS cells named SAOS400, recently described for their radiation resistance and higher expression of therapy-induced senescence (TIS) markers. SAOS400 cells, incubated with CEN, activated a "cytostatic" form of autophagy confirmed by cell cycle arrest in the G2/M phase and increased expression of autophagic proteins. Treatment of SAOS400 cells with CEN also resulted in decreased expression of the senescence marker p16INK4. However, when SAOS400 cells were γ-irradiated in combination with CEN, the threshold for cell death was reached (>60% after 96 h). We showed that this type of cell death corresponded to 'cytotoxic' or 'lethal' autophagy and that the combined treatment of CEN plus γ-rays was synergistic, with the combination index < 1. Since CEN contained ß-carotene, the pure compound was used in SAOS400 cells at the same concentration present in CEN and up to 10 times higher. However, no radio-sensitizing effect of ß-carotene was observed, suggesting that the biological effect of CEN was due to less abundant but more bioactive molecules, or to the synergistic activity of multiple components present in the extracts, confirming the functional pleiotropy of natural extracts enriched in bioactive molecules.

Cannabis Bioactive Compound-Based Formulations: New Perspectives for the Management of Orofacial Pain.

The management of orofacial pain to alleviate the quality of life of affected patients is becoming increasingly challenging for scientific research and healthcare professionals. From this perspective, in addition to conventional therapies, new alternatives are being sought, increasingly looking at the use of both natural and synthetic products. Cannabis sativa L. represents an interesting source of bioactive compounds, including non-psychoactive cannabinoids, flavonoids, and terpenes, many of which are effective in improving pain intensity. Here, we aim to analyze the possible mechanisms of action of the bioactive natural and synthetic hemp-derived compounds responsible for the modulatory effects on pain-related pathways. The ability of these compounds to act on multiple mechanisms through a synergistic effect, reducing both the release of inflammatory mediators and regulating the response of the endocannabinoid system, makes them interesting agents for alternative formulations to be used in orofacial pain.

Biochemical and Cellular Characterization of New Radio-Resistant Cell Lines Reveals a Role of Natural Flavonoids to Bypass Senescence.

Cancer is one of the main causes of death worldwide, and, among the most frequent cancer types, osteosarcoma accounts for 56% of bone neoplasms observed in children and colorectal cancer for 10.2% of tumors diagnosed in the adult population. A common and frequent hurdle in cancer treatment is the emergence of resistance to chemo- and radiotherapy whose biological causes are largely unknown. In the present work, human osteosarcoma (SAOS) and colorectal adenocarcinoma (HT29) cell lines were γ-irradiated at doses mimicking the sub-lethal irradiation in clinical settings to obtain two radio-resistant cellular sub-populations named SAOS400 and HT500, respectively. Since "therapy-induced senescence" (TIS) is often associated with tumor response to radiotherapy in cancer cells, we measured specific cellular and biochemical markers of senescence in SAOS400 and HT500 cells. In detail, both cell lines were characterized by a higher level of expression of cyclin-dependent kinase inhibitors p16INK4 and p21CIP1 and increased positivity to SAß-gal (senescence-associated ß-galactosidase) with respect to parental cells. Moreover, the intracellular levels of reactive oxygen species in the resistant cells were significantly lower compared to the parental counterparts. Subsequently, we demonstrated that senolytic agents were able to sensitize SAOS400 and HT500 to cell death induced by γ-irradiation. Employing two natural flavonoids, fisetin and quercetin, and a BH3-mimetic, ABT-263/navitoclax, we observed that their association with γ-irradiation significantly reduced the expression of p16INK4, p21CIP1 and synergistically (combination index < 1) increased cell death compared to radiation mono-alone treatments. The present results reinforce the potential role of senolytics as adjuvant agents in cancer therapy.

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.

Phytochemical Characterization and Effects on Cell Proliferation of Lentisk (Pistacia lentiscus) Berry Oil: a Revalued Source of Phenolics.

The ethno-pharmaceutical use of the edible fixed oil produced from lentisk (Pistacia lentiscus) berries covers a long tradition in several Mediterranean regions. Many of the health-promoting properties of lentisk berry oil (LBO) have been associated with the content of polar (poly)phenolic compounds. However, the polar fraction (methanol 80%, v/v) of LBO (LBO-pf) remains poorly and inadequately characterized. We assessed the phytochemical composition (fatty acids, phytosterols and polyphenols) of cold-pressed LBO produced in Cilento (Campania region, Italy) over four years of production (2015-2018). Main phenolic compounds present in LBO-pf were identified and semi-quantified combining ultra-high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) and HPLC with diode array detection. Phenolic compounds, also responsible for oil stability and antioxidant properties, are relatively abundant in LBO, compared to other edible oils. LBO-pf induced clear dose-dependent effects on the growth of HT-29 cell line derived from human colorectal adenocarcinoma, as evidenced by the cell cycle arrest. Our data support the health-promoting properties of cold-pressed LBO, which is obtained with good yield from spontaneous plants growing in semiarid regions.

Comparative Study of Chemical, Biochemical Characteristic and ATR-FTIR Analysis of Seeds, Oil and Flour of the Edible Fedora Cultivar Hemp (Cannabis sativa L.).

A series of chemical and biochemical parameters of edible hemp resources (seeds, oil, and flour) from the monoecious EU registered hemp genotype Fedora, was determined, including fatty acid profile, phytosterol composition, total phenolics, antioxidant activity, macro- and micro-elements. The fatty acid ω-3/ω-6 approached the nutritionally optimal 3/1 ratio. ß-sitosterol and other phytosterols sterols dominated the unsaponifiable fraction. Hemp seeds, flour, and oil contained 767 ± 41, 744 ± 29, and 21 ± 5 mg GAE kg-1 total polyphenols, respectively. The antioxidant potential of Fedora flour and seeds, evaluated through the DPPH (2,2-Diphenyl-1-picrylhydrazyl) assay, was higher than that of oil. K and Mg were the most abundant macro-elements, particularly in flour, while the concentration of trace elements was Fe > Cu > Ni > Mn. The presence of an array of bioactive compound candidate Fedora products as health-promoting food matrices. The ATR-FTIR spectra of hemp-derived products indicated the proximate composition of macro-nutrients.

Potential anti-inflammatory effects of the hydrophilic fraction of pomegranate (Punica granatum L.) seed oil on breast cancer cell lines.

In this work, we characterized conjugated linolenic acids (e.g., punicic acid) as the major components of the hydrophilic fraction (80% aqueous methanol extract) from pomegranate (Punica granatum L.) seed oil (PSO) and evaluated their anti-inflammatory potential on some human colon (HT29 and HCT116), liver (HepG2 and Huh7), breast (MCF-7 and MDA-MB-231) and prostate (DU145) cancer lines. Our results demonstrated that punicic acid and its congeners induce a significant decrease of cell viability for two breast cell lines with a related increase of the cell cycle G0/G1 phase respect to untreated cells. Moreover, the evaluation of a great panel of cytokines expressed by MCF-7 and MDA-MB-231 cells showed that the levels of VEGF and nine pro-inflammatory cytokines (IL-2, IL-6, IL-12, IL-17, IP-10, MIP-1α, MIP-1ß, MCP-1 and TNF-α) decreased in a dose dependent way with increasing amounts of the hydrophilic extracts of PSO, supporting the evidence of an anti-inflammatory effect. Taken together, the data herein suggest a potential synergistic cytotoxic, anti-inflammatory and anti-oxidant role of the polar compounds from PSO.

Editorial to the Special Issue "Plant Extracts: From Extract Technology to Health Benefits".

The valorization of food industry waste is essential to the sustainable development of the agro-food industry, starting from the extraction of plant special metabolites, a challenge that still exists today [...].

Application of PLA-Based Films to Preserve Strawberries' Bioactive Compounds.

Poly-(Lactic Acid) (PLA) is regarded as one of the most promising bio-based polymers due to its biocompatibility, biodegradability, non-toxicity, and processability. The investigation of the potential of PLA films in preserving the quality of strawberries is fully in line with the current directives on the sustainability of food packaging. The study aims to investigate the effects of PLA films on strawberries' physical and chemical properties, thereby determining whether they can be used as a post-harvest solution to control antioxidant loss, reduce mold growth, and extend the shelf-life of strawberries. Well-designed PLA films with different-sized holes obtained by laser perforation (PLA0, PLA16 and PLA23) were tested against a conventional packaging polypropylene (PP) tray for up to 20 days of storage. Weight loss and mold growth were significantly slower in strawberries packed in PLA films. At the same time, PLA-based films effectively preserved the deterioration of vitamin C content, polyphenols and antioxidant activity compared to the control. Furthermore, among all, the micro-perforated PLA film (PLA23) showed better preservation in the different parameters evaluated. These results could effectively inhibit the deterioration of fruit quality, showing promising expectations as an effective strategy to extend the shelf-life of strawberries.

Senolytic Flavonoids Enhance Type-I and Type-II Cell Death in Human Radioresistant Colon Cancer Cells through AMPK/MAPK Pathway.

Resistance to cancer therapies remains a clinical challenge and an unsolved problem. In a previous study, we characterized a new colon cancer cell line, namely HT500, derived from human HT29 cells and resistant to clinically relevant levels of ionizing radiation (IR). Here, we explored the effects of two natural flavonoids, quercetin (Q) and fisetin (F), well-known senolytic agents that inhibit genotoxic stress by selectively removing senescent cells. We hypothesized that the biochemical mechanisms responsible for the radiosensitising effects of these natural senolytics could intercept multiple biochemical pathways of signal transduction correlated to cell death resistance. Radioresistant HT500 cells modulate autophagic flux differently than HT29 cells and secrete pro-inflammatory cytokines (IL-8), commonly associated with senescence-related secretory phenotypes (SASP). Q and F inhibit PI3K/AKT and ERK pathways, which promote p16INK4 stability and resistance to apoptosis, but they also activate AMPK and ULK kinases in response to autophagic stress at an early stage. In summary, the combination of natural senolytics and IR activates two forms of cell death: apoptosis correlated to the inhibition of ERKs and lethal autophagy dependent on AMPK kinase. Our study confirms that senescence and autophagy partially overlap, share common modulatory pathways, and reveal how senolytic flavonoids can play an important role in these processes.

A Comparative Study between Microwave Hydrodiffusion and Gravity (MHG) and Ultrasound-Assisted Extraction (UAE): Chemical and Biological Characterization of Polyphenol-Enriched Extracts from Aglianico Grape Pomace.

The wine industry produces large amounts of grape pomace (GP), a waste that needs to be disposed of properly. Bioactive compounds with high added value can be recovered from GP as an interesting strategy to reduce the environmental impact. Here, two different technologies were employed to recover polyphenol compounds from GP: microwave hydrodiffusion and gravity (MHG) and ultrasound-assisted extraction (UAE). The further purification of UAE and MHG extracts was carried out through solid-phase extraction (SPE) to obtain three fractions, F1, F2 and F3. ATR-FTIR analysis confirmed the presence of sugar and polysaccharide components in F1, as well as non-anthocyanin and anthocyanin compounds in F2 and F3, respectively. Also, the chemical profile was determined by HPLC-UV-DAD, identifying the presence of catechin in F2, and malvidin-3-O-glucoside chloride and cyanidin chloride derivative as the main anthocyanin compounds in F3. The fractions and their parental extracts were characterized for total phenolic content (TPC) and scavenger activity by in vitro assays. We found that F2-MHG and F3-MHG contained phenol contents 6.5 and 8.5 times higher than those of the parental non-fractionated extracts. Finally, F3-MHG (100 µg/mL, w/v) was shown to reduce the proliferation of HT-29 cells.

Molecular Docking of Natural Compounds for Potential Inhibition of AhR.

The aryl hydrocarbon receptor (AhR) is a highly conserved environmental sensor, historically known for mediating the toxicity of xenobiotics. It is involved in numerous cellular processes such as differentiation, proliferation, immunity, inflammation, homeostasis, and metabolism. It exerts a central role in several conditions such as cancer, inflammation, and aging, acting as a transcription factor belonging to the basic helix-loop-helix/Per-ARNT-Sim (bHLH-PAS) protein family. A key step in the canonical AhR activation is AhR-ARNT heterodimerization followed by the binding to the xenobiotic-responsive elements (XREs). The present work aims to investigate the potential AhR inhibitory activity of selected natural compounds. Due to the absence of a complete structure of human AhRs, a model consisting of the bHLH, the PAS A, and the PAS B domains was constructed. Blind and focused docking simulations revealed the presence of further binding pockets, different from the canonical one presented in the PAS B domain, which could be important for AhR inhibition due to the possibility to impede AhR:ARNT heterodimerization, either preventing conformational changes or masking crucial sites necessary for protein-protein interaction. Two of the compounds retrieved from the docking simulations, i.e., ß-carotene and ellagic acid, confirmed their capacity of inhibiting benzo[a]pyrene (BaP)-induced AhR activation in in vitro tests on the human hepatoma cell line HepG2, validating the efficacy of the computational approach.

Ancient Grain Flours with Different Degrees of Sifting: Advances in Knowledge of Nutritional, Technological, and Microbiological Aspects.

Ancient grains have gained considerable attention in recent years, as some research suggests they may be healthier than modern wheat. The present study aims to evaluate the chemical, rheological, and microbiological features of three Southern Italian cultivated ancient wheat varieties: Risciola, Carosella, and Saragolla. ATR-FTIR analyses were performed on the finely ground grain samples of the three varieties. The selected grains were ground with a stone mill, and different sifting degrees (whole-100%, type 1-80%, and type 0-72%) were evaluated. The flours showed a good nutritional profile, a higher amylose/amylopectin ratio, and a lower glycemic index than the literature. The gluten index of the samples was in the range 2.6-28.9%, and the flours can be classified as weak, having a value <30%. The farinographic test showed a short development time, low dough stability, a high softening degree, and water absorption, which increased with the degree of sifting. Microbiological analyses performed on flours from ancient grains at different degrees of sifting show their safety, according to their microbiological parameters, which fall within the legal microbiological requirements established by the European Commission Regulation (EC).

Phenolic Extract from Extra Virgin Olive Oil Induces Different Anti-Proliferative Pathways in Human Bladder Cancer Cell Lines.

Regular consumption of olive oil is associated with protection against chronic-degenerative diseases, such as cancer. Epidemiological evidence indicates an inverse association between olive oil intake and bladder cancer risk. Bladder cancer is among the most common forms of cancer; in particular, the transitional cell carcinoma histotype shows aggressive behavior. We investigated the anti-proliferative effects of a phenolic extract prepared from an extra virgin olive oil (EVOOE) on two human bladder cancer cell lines, namely RT112 and J82, representing the progression from low-grade to high-grade tumors, respectively. In RT112, the EVOOE reduced cell viability (IC50 = 240 µg/mL at 24 h), triggering a non-protective form of autophagy, evidenced by the autophagosome formation and the increase in LC-3 lipidation. In J82, EVOOE induced a strong decrease in cell viability after 24 h of treatment (IC50 = 65.8 µg/mL) through rapid and massive apoptosis, assessed by Annexin V positivity and caspase-3 and -9 activation. Moreover, in both bladder cancer cell lines, EVOOE reduced intracellular reactive oxygen species, but this antioxidant effect was not correlated with its anti-proliferative outcomes. Data obtained suggest that the mixture of phenolic compounds in extra virgin olive oil activates different anti-proliferative pathways.

Redox regulation by carotenoids: Evidence and conflicts for their application in cancer.

Carotenoids have been constantly investigated since the early fifty for their chemical, biochemical and biological properties being presence in foods. Among the more than 1100 carotenoids synthesized by plants and microorganisms, approximately 50 are present in the human diet, and about 20 can be detected in human blood and tissues. Review articles that discuss the anticancer and cancer preventing activity of phytochemicals have often in common the difficulty to find a coherency between the results deriving from experimental studies and the controversial or weak clinical indications arising from epidemiological and interventional studies. In this scenario, the class of carotenoids does not represent an exception. In fact, according with World Cancer Research Fund, strong evidence exists that high-dose supplementation of ß-carotene increases the risk of lung cancer, while for other types of cancer, the protective or harmful effects of food-containing carotenoids or carotenoid supplements have been considered limited, suggestive or unlikely. The analysis of the mechanistic evidence is complicated by the double nature of carotenoids being molecules acting either as antioxidant or pro-oxidant compounds. The present review analyzes the ambiguity and the unexpected results deriving from the epidemiological and interventional studies and discusses how the effects of carotenoids on cancer risk can be explained by understanding their capacity to modulate the cellular antioxidant response, depending on the concentration applied and the cellular metabolism. In the final part, a new global approach is proposed to study the contribution of carotenoids, but also of other phytochemicals, to disease prevention, including cancer.

Nutritional Quality of Pasta Sold on the Italian Market: The Food Labelling of Italian Products (FLIP) Study.

Pasta represents a staple food in many populations and, in recent years, an increasing number of pasta items has been placed on the market to satisfy needs and trends. The aims of this work were: (i) to investigate the nutritional composition of the different types of pasta currently sold in Italy by collecting the nutrition facts on their packaging; (ii) to compare energy, nutrient and salt content per 100 g and serving in fresh and dried pasta; (iii) to compare the nutrition declaration in pairs of products with and without different declarations (i.e., gluten free (GF), organic, and nutrition claims (NC)). A total of 756 items, made available by 13 retailers present on the Italian market, were included in the analysis. Data showed a wide difference between dried and fresh pasta, with high inter-type variability. A negligible amount of salt was observed in all types of pasta, except for stuffed products, which had a median high quantity of salt (>1 g/100 g and ~1.5 g/serving). Organic pasta had higher fibre and lower protein contents compared to conventional pasta. GF products were higher in carbohydrate and fat but lower in fibre and protein than not-GF products, while only a higher fibre content was found in pasta with NC compared to products not boasting claims. Overall, the results show high variability in terms of nutrition composition among the pasta items currently on the market, supporting the importance of reading and understanding food labels for making informed food choices.

The Pro-Oxidant Activity of Red Wine Polyphenols Induces an Adaptive Antioxidant Response in Human Erythrocytes.

The protective effect of dealcoholized red wine on human health has been partially associated with its polyphenolic components, suggesting that the pool of polyphenols, including flavonoids and anthocyanins, can be responsible for the functional effects of this beverage. We hypothesize a new role of red wine polyphenols (RWp) in modulating the antioxidant potential of erythrocytes, protecting them against oxidative stress. We previously demonstrated that RWp activated the Plasma Membrane Redox System (PMRS), which is involved in neutralizing plasma free radicals. Here, we investigated the underlying mechanism triggered by RWp in the activation of PMRS via the involvement of GSH. Hence, treatment of human erythrocytes with RWp (73 µg/mL Gallic Acid Equivalents) increased GSH intracellular concentration, which depends upon the activation of glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PD), whose enzymatic activities increase of about 30% and 47%, respectively. Changes in the GSH pathway induced by RWp were associated with a slight but significant increase in reactive oxygen species (ROS). We conclude that the pro-oxidant effect of RWp promoted an adaptive stress response in human erythrocytes, which enhances their antioxidant defense.

Roles of flavonoids against coronavirus infection.

In terms of public health, the 21st century has been characterized by coronavirus pandemics: in 2002-03 the virus SARS-CoV caused SARS; in 2012 MERS-CoV emerged and in 2019 a new human betacoronavirus strain, called SARS-CoV-2, caused the unprecedented COVID-19 outbreak. During the course of the current epidemic, medical challenges to save lives and scientific research aimed to reveal the genetic evolution and the biochemistry of the vital cycle of the new pathogen could lead to new preventive and therapeutic strategies against SARS-CoV-2. Up to now, there is no cure for COVID-19 and waiting for an efficacious vaccine, the development of "savage" protocols, based on "old" anti-inflammatory and anti-viral drugs represents a valid and alternative therapeutic approach. As an alternative or additional therapeutic/preventive option, different in silico and in vitro studies demonstrated that small natural molecules, belonging to polyphenol family, can interfere with various stages of coronavirus entry and replication cycle. Here, we reviewed the capacity of well-known (e.g. quercetin, baicalin, luteolin, hesperetin, gallocatechin gallate, epigallocatechin gallate) and uncommon (e.g. scutellarein, amentoflavone, papyriflavonol A) flavonoids, secondary metabolites widely present in plant tissues with antioxidant and anti-microbial functions, to inhibit key proteins involved in coronavirus infective cycle, such as PLpro, 3CLpro, NTPase/helicase. Due to their pleiotropic activities and lack of systemic toxicity, flavonoids and their derivative may represent target compounds to be tested in future clinical trials to enrich the drug arsenal against coronavirus infections.

Mechanisms of aging and potential role of selected polyphenols in extending healthspan.

Aging became a priority in medicine due to the rapid increase of elderly population and age-related diseases in the Western countries. Nine hallmarks have been identified based on their alteration during aging and their capacity to increase longevity. The pathways and the molecular mechanisms to improve lifespan and healthspan are controlled by behavioral, pharmacologic and dietary factors, which remain largely unknown. Among them, naturally occurring compounds, such as polyphenols, are considered potential antiaging agents, because of their ability to modulate some of the evolutionarily conserved hallmarks of aging, including oxidative damage, inflammation, cell senescence, and autophagy. Initially, these compounds gained researchers' attention due to their ability to extend the lifespan of simple model organisms. More recently, some of them have been proposed as senolytic agents to protect against age-related disorders, such as cancer, cardiovascular and neurodegenerative diseases. The intent of this review is to present the most validated molecular mechanisms regulating ageing and longevity and critically analyze how selected polyphenols, namely resveratrol, quercetin, curcumin and catechins, can interfere with these mechanisms.