Reassessing the role of phytochemicals in cancer chemoprevention.

In this comprehensive review we tried to reassess the role of phytochemicals in cancer chemoprevention. The exploration of the "synergistic effect" concept, advocating combined chemopreventive agents, faces challenges like low bioavailability. The review incorporates personal, occasionally controversial, viewpoints on natural compounds' cancer preventive capabilities, delving into mechanisms. Prioritizing significant contributions within the vast research domain, we aim stimulating discussion to provide a comprehensive insight into the evolving role of phytochemicals in cancer prevention. While early years downplayed the role of phytochemicals, the late nineties witnessed a shift, with leaders exploring their potential alongside synthetic compounds. Challenges faced by chemoprevention, such as limited pharmaceutical interest and cost-effectiveness issues, persist despite successful drugs. Recent studies, including the EPIC study, provide nuanced insights, indicating a modest risk reduction for increased fruit and vegetable intake. Phytochemicals, once attributed to antioxidant effects, face scrutiny due to low bioavailability and conflicting evidence. The Nrf2-EpRE signaling pathway and microbiota-mediated metabolism emerge as potential mechanisms, highlighting the complexity of understanding phytochemical mechanisms in cancer chemoprevention.

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.

Stillage Waste from Strawberry Spirit Production as a Source of Bioactive Compounds with Antioxidant and Antiproliferative Potential.

The production of fruit distillates generates solid residues which are potentially rich in bioactive compounds worthy of valorization and exploitation. We report herein the in vitro antioxidant and antiproliferative properties of an extract obtained from the waste of fermented strawberry distillate production. The main low molecular weight phenolic components of the extract were identified as ellagic acid and p-coumaric acid using spectroscopic and chromatographic analysis. The extract exhibited high antioxidant properties, particularly in the ferric reducing/antioxidant power (FRAP) assay, and a high total phenolic content (TPC). It was also able to induce an antiproliferative effect on different human cancer cell lines. A strong decrease in viability in human promyelocytic leukemia (HL-60) cells through a rapid and massive apoptosis were observed. Moreover, at an early time (<30 min), reactive oxygen species (ROS) production and inactivation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinases (MAPK) pathway were detected. Notably, the antiproliferative activity of the sample was comparable to that observed with an analogous extract prepared from unfermented, fresh strawberries. These results bring new opportunities for the valorization of fruit distillery by-products as low-cost resources for the design of bioactive formulations of comparable value to that from fresh food.

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.

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.

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.

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.

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.

Antioxidant and Chemopreventive Effect of Aliophen® Formulation Based on Malts and Hops.

Experimental and clinical studies evidenced the health effects of moderate consumption of beer, mainly due to the presence of bioactive compounds, such as polyphenols, vitamins, or fibers. To exploit the potential beneficial effect on health and in disease prevention of these compounds, a new beverage based on barley malts and hops named Aliophen® has been designed, through a patented production process, with a high total polyphenolic amount compared to alcohol-free beer and similar to the one present in light and dark beers. In the present study, the antioxidant activity of Aliophen® against low-density lipoprotein (LDL) oxidation and its ability to protect erythrocytes from hemolysis have been characterized. Moreover, the chemopreventive effect of Aliophen® against colon cancer has been assessed, employing a mouse model of chemically induced carcinogenesis using azoxymethane (AOM). Data obtained showed that Aliophen at a low dose (3 mg/kg) inhibited the formation of preneoplastic lesions, polyps, and tumors. At higher doses (300 mg/kg) the protective effect was measured in the first phase of the onset of cancer. The antioxidant properties of Aliophen® were also observed in AOM-treated mice where it increased the serum antioxidant capacity. Based on the data presented, Aliophen® can exert promising health effects, including an anticancer capacity presumably associated with its antioxidant properties.

Nrf2 targeting by sulforaphane: A potential therapy for cancer treatment.

In the past decades, extensive studies have reported the potential chemopreventive activity of sulforaphane, an isothiocyanate derived from glucoraphanin, occurring in large amounts in Brassica genus plants. Sulforaphane was found to be active against several forms of cancer. A growing body of data shows that sulforaphane acts against cancer at different levels, from development to progression, through pleiotropic effects. In this review, we discuss the available experimental and clinical data on the potential therapeutic role of sulforaphane against cancer. Its effects range from the protection of cells from DNA damage to the modulation of the cell cycle via pro-apoptotic, anti-angiogenesis and anti-metastasis activities. At molecular level, sulforaphane modulates cellular homeostasis via the activation of the transcription factor Nrf2. Although data from clinical studies are limited, sulforaphane remains a good candidate in the adjuvant therapy based on natural molecules against several types of cancer.

Antioxidant polyphenols in cancer treatment: Friend, foe or foil?

Cancer prevention can be probably obtained with easier, faster and less financial strains by pursuing educational programs aimed to induce changes in lifestyle, starting from dietary habits. In the past decades, observational and case-control studies tried to establish a functional relationship between cancer mortality and morbidity and diet. The field becomes even more intricate when scientists investigated which dietary components are responsible for the putative, protective effects of fruits and vegetables against cancer. A relevant part of the literature focused on the positive role of "antioxidant" compounds in foods, including polyphenols. The present review critically evaluate clinical and pre-clinical studies based on polyphenol administration, which contributed to support the concept, deeply rooted in the general population, that antioxidant polyphenols can fight cancer. The controversial and contradictory issues related to the pros and cons on the use of polyphenols against cancer reflect the confounding assumption that cancer treatment and cancer prevention may overlap. We conclude that a clear cut must be done between these two concepts and that the experimental approaches to investigate one or the other should be significantly different, starting from adequate and specifically selected cellular models.

CK2 and PI3K are direct molecular targets of quercetin in chronic lymphocytic leukaemia.

Despite the encouraging results of the innovative therapeutic treatments, complete remission is uncommon in patients affected by chronic lymphocytic leukaemia, which remains an essentially incurable disease. Recently, clinical trials based on BH3-mimetic drugs showed positive outcomes in subjects with poor prognostic features. However, resistance to treatments occurs in a significant number of patients. We previously reported that the multi-kinase inhibitor quercetin, a natural flavonol, restores sensitivity to ABT-737, a BH3-mimetic compound, in both leukemic cell lines and B-cells isolated from patients. To identify the molecular target of quercetin, we employed a new cell line, HG3, obtained by immortalization of B-cells from a chronic lymphocytic leukaemia patient at the later stage of disease. We confirmed that quercetin in association with ABT-737 synergistically enhances apoptosis in HG3 (combination index < 1 for all fractions affected). We also reported that the cellular uptake of quercetin is extremely rapid, with an intracellular concentration of about 38.5 ng/106 cells, after treatment with 25 µM for 5 min. We demonstrated that the activity of protein kinase CK2, which positively triggers PI3K/Akt pathway by inactivating PTEN phosphatase, is inhibited by quercetin immediately after its addition to HG3 cells (0-2 min). PI3K activity was also inhibited by quercetin within 60 min from the treatment. The combined inhibition of CK2 and PI3K kinase activities by quercetin restored ABT-737 sensitivity and increased lethality in human leukemia cells.

A Carotenoid Extract from a Southern Italian Cultivar of Pumpkin Triggers Nonprotective Autophagy in Malignant Cells.

Carotenoids, including ß-carotene, lycopene, and derivatives, such as retinoic acid, have been studied for their significant antiproliferative and differentiating activity on cancer cells in experimental models and in clinics. We are presenting here data on the mechanism of action of a carotenoid-enriched extract obtained from the pumpkin Cucurbita moschata, variety "long of Naples," on two malignant human cell lines, Caco-2 and SAOs, derived from a colon adenocarcinoma and an osteosarcoma, respectively. The carotenoid extract has been obtained from pumpkin pulp and seeds by supercritical CO2 extraction and employed to prepare oil-in-water nanoemulsions. The nanoemulsions, applied at a final carotenoid concentration of 200-400 µg/ml, were not cytotoxic, but induced a delay in cell growth of about 40% in both SAOs and Caco-2 cell lines. This effect was associated with the activation of a "nonprotective" form of autophagy and, in SAOs cells, to the induction of cell differentiation via a mechanism that involved AMPK activation. Our data suggest the presence of a pool of bioactive compounds in the carotenoid-enriched extract, acting additively, or synergistically, to delay cell growth in cancer cells.

A Phenolic Extract Obtained from Methyl Jasmonate-Treated Strawberries Enhances Apoptosis in a Human Cervical Cancer Cell Line.

In the present study, we evaluated the effect of methyl jasmonate (MeJA) treatment on strawberry phenolic composition. Strawberry extracts contain a mixture of phenolic compounds possessing several biological properties. We demonstrated that these extracts were more effective in inducing apoptosis in HeLa cells compared to phenolic preparations derived from untreated strawberries. Treatment of strawberries with 0.5% MeJA resulted in increased polyphenols content (from 7.4 to 8.6 mM quercetin equivalents) and antioxidant properties (from 3.9 to 4.6 mM quercetin equivalents). The identification and quantification of phenolic compounds by liquid chromatography-mass spectrometry in the strawberry extracts showed that cyanidin glucoside, pelargonidin glucoside, and ellagic glucoside acid were significantly higher in strawberries treated with MeJA. Phenolic extracts from MeJA-treated strawberries significantly decreased the cell viability in HeLa cells, compared to extracts derived from untreated fruits. We hypothesized that the enhanced apoptotic activity of MeJA-treated strawberries was due to a synergistic or additive effect of different phenolic compounds present in the extract, rather than the activity of a single molecule.

Red Wine Inhibits Aggregation and Increases ATP-diphosphohydrolase (CD39) Activity of Rat Platelets in Vitro.

Moderate consumption of red wine has been shown to exert a peculiar cardioprotective effect compared with other alcoholic beverages; inhibition of platelet aggregation seems to be one of the mechanisms underlying this beneficial effect. CD39/ATP-diphosphohydrolase is an integral membrane glycoprotein metabolizing ATP and ADP to AMP; in concert with CD73/ecto-5'-nucleotidase, it contributes to extracellular adenosine accumulation. CD39 is considered a key modulator of thrombus formation; it inhibits platelet aggregation by promoting ADP hydrolysis. There is evidence that red wine consumption increases CD39 activity in platelets from streptozotocin-induced diabetic rats. Here we show that two kinds of Aglianico red wines inhibit aggregation and increase ATP--and ADPase activity in rat platelets.

Nrf2 as molecular target for polyphenols: A novel therapeutic strategy in diabetic retinopathy.

Diabetic retinopathy is a microvascular complication of diabetes that is considered one of the leading causes of blindness among adults. More than 4.4 million people suffer from this disorder throughout the world. Growing evidence suggests that oxidative stress plays a crucial role in the pathophysiology of diabetic retinopathy. Nuclear factor erythroid 2-related factor 2 (Nrf2), a redox sensitive transcription factor, plays an essential protective role in regulating the physiological response to oxidative and electrophilic stress via regulation of multiple genes encoding antioxidant proteins and phase II detoxifying enzymes. Many studies suggest that dozens of natural compounds, including polyphenols, can supress oxidative stress and inflammation through targeting Nrf2 and consequently activating the antioxidant response element-related cytoprotective genes. Therefore, Nrf2 may provide a new therapeutic target for treatment of diabetic retinopathy. In the present article, we will focus on the role of Nrf2 in diabetic retinopathy and the ability of polyphenols to target Nrf2 as a therapeutic strategy.

Neuroprotective Role of Natural Polyphenols.

Neurodegenerative diseases cause a progressive functional alteration of neuronal systems, resulting in a state of dementia which is considered one of the most common psychiatric disorders of the elderly. Dementia implies an irreversible impairment of intellect that increases with age causing alteration of memory, language and behavioral problems. The most common form, which occurs in more than half of all cases, is Alzheimer's disease, characterized by accumulation of amyloid plaques and neurofibrillary tangles. Neuroinflammation and oxidative stresses have been considered as a hallmark of Alzheimer disease, playing a crucial role in neurotoxicity. For this reason, an adequate antioxidant strategy may improve the treatment of neurodegenerative diseases and dementia. Several studies support the neuroprotective abilities of polyphenolic compounds resulting in neuronal protection against injury induced by neurotoxins, ability to suppress neuroinflammation and the potential to promote memory, learning and cognitive functions. We critically reviewed here the therapeutic potential of pure herbal compounds (e.g., green tea polyphenol (-)- epigallocatechin-3-gallate, resveratrol, curcumin, quercetin and others) and extracts enriched in polyphenols showing the most promising neuroprotective effects. We are also presenting data on the ability of an extract derived from elderberry, Sambucus nigra, possessing elevated polyphenolic content and antioxidant capacity to protect neuronal cells against oxidizing agents.

Red wine activates plasma membrane redox system in human erythrocytes.

In the present study, we report that polyphenols present in red wine obtained by a controlled microvinification process are able to protect human erythrocytes from oxidative stress and to activate Plasma Membrane Redox System (PMRS). Human plasma obtained from healthy subjects was incubated in the presence of whole red wine at a concentration corresponding to 9.13-73 µg/ml gallic acid equivalents to verify the capacity to protect against hypochlorous acid (HOCl)-induced plasma oxidation and to minimize chloramine formation. Red wine reduced hemolysis and chloramine formation induced by HOCl of 40 and 35%, respectively. PMRS present on human erythrocytes transfers electrons from intracellular molecules to extracellular electron acceptors. We demonstrated that whole red wine activated PMRS activity in human erythrocytes isolated from donors in a dose-dependent manner with a maximum at about 70-100 µg/ml gallic acid equivalents. We also showed that red wine increased glutathione (GSH) levels and erythrocytic antioxidant capacity, measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) quenching assay. Furthermore, we reported that GSH played a crucial role in regulating PMRS activity in erythrocytes. In fact, the effect of iodoacetamide, an alkylating agent that induces depletion of intracellular GSH, was completely counteracted by red wine. Bioactive compounds present in red wine, such as gallic acid, resveratrol, catechin, and quercetin were unable to activate PMRS when tested at the concentrations normally present in aged red wines. On the contrary, the increase of PMRS activity was associated with the anthocyanin fraction, suggesting the capacity of this class of compounds to positively modulate PMRS enzymatic activity.